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EP4602174A1 - Viral particles targeting hematopoietic stem cells - Google Patents

Viral particles targeting hematopoietic stem cells

Info

Publication number
EP4602174A1
EP4602174A1 EP23802116.6A EP23802116A EP4602174A1 EP 4602174 A1 EP4602174 A1 EP 4602174A1 EP 23802116 A EP23802116 A EP 23802116A EP 4602174 A1 EP4602174 A1 EP 4602174A1
Authority
EP
European Patent Office
Prior art keywords
protein
seq
particle
niv
biologically active
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23802116.6A
Other languages
German (de)
French (fr)
Inventor
Luca BIASCO
Patricia CRUITE
Matteo STOPPATO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sana Biotechnology Inc
Original Assignee
Sana Biotechnology Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sana Biotechnology Inc filed Critical Sana Biotechnology Inc
Publication of EP4602174A1 publication Critical patent/EP4602174A1/en
Pending legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
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    • C12N2510/00—Genetically modified cells
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/11011—Alpharetrovirus, e.g. avian leucosis virus
    • C12N2740/11022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16045—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18211—Henipavirus, e.g. hendra virus
    • C12N2760/18222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Lipid particles including viral and virus-like particles, such as lentiviral particles, are commonly used for delivery of exogenous agents to cells.
  • delivery to certain target cells can be challenging.
  • Improved lipid particles, such as lentiviral particles, and methods for delivery are needed. The provided disclosure addresses this need.
  • the embodiments herein provide for lipid particles that allow for efficient targeting and delivery of an exogenous agent contained therein to hematopoietic stem/progenitor cells (HSPC), including to naive hematopoietic stem cells (HSCs).
  • the targeting can be to such stem or progenitor cells in a human subject, such as human HSPC.
  • the lipid particles can include viral vectors such as lentiviral vectors that are pseudotyped with a fusogen with a preferential ability to target HSPC.
  • the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof, such as the exemplarly fusogens BaEVTR or BaEVRLess.
  • the lipid particle includes a targeting agent to target the fusogen to HSPCs, such as to CD34+ cells.
  • the targeting agent is fused to a fusogen, such as a viral envelope attachment glycoprotein, exposed on the lipid bilayer to provide a retargeted fusogen.
  • a targeted lipid particle comprising a lipid bilayer, a fusogen, and a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3, wherein the fusogen and the targeting agent are exposed on the surface of the lipid bilayer.
  • the targeting agent is fused to a transmembrane domain incorporated into the lipid bilayer. In some embodiments, the targeting agent is fused to the fusogen.
  • the particle is a viral particle or virus-like particle.
  • the lipid bilayer is derived from a membrane of a producer cell used for producing a viral particle or virus-like particle.
  • the viral particle or virus-like particle is a retroviral particle or retrovirus-like particle.
  • the viral particle or virus-like particle is a lentivirus-like particle.
  • the viral particle or virus-like particle is a lentiviral particle.
  • a targeted lentiviral particle comprising a fusogen and a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3, wherein the fusogen and the targeting agent are exposed on the surface of the lentiviral particle.
  • the particle is pseudotyped with the fusogen.
  • the targeting agent is fused to the fusogen. In some of any embodiments, the targeting agent is fused to the fusogen directly. In some of any embodiments, the targeting agent is fused to the fusogen via a linker.
  • the targeting agent is fused to a transmembrane domain incorporated into the viral envelope.
  • the particle is prepared by a method including transducing a producer cell with plasmids encoding the fusogen and targeting agent and a Gag-pol and Rev.
  • the producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells.
  • the producer cell is a 293T cell.
  • the particle is replication defective.
  • the viral nucleic acid comprises one or more, optionally all, of the following nucleic acid sequences: 5’ LTR (e.g., containing U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT)/central termination sequence (CTS) (e.g., DNA flap), Poly A tail sequence, a posttranscriptional regulatory element (e.g., WPRE), a Rev response element (RRE), and 3’ LTR (e.g., containing U5 and lacking a functional U3).
  • 5’ LTR e.g., containing U5 and lacking a functional U3 domain
  • Psi packaging element Psi packaging element
  • cPPT Central polypurine tract
  • CTS central termination sequence
  • Poly A tail sequence e.g., DNA flap
  • WPRE posttranscriptional regulatory element
  • RRE Rev response element
  • 3’ LTR e.g., containing U5 and lacking a functional U3
  • the particle is devoid of viral genomic nucleic acids.
  • the target molecule is ASCT2. In some of any embodiments, the target molecule is CD 117.
  • the fusogen is a VSV-G protein or a functional variant thereof.
  • the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
  • the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild-type BaEV inhibitory R peptide.
  • the fusogen is set forth in any of SEQ ID NO:254-260.
  • the fusogen is set forth in SEQ ID NO:261 (BaEVRLess).
  • the fusogen is set forth in SEQ ID NO:262 (BaEVTR).
  • a targeted lipid particle comprising a lipid bilayer, a fusogen, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof and is exposed on the surface of the lipid bilayer.
  • the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof and is exposed on the surface of the lipid bilayer.
  • BaEV baboon endogenous virus
  • the fusogen is a Cocal virus G protein or a functional variant thereof.
  • the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
  • the fusogen is a Paramyxovirus fusion protein (e.g., a Morbilli virus or Henipavirus) or a functional variant thereof.
  • the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbilli virus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
  • Morbillivirus fusion protein e.g., measles virus (MeV), canine distemper virus, Cetacean morbilli virus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus
  • the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
  • the fusogen is a Nipah virus fusion protein or a functional variant thereof.
  • the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
  • the targeting agent is fused to the NiV-G protein or biologically active portion thereof. In some of any embodiments, the targeting agent is fused to the C-terminus of the NiV-G protein or biologically active portion thereof.
  • the NiV-G or biologically active portion thereof is a wildtype NiV-G protein or a functionally active variant or biologically active portion thereof. In some of any embodiments, the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4.
  • the NiV-G protein or biologically active portion thereof does not include an initial methionine. In some of any embodiments, NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
  • the fusogen comprises one or modifications to reduce binding to its native receptor.
  • the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
  • the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
  • the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
  • the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.
  • the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30.
  • the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30.
  • the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16.
  • the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
  • the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
  • the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule.
  • the targeting agent is a single domain antibody that binds to the target molecule.
  • the targeting agent is a VHH that binds to the target molecule.
  • the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
  • the target molecule is expressed on a target cell.
  • the particle comprises an exogenous agent for delivery to a target cell.
  • the target molecule is expressed on the target cell.
  • the particle comprises a fusion protein between a viral structural protein and an exogenous agent.
  • the fusion protein is a cleavable fusion protein comprising a cleavable linker positioned between the viral structural protein and the exogenous agent.
  • the fusion protein comprises one or more nuclear localization sequences. In some of any of the provided embodiments, the fusion protein comprises one or more nuclear export sequences.
  • the exogenous agent is present in the lumen.
  • the exogenous agent is a protein or a nucleic acid.
  • the exogenous agent is or encodes a therapeutic agent or a diagnostic agent.
  • the exogenous agent is or encodes a factor associated with gene editing.
  • the exogenous agent is or encodes a genome-modifying protein for gene editing a target gene encoding an endogenous protein.
  • the genome-modifying protein is associated with gene editing by a sequence-specific nuclease, a CRISPR- associated transposase (CAST), prime editing, or Programmable Addition via Site-specific Targeting Elements (PASTE).
  • CAST CRISPR- associated transposase
  • PASTE Programmable Addition via Site-specific Targeting Elements
  • the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
  • the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild-type BaEV inhibitory R peptide.
  • the fusogen is set forth in any of SEQ ID NO:254-260.
  • the fusogen is set forth in SEQ ID NO:261 (BaEVRLess).
  • the fusogen is set forth in SEQ ID NO:262 (BaEVTR).
  • the NiV-G protein or biologically active portion thereof does not include an initial methionine. In some of any embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
  • the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
  • the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
  • the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.
  • the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30.
  • the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C- terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30.
  • the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
  • the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
  • the first and/or second polynucleotide is a vector.
  • the first and/or second polynucleotide is codon optimized.
  • the first polynucleotide comprises at least one promoter that is operatively linked to control expression of the fusogen; and/or the second polynucleotide comprises at least one promoter that is operatively linked to control expression of the targeting agent.
  • the promoter of the first and/or second polynucleotide is a constitutive promoter. In some embodiments, the promoter of the first and/or second polynucleotide is an inducible promoter.
  • the source cell is a mammalian cell. In some of any embodiments, the source cell is a producer cell used for producing a viral particle or virus-like particle. In some embodiments, the particle is a viral particle or virus-like particle. In some of any embodiments, the viral particle or virus-like particle is a retroviral particle or retrovirus-like particle. In some of any embodiments, the viral particle or virus-like particle is a lentivirus-like particle. In some of any embodiments, the viral particle or virus-like particle is a lentiviral particle. In some of any embodiments, the particle is pseudotyped with the fusogen.
  • the producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells.
  • the producer cell is a 293T cell.
  • the target molecule is ASCT2. In some of any embodiments, the target molecule is CD 117. [0094] In some of any embodiments, the fusogen is a viral fusion protein. In some of any embodiments, the fusogen is a viral envelope protein.
  • the fusogen is a VSV-G protein or a functional variant thereof.
  • the fusogen is a Cocal virus G protein or a functional variant thereof.
  • the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
  • the fusogen is a Nipah virus fusion protein or a functional variant thereof.
  • the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
  • the NiV-G or biologically active portion thereof is a wild-type NiV- G protein or a functionally active variant or biologically active portion thereof.
  • the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4.
  • the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4.
  • the NiV-G protein or biologically active portion thereof does not include an initial methionine. In some of any embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
  • the fusogen comprises one or modifications to reduce binding to its native receptor.
  • the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
  • the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
  • the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
  • the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.
  • the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30.
  • the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C- terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30.
  • the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16.
  • the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21. [0104] In some of any embodiments, the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
  • the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule.
  • the targeting agent is a single domain antibody that binds to the target molecule.
  • the targeting agent is a VHH that binds to the target molecule.
  • the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
  • a targeted lipid particle produced by the method of some of any embodiments.
  • a targeted lentiviral particle produced by the method of some of any embodiments.
  • composition comprising a plurality of the lipid particle of some of any embodiments.
  • composition comprising a plurality of the lentiviral particle of some of any embodiments.
  • the composition further comprises a pharmaceutically acceptable excipient.
  • a method of transducing a target cell the method containing contacting a target cell with the lentiviral particle of some of any embodiments or the composition of some of any embodiments.
  • the particle comprises an exogenous agent, and the transduction introduces the exogenous agent into the target cell.
  • a method of delivering an exogenous agent to a target cell the method containing contacting a target cell with the particle of some of any embodiments or the composition of some of any embodiments, the particle or plurality of particles containing an exogenous agent for delivery of the target cell.
  • the contacting is in vitro or ex vivo.
  • a method of delivering an exogenous agent to a target cell in a subject which includes administering to a subject the particle of some of any embodiments or the composition of some of any embodiments, the particle or plurality of particles containing an exogenous agent for delivery to a target cell in the subject.
  • the exogenous agent is or encodes a therapeutic agent or a diagnostic agent.
  • the exogenous agent is for treating a disease or condition in the subject.
  • a method of treating a disease or condition in a subject which includes administering to a subject the particle of some of any embodiments or the composition of some of any embodiments, the particle or plurality of particles containing an exogenous agent for treating a disease or condition in the subject.
  • the method comprises administering to the subject one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood.
  • the subject has previously been administered one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood.
  • a method of treating a disease or condition in a subject which includes administering to a subject: (a) one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood; and (b) a lipid particle containing a lipid bilayer, a fusogen exposed on the surface of the lipid bilayer, and an exogenous agent for treating a disease or condition in the subject.
  • the administration of at least one of the one or more agents that stimulate mobilization is initiated prior to the administration of the particle.
  • the fusogen is a viral fusion protein. In some of any embodiments, the fusogen is a viral envelope protein.
  • the fusogen is a VSV-G protein or a functional variant thereof.
  • the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
  • the fusogen is a Nipah virus fusion protein or a functional variant thereof.
  • the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
  • the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
  • the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
  • the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18. [0140] In some of any embodiments, the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof. In some of any embodiments, the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30.
  • the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C- terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16.
  • the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
  • the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
  • the bone marrow cells are CD34+. In some of any embodiments, the bone marrow cells are CD34+ progenitor cells. In some of any embodiments, the bone marrow cells are hematopoietic stem cells.
  • the one or more agents that stimulate mobilization are selected from the group consisting of stem cell factor (SCF), small molecule VLA-4 inhibitor BI05192, BOP (N-(benzenesnlfonyl)-L-prolyl-L-0-(l-pyrrolidinylcarbonyl)tyrosine), heparin, granulocyte colonystimulating factor (G-CSF), MGTA-145, and plerixafor (AMD3100).
  • SCF stem cell factor
  • VLA-4 inhibitor BI05192 small molecule VLA-4 inhibitor
  • BOP N-(benzenesnlfonyl)-L-prolyl-L-0-(l-pyrrolidinylcarbonyl)tyrosine
  • heparin heparin
  • G-CSF granulocyte colonystimulating factor
  • MGTA-145 MGTA-145
  • plerixafor AMD3100
  • the one or more agents that stimulate mobilization include G- CSF.
  • the G-CSF is administered to the subject daily on the two days, three days, four days, or five days prior to the administration of the first dose of the particle.
  • the G-CSF is administered to the subject on the day of the administration of the first dose of the particle.
  • the G-CSF is administered to the subject on the day of administration of a second or later dose of the particle.
  • the one or more mobilization agents comprise plerixafor.
  • the plerixafor is administered to the subject on the day of the administration of the first dose of the particle.
  • the plerixafor is administered to the subject on the day of the administration of a second or later dose of the particle.
  • the one or more agents that stimulate mobilization are G-CSF and plerixafor.
  • the G-CSF is administered to the subject daily on the four days prior to the administration of the first dose of the particle; and the plerixafor is administered to the subject on the day of the administration of the first dose of the particle.
  • the G-CSF is administered to the subject (i) daily on the two days prior to the administration of the particle; (ii) on the day of the administration of the first dose of the particle; and (iii) on the day of administration of a second or later dose of the particle; and the plerixafor is administered to the subject on the day of administration of a second or later dose of the particle.
  • the method comprises administering to the subject a transduction adjuvant.
  • the transduction adjuvant is administered to the subject on the day of the administration of at least one dose of the particle.
  • the transduction adjuvant is administered to the subject on the days of the administration of at least two doses of the particle.
  • the transduction adjuvant is a cationic peptide. In some embodiments, the transduction adjuvant is a histidine-rich cationic peptide. In some of any embodiments, the transduction adjuvant is a cationic amphipathic peptide. In some of any embodiments, the transduction adjuvant is derived from the LAH4 peptide family. In some of any embodiments, the transduction adjuvant is Vectofusin-1.
  • the target molecule is expressed on the target cell.
  • the target cell is a hematopoietic cell.
  • the target cell is CD34+.
  • the target cell is a CD34+ progenitor cell.
  • the hematopoietic cell is a hematopoietic stem cell.
  • the exogenous agent is present in the lumen.
  • the exogenous agent is a protein or a nucleic acid.
  • the exogenous agent is or encodes a factor associated with gene editing. In some of any embodiments, the exogenous agent is or encodes a genome-modifying protein for gene editing a target gene encoding an endogenous protein. In some embodiments, the exogenous agent is a fusion protein between a viral structural protein and an exogenous agent. In some of any of the provided embodiments, the fusion protein is a cleavable fusion protein comprising a cleavable linker positioned between the viral structural protein and the exogenous agent. In some of any of the provided embodiments, the fusion protein comprises one or more nuclear localization sequences. In some of any of the provided embodiments, the fusion protein comprises one or more nuclear export sequences.
  • the genome-modifying protein is associated with gene editing by a sequence-specific nuclease, a CRISPR-associated transposase (CAST), prime editing, or Programmable Addition via Site-specific Targeting Elements (PASTE).
  • CAST CRISPR-associated transposase
  • PASTE Programmable Addition via Site-specific Targeting Elements
  • the genome -modifying protein is a sequence-specific nuclease.
  • the sequence-specific nuclease is selected from the group consisting of an RNA-guided nuclease, a meganuclease, a transcription activator-like effector nuclease (TALEN), and a zinc-finger nuclease (ZFN).
  • the sequence-specific nuclease is an RNA-guided nuclease.
  • the RNA-guided nuclease is TnpB.
  • the RNA-guided nuclease comprises a Cas nuclease and a guide RNA (CRISPR-Cas combination).
  • CRISPR-Cas combination is a ribonucleoprotein (RNP) complex containing the gRNA and the Cas nuclease.
  • RNP ribonucleoprotein
  • the Cas nuclease is a Type II or Type V Cas protein.
  • the genome-modifying protein is selected from the group consisting of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxll, Csyl, Csy2, Csy3, Mad7.
  • the exogenous agent is or encodes a factor associated with base editing or prime editing (e.g., target-primed reverse transcription (TPRT)).
  • the exogenous agent is or encodes a transposase, integrase, or recombinase.
  • the exogenous agent is or encodes a DNA polymerase, RNA polymerase, or reversetranscriptase.
  • the exogenous agent is for use in gene therapy to correct a genetic deficiency in the target cell.
  • the exogenous agent is a nucleic acid containing a payload gene for correcting the genetic deficiency.
  • the exogenous agent is or encodes a membrane protein.
  • the membrane protein is an antigen receptor.
  • the antigen receptor binds to an antigen expressed on cells associated with a disease or condition in the subject.
  • the disease or condition is a cancer.
  • the antigen receptor binds to an antigen expressed on tumor cells in the subject.
  • the antigen receptor is a chimeric antigen receptor (CAR).
  • the antigen receptor is an engineered T cell receptor (TCR).
  • the delivery to target cells expressing the target molecule is increased by or by greater than 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5- fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, or more compared to delivery to non-target cells not expressing the target molecule.
  • the subject is a human subject.
  • the target molecule is CD 133.
  • the fusogen is fused to a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3.
  • the target molecule is ASCT2.
  • the target molecule is CD 133.
  • the targeting agent is a CD 133 binding agent that is an antibody or antigen-binding fragment comprising a variable heavy (VH) chain and a variable light (VL) chain selected from a) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 271, 272, 273, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 280, 281, 282, respectively, and a VL chain comprising a CDR-L
  • the targeting agent is a CD 133 binding agent that is an antibody or antigen-binding fragment comprising a variable heavy (VH) chain and a variable light (VL) chain selected from a) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 314, 315, and 273, respectively, and a VL comprising a CDR- Ll, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 316, 317, and 282, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequence
  • the CD 133 binding agent is selected from a) a CD 133 binding agent comprising a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 270, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 274, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; b) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 279, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of
  • the CD 133 binding agent is a scFv.
  • the CD 133 binding agent comprises the amino acid sequence set forth in any one of SEQ ID NOs: 269, 278, 287, 296 or 305, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the targeting agent is a CD117 binding agent that is a VHH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent comprises the amino acid sequence set froth in any one of SEQ ID NOS: 324-374.
  • an anti-CDl 17 binding agent comprising a VHH single domain antibody comprising a CDR-H1, a CDR-H2, and a CDR-H3 contained within an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374.
  • an anti-CDl 17 binding agent comprising a VHH single domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOS: 324-374.
  • a viral fusion protein comprising a viral envelope protein and an antiCD 133 binding agent
  • the anti-CDl 33 binding agent is an antibody or antigen-binding fragment comprising a variable heavy (VH) chain and a variable light (VL) chain selected from a) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 314, 315, and 273, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 316, 317, and 282, respectively, and a VL comprising a CDR-L1, a C
  • the CD 133 binding agent is selected from a) a CD 133 binding agent comprising a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 270, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 274, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; b) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 279, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of
  • the CD 133 binding agent is a scFv.
  • the CD 133 binding agent comprises the amino acid sequence set forth in any one of SEQ ID NOs: 269, 278, 287, 296 or 305, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the viral envelope protein is a VSV-G protein or a functional variant thereof. In some of any of the provided embodiments, the viral envelope protein is a Cocal virus G protein or a functional variant thereof.
  • the viral envelope protein is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
  • the viral envelope protein is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof.
  • the viral envelope protein is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
  • the viral envelope protein is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
  • a Henipavirus fusion protein e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus
  • the viral envelope protein is a Nipah virus fusion protein or a functional variant thereof.
  • the viral envelope protein comprises a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
  • the binding agent is fused to the C-terminus of the NiV-G protein or biologically active portion thereof.
  • the NiV-G or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof.
  • the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof does not include an initial methionine.
  • the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 42. In some of any of the provided embodiments, the viral envelope protein comprises one or more modifications to reduce binding to its native receptor.
  • the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
  • the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
  • the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17. In some of any of the provided embodiments, the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18. Brief Description of The Drawings
  • FIG. 1A depicts a schematic of experiments performed to assess the capacity of VSV-G and BaEVTR vectors to transduce human hematopoietic stem cells (HSCs), blood lineage cells, and hepatocytes in vitro.
  • HSCs human hematopoietic stem cells
  • FIG. 1A depicts a schematic of experiments performed to assess the capacity of VSV-G and BaEVTR vectors to transduce human hematopoietic stem cells (HSCs), blood lineage cells, and hepatocytes in vitro.
  • HSCs human hematopoietic stem cells
  • FIG. IB shows results for transduction of hematopoietic cells with VSV-G and BaEVTR lentiviral vectors.
  • FIG. 1C shows results of transduction of hematopoietic stem cells with increasing dilutions with a BaEVTR or VSV-G lentiviral vector.
  • FIG. ID shows additional results of transduction of hematopoietic stem cells with increasing dilutions with a BaEVTR or VSV-G lentiviral vector.
  • FIG. IE shows results of transduction of hematopoietic stem cells with two different Multiplicity of infection values of BaEVTR vector.
  • FIG. 2 shows results for the ex vivo transduction of hematopoietic cells and subsequent transgene engraftment in a mouse model.
  • FIG. 3 shows the phenotypic distribution of hematopoietic cells following humanization of two mouse models.
  • FIG.4A shows an exemplary humanization protocol for mouse experiments described herein in Example 4.
  • FIG. 4B shows distribution of hematopoietic cells following early-stage humanization of a mouse models.
  • FIG. 5A shows the effect of dose schedules on transduction of hematopoietic cells with a BaEVTR vector or a VSV-G vector.
  • FIG.5B shows the effect of mobilization on transduction of hematopoietic cells with a BaEVTR vector.
  • FIG. 6A shows the effect of mobilization on in vivo distribution of hematopoietic cells following early-stage humanization.
  • FIG.6B shows additional results of the effect of mobilization on in vivo distribution of hematopoietic cells following early-stage humanization.
  • FIG.6D shows additional results of the effect of mobilization on transduction of hematopoietic cells with a BaEVTR vector in early-stage humanized mice.
  • FIG.8B shows the effect of co-infusion of BaEVTR vectors blood CD34+ cells on stable transduction of hematopoietic cells after 16 weeks.
  • FIG.8C shows additional results of the effect of co-infusion of BaEVTR vectors and cord-blood CD34+ cells on hematopoietic cell and progenitor cells sub-populations.
  • FIG. 9 shows the effect of different administration schedules on transduction of hematopoietic cells with VSV-G and BaEVTR vectors.
  • FIG. 10A shows results for transduction of hematopoietic cells with VSV-G and BaEVTR vectors and retargeted Nipah vectors.
  • FIG. 10B shows additional results for transduction of hematopoietic cells with VSV-G and BaEVTR vectors and retargeted Nipah vectors.
  • FIG. 15A shows a representative flow plot of B2M gene editing in human hematopoietic cells with the BaEVTR virus-like particles.
  • FIG. 16 C shows , the in vivo knockout of B2M gene (e.g., B2M editing) in different HSPC subpopulations.
  • FIG. 16D shows an exemplary long-term humanization protocol for mice experiments herein in Example 12.
  • FIG. 16E shows the level of editing in long-term humanized mice.
  • FIG. 16F shows true in vivo B2M editing in huCD45+ cells.
  • FIG. 18 A model study design for non-human primates is shown in FIG. 18. Detailed Description
  • the target cell is a hematopoietic stem/progenitor cell (HSPC), such as a CD34+ cell.
  • the lipid particle is a targeted lipid particle that preferentially targets a target molecule expressed on the target cell.
  • the target molecule is expressed on a target cell.
  • the provided particle preferentially targets a target cell compared to a non-target cell.
  • the lipid particles allow for targeted delivery to hematopoietic stem/progenitor cells (HSPC), including to naive hematopoietic cells (HSCs).
  • Exogenous agent delivery that is preferentially targeted to certain target cells can result in long-term and/or systemic genetic engineering of the target cells and/or cells derived therefrom, for instance when engineered target cells are able to differentiate into a variety of cell types.
  • CD34+ progenitor cells which are a heterogeneous cell population that include a subpopulation of hematopoietic stem cells (HSCs), pluripotent stem cells, and cells in the early stages of lineage commitment, can differentiate to produce all hematopoietic cell types found in circulation.
  • CD34+ progenitor cells that are HSCs or multipotent progenitors (MPPs) at the top of the hematopoietic tree may be the most appealing cell targets for achieving long-term and/or systemic genetic engineering across a broad range of hematopoietic cell types.
  • MPPs multipotent progenitors
  • myeloid progenitors and lymphoid progenitors such as myelo-lymphoid progenitors (MLPs), early T progenitors (ETPs), megakaryo-erythroid progenitors (MEPs), common myeloid progenitors (CMPs), and granulo-monocyte progenitors (GMPs).
  • MLPs myelo-lymphoid progenitors
  • EDPs early T progenitors
  • MEPs megakaryo-erythroid progenitors
  • CMPs common myeloid progenitors
  • GMPs granulo-monocyte progenitors
  • the lipid particles can include viral vectors such as lentiviral vectors that are pseudotyped with a fusogen with a preferential ability to target HSPC.
  • the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof, such as the exemplarly fusogens BaEVTR or BaEVRLess.
  • the lipid particle includes a targeting agent to target the fusogen to HSPCs, such as to CD34+ cells.
  • the targeting agent is fused to a fusogen, such as a viral envelope attachment glycoprotein, exposed on the lipid bilayer to provide a retargeted fusogen.
  • a fusogen such as a viral envelope attachment glycoprotein
  • results herein demonstrate that such lipid particles can efficiently target HSPC, including naive HSC.
  • results show that the lipid particles can be delivered in vivo not only to cells in the peripheral blood but that they also can establish basal accesss to human HPSC in the bone marrow (BM) and peripheral blood without relying on high vector doses or selective enrichment.
  • the provided delivery methods also can be combined with mobilization agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood in the subject.
  • results herein show that improved transduction of cells in peripheral blood can be achieved when combined with mobilization.
  • combined mobilization strategies also allow transduction to naive HSC in the bone marrow even where there is not substantial egress of human HSPC into the peripheral blood.
  • examples herein show that embodiments using a provided lipid particle able to target HSPC (e.g. BaEVTR fusogen) allow about 2% targeting of phenotypic HSC compared to undetectable transduction without mobilization.
  • the fusogen of a provided lipid particle is not fused to a targeting agent.
  • a lipid particle e.g. lentiviral vector
  • BaEV baboon endogenous virus
  • such lipid particles can additionally include a targeting agent.
  • the fusogen of a provided lipid particle is a retargeted fusogen in which a viral envelope attachment glycoprotein is linked or fused to a targeted agent.
  • a retargeted fusogen is engineered to recognize receptors on chosen target cell types resulting in highly cell-specific in vivo gene transfer.
  • lipid particles e.g. lentiviral vector
  • the use of a retargeted fusogen can reduce off-target delivery.
  • a retargeted fusogen as shown herein can exhibit striking (e.g. lOOx) specificity increase over other broadly tropic pseudotyped lipid particles in targeting a receptor-positive population, including in target cells exclusively localized in the bone marrow even where such cells make up as little as 0.15% of total human cells.
  • the target molecule bound by the targeting agent of the provided particle is expressed on CD34+ progenitor cells.
  • the target cell is a CD34+ progenitor cell.
  • the target molecule is expressed on all CD34+ progenitor cells.
  • the target molecule is expressed on at least a subset of CD34+ progenitor cells.
  • the target molecule is expressed on HSCs.
  • the target molecule is expressed on MPPs.
  • the target molecule is selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3.
  • CD34+ progenitor cells e.g., subpopulations thereof that included highly pluripotent HSCs and MPPs, as well as myeloid progenitors that included MEPs, CMPs, and GMPs
  • exemplary targeted lipid particles e.g., targeted lentiviral particles, having a targeting agent that binds to a target molecule selected from the foregoing, e.g., CD117.
  • exemplary targeted lipid particles e.g., targeted lentiviral particles, having a targeting agent that binds to a target molecule selected from the foregoing, e.g., ASCT2.
  • the provided particle is any described in Section II.
  • the provided particle is a lipid particle, such as any described in Section II-A.
  • the provided particle is a lipid particle that is a viral particle, such as any described in Section II-A-1.
  • the provided particle is a targeted lentiviral particle.
  • the provided particle is a lipid particle that is a virus-like particle, such as any described in Section II-A-2.
  • the provided particle is a lipid particle that is a cell-based particle, such as any described in Section II-A-3.
  • the provided particle has a fusogen, such as any described in Section II-B.
  • the fusogen contains a mammalian protein, such as any described in Section II-B-1.
  • the fusogen contains a viral protein, such as any described in Section II-B- 2.
  • the fusogen is a baboon endogenous retrovirus, such as BaEVTR.
  • the targeting agent of the provided particle is any described in Section II-C.
  • the target molecule or target cell for any of the provided particles is any described in Section II-C.
  • the provided particle contains an exogenous agent.
  • the exogenous agent is for delivery to the target cell.
  • the provided particle exhibits preferential delivery of the exogenous agent to a target cell compared to a non-target cell.
  • the delivery to target cells is increased by or by greater than 1.1 -fold, 1.2- fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4- fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, or more compared to delivery to non-target cells.
  • the exogenous agent of the provided particle is any described in Section II-D.
  • the exogenous agent is a nucleic acid, such as any described in Section II-D-1.
  • the nucleic acid contains a payload gene encoding a payload agent, for instance a protein, such as any described in Section II-D-2.
  • the exogenous agent is a protein, such as any described in Section II-D-2.
  • the protein is a payload agent.
  • the exogenous agent is a small molecule, such as any described in Section II-D-3.
  • the pay load agent encoded or contained by the exogenous agent is any described in Section III.
  • the exogenous agent is or encodes an engineered receptor, such as any described in Section III-A.
  • the exogenous agent is or encodes a gene-editing agent, such as any described in Section III-B.
  • polynucleotides such as any described in Section IV-A, encoding the fusogen and targeting agent of any of the provided particles.
  • cells such as any described in Section IV-B, containing polynucleotides encoding one or both of the fusogen and targeting agent of any of the provided particles, for instance any of the provided polynucleotides, such as any described in Section IV-A.
  • Also provided herein in some embodiments are methods, such as any described in Section IV-B, of making a targeted lipid particle, such as any described in Section II, using polynucleotides encoding one or both of the fusogen and targeting agent of any of the provided particles, for instance any of the provided polynucleotides, such as any described in Section IV-A.
  • the provided methods are for making any of the provided particles containing an exogenous agent, such as any described in Section II-D.
  • the articles “a” and “an” refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • Exemplary lipid particles also include viral-based particles, such as virus-like particles or viral particles (e.g., lentiviral particles), exosomes, enucleated cells, various vesicles, such as a microvesicle, a membrane vesicle, an extracellular membrane vesicle, a plasma membrane vesicle, a giant plasma membrane vesicle, an apoptotic body, a mitoparticle, a pyrenocyte, or a lysosome.
  • a lipid particle can be a fusosome.
  • the lipid particle is not a platelet.
  • the lipid particle is derived from a source cell.
  • a lipid particle also may include an exogenous agent, which may be present in the lumen of the lipid particle.
  • viral vector can be any type of lipid particle which comprises one or more viral structural proteins in addition to at least one non-structural viral genomic component or functional fragment thereof (e.g., a polymerase, an integrase, a protease or other non-structural component).
  • non-structural viral genomic component e.g., a polymerase, an integrase, a protease or other non-structural component.
  • retroviral particle refers to a viral particle that contains retroviral nucleic acid or is derived from a retrovirus.
  • a retroviral particle includes the following components: a vector genome (retrovirus nucleic acid), a nucleocapsid encapsidating the nucleic acid, and a membrane envelope surrounding the nucleocapsid.
  • a retroviral particle contains sufficient retroviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle capable of infecting a target cell. Infection of the target cell may include reverse transcription and integration into the target cell genome.
  • a retroviral particle may be a recombinant retroviral particle that is replication defective and lacks genes essential for replication, such as a functional gag-pol and/or env gene and/or other genes essential for replication.
  • a retroviral particle also may be a self-inactivating (SIN) vector.
  • a “lentiviral particle” or LV refers to a viral particle that contains lentiviral nucleic acid or is derived from a lentivirus.
  • a lentiviral particle includes the following components: a vector genome (lentivirus nucleic acid), a nucleocapsid encapsidating the nucleic acid, and a membrane stirrounding the nucleocapsid.
  • a lentiviral particle contains sufficient lentiviral genetic information to allow packaging of an RN genome, in the presence of packaging components, Into a viral particle capable of infecting a target cell. Infection of the target cell may include reverse transcription and integration into the target cell genome.
  • a lentiviral particle may be a recombinant lentiviral particle that is replication defective and lacks genes essential for replication, such as a functional gag-pol and/or env gene and/or other genes essential for replication.
  • a lentiviral particle also may be a self-inactivating (SIN) vector.
  • a “retroviral nucleic acid,” refers to a nucleic acid containing at least the minimal sequence requirements for packaging into a retroviral particle, alone or in combination with a helper cell, helper virus, or helper plasmid.
  • lentiviral nucleic acid the nucleic acid refers to at least the minimal sequence requirements for packaging into a lentiviral particle, alone or in combination with a helper cell, helper virus, or helper plasmid.
  • the viral nucleic acid comprises one or more of (e.g., all of) a 5’ LTR (e.g., to promote integration), U3 (e.g., to activate viral genomic RNA transcription), R (e.g., a Tat-binding region), U5, a 3’ LTR (e.g., to promote integration), a packaging site (e.g., psi ( )), RRE (e.g., to bind to Rev and promote nuclear export).
  • the viral nucleic acid can comprise RNA (e.g., when part of a virion) or DNA (e.g., when being introduced into a source cell or after reverse transcription in a recipient cell).
  • the viral nucleic acid is packaged using a helper cell, helper virus, or helper plasmid which comprises one or more of (e.g., all of) gag, pol, and env.
  • fusosome refers to a particle containing a bilayer of amphipathic lipids enclosing a lumen or cavity and a fusogen that interacts with the amphipathic lipid bilayer.
  • the fusosome comprises an exogenous agent.
  • the exogenous agent is a nucleic acid (e.g., DNA or RNA), a peptide, or a protein.
  • the fusosome is a membrane enclosed preparation.
  • the fusosome is derived from a source cell.
  • fusosome composition refers to a composition comprising one or more fusosomes.
  • fusogen refers to an agent or molecule that creates an interaction between two membranes, including membrane enclosed lumens.
  • the fusogen facilitates fusion of the membranes.
  • the fusogen creates a connection, e.g., a pore, between two membranes or lumens (e.g., a lumen of a retroviral particle and a cytoplasm of a target cell).
  • the fusogen comprises a complex of two or more proteins, e.g., wherein neither protein has fusogenic activity alone.
  • the fusogen comprises a targeting domain.
  • a “re-targeted fusogen” refers to a fusogen that comprises a targeting moiety having a sequence that is not part of the naturally-occurring form of the fusogen.
  • the fusogen comprises a different targeting agent relative to the targeting agent in the naturally-occurring form of the fusogen.
  • the naturally-occurring form of the fusogen lacks a targeting domain, and the re-targeted fusogen comprises a targeting agent that is absent from the naturally- occurring form of the fusogen.
  • the fusogen is modified to comprise a targeting agent.
  • a “target cell” refers to a cell of a type to which it is desired that a targeted lipid particle or viral vector delivers an exogenous agent.
  • a target cell is a cell of a specific tissue type or class.
  • the targeting agent or fusogen e.g., re-targeted fusogen, leads to preferential delivery of the exogenous agent to a target cell compared to a non-target cell.
  • a “non-target cell” refers to a cell of a type to which it is not desired that a targeted lipid particle or viral particle delivers an exogenous agent.
  • a non-target cell is a cell of a specific tissue type or class.
  • the targeting agent or fusogen e.g., re-targeted fusogen leads to lower delivery of the exogenous agent to a non-target cell compared to a target cell.
  • a biologically active portion of an F protein retains fusogenic activity in conjunction with the G protein when each are embedded in a lipid bilayer.
  • a biologically active portion of the G protein retains fusogenic activity in conjunction with an F protein when each is embedded in a lipid bilayer.
  • the retained activity can include 10%-150% or more of the activity of a full-length or wild-type F protein or G protein.
  • biologically active portions of F and G proteins include proteins with truncations of the cytoplasmic domain, such as any of the described NiV-F with a truncated cytoplasmic tail.
  • percent (%) amino acid sequence identity and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • amino acid substitution may include but are not limited to the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 19. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, for example, retained/improved binding.
  • Amino acids may be grouped according to common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions in a disclosed sequence refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm.
  • corresponding residues of a similar sequence e.g. fragment or species variant
  • structural alignment methods By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.
  • isolated refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or produced.
  • a polypeptide is referred to as “isolated” when it is separated from at least some of the components of the cell in which it was produced.
  • a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be “isolating” the polypeptide.
  • a polynucleotide is referred to as “isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, for example, in the case of an RNA polynucleotide.
  • a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as “isolated”.
  • the term “effective amount” as used herein means an amount of a pharmaceutical composition which is sufficient enough to significantly and positively modify the symptoms and/or conditions to be treated (e.g., provide a positive clinical response).
  • the effective amount of an active ingredient for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically-acceptable excipient(s) and/or carrier(s) utilized, and like factors with the knowledge and expertise of the attending physician.
  • an “exogenous agent” as used herein with reference to a viral particle refers to an agent that is neither comprised by nor encoded in the corresponding wild-type virus or fusogen made from a corresponding wild-type source cell.
  • the exogenous agent does not naturally exist, such as a protein or nucleic acid that has a sequence that is altered (e.g., by insertion, deletion, or substitution) relative to a naturally occurring protein.
  • the exogenous agent does not naturally exist in the source cell.
  • the exogenous agent exists naturally in the source cell but is exogenous to the virus.
  • the exogenous agent does not naturally exist in the recipient cell.
  • the exogenous agent exists naturally in the recipient cell, but is not present at a desired level or at a desired time.
  • the exogenous agent comprises nucleic acids or protein.
  • a “promoter” refers to a cis- regulatory DNA sequence that, when operably linked to a gene coding sequence, drives transcription of the gene.
  • the promoter may comprise a transcription factor binding sites.
  • a promoter works in concert with one or more enhancers which are distal to the gene.
  • operably linked or “operably associated” includes reference to a functional linkage of at least two sequences.
  • operably linked includes linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
  • Operably associated includes linkage between an inducing or repressing element and a promoter, wherein the inducing or repressing element acts as a transcriptional activator of the promoter.
  • composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, nonaqueous or any combination thereof.
  • the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • the term “pharmaceutical composition” refers to a mixture of at least one particle with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
  • the pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
  • a “disease” or “disorder” as used herein refers to a condition where treatment is needed and/or desired.
  • the terms “treat,” “treating,” or “treatment” refer to ameliorating a disease or disorder, e.g., slowing or arresting or reducing the development of the disease or disorder or reducing at least one of the clinical symptoms thereof.
  • ameliorating a disease or disorder can include obtaining a beneficial or desired clinical result that includes, but is not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (for example, metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total).
  • a beneficial or desired clinical result that includes, but is not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (for example, metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or
  • an “individual” or “subject” refers to an individual or subject in need of treatment for a disease or disorder.
  • the subject to receive the treatment can be a patient, designating the fact that the subject has been identified as having a disorder of relevance to the treatment, or being at adequate risk of contracting the disorder.
  • the subject is a human, such as a human patient.
  • the terms “effective amount” and “pharmaceutically effective amount” refer to a nontoxic but sufficient amount of an agent or drug to provide the desired biological result. That result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease or disorder, imaging or monitoring of an in vitro or in vivo system (including a living organism), or any other desired alteration of a biological system.
  • An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • nuclear export sequence (NES) or “nuclear export signal” (NES) refer to a nuclear export signal or other sequence or domain that is present in a protein and capable of targeting the protein for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport.
  • a nuclear export domain can be fused (e.g., fused in-frame) with a polypeptide.
  • nuclear localization siequence (NLS) or “nuclear localization sequence” (NLS) refer to a nuclear localization signal or other seuqence or domain that is present in a protien and capable of targeting the protein for import from the cytoplams to the cell nucleus through the nuclear pore complex using nuclear transport.
  • a nucelar lozalization can be fused (e.g., fused in-frame) with a polypeptide.
  • lipid particles that allow for efficient targeting and delivery of an exogenous agent contained therein to hematopoietic stem/progenitor cells (HSPC), including to naive hematopoietic stem cells (HSCs).
  • the targeting can be to such stem or progenitor cells in a human subject, such as human HSPC.
  • the lipid particles can include viral vectors such as lentiviral vectors that are pseudotyped with a fusogen with a preferential ability to target HSPC.
  • the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof, such as the exemplarly fusogens BaEVTR or BaEVRLess.
  • the lipid particle includes a targeting agent to target the fusogen to HSPCs, such as to CD34+ cells.
  • the targeting agent is fused to a fusogen, such as a viral envelope attachment glycoprotein, exposed on the lipid bilayer to provide a retargeted fusogen.
  • targeted particles having a targeting agent that binds to a target molecule.
  • the target molecule is expressed on a target cell.
  • the provided particle preferentially targets a target cell compared to a non-target cell.
  • the provided particle is a lipid particle, such as any described in Section II-A.
  • the provided particle is a lipid particle that is a viral particle, such as any described in Section II-A-1.
  • the provided particle is a targeted lentiviral particle.
  • the provided particle is a lipid particle that is a virus-like particle, such as any described in Section II-A-2.
  • the provided particle is a lipid particle that is a cell-based particle, such as any described in Section II-A-3.
  • the provided particle has a fusogen, such as any described in Section II-B.
  • the fusogen promotes mixing between lipids in the particle and lipids in the target cell.
  • the fusogen facilitates the fusion of the particle to a membrane of the target cell.
  • the particle integrates into the membrane of the target cell.
  • the membrane is the plasma membrane of the target cell.
  • the fusogen promotes formation of one or more pores between the interior of the particle and the cytosol of the target cell.
  • the fusogen is endogenous to the particle.
  • the particle is pseudotyped with the fusogen, such as when the particle is a viral particle or virus-like particle.
  • the particle is a viral particle or a virus-like particle.
  • the fusogen is exposed on the surface of the viral envelope of the particle. In some embodiments, a portion of the fusogen is embedded in the viral envelope of the particle.
  • the fusogen contains a mammalian protein, such as any described in Section II-B-1. In some embodiments, the fusogen contains a viral protein, such as any described in Section II-B-2.
  • the lipid particle includes a targeting agent to target the fusogen to HSPCs.
  • the targeting agent of the provided particle is any described in Section II- C. Exemplary target molecules and target cells for any of the provided particles are also described in Section II-C.
  • the fusogen is fused to the targeting agent of the particle. In some aspects, the fusogen is retargeted by the targeting agent to display altered tropism. In other embodiments, the targeting agent is fused to a separate transmembrane domain incorporated into the lipid bilayer, not the fusogen.
  • the provided particle contains an exogenous agent.
  • the exogenous agent is in the lumen of the provided particle.
  • the sequence of the exogenous agent is exogenous to the particle.
  • the sequence of the exogenous agent is non-viral.
  • the exogenous agent is for delivery to the target cell.
  • the exogenous agent of the provided particle is any described in Section II-D.
  • the exogenous agent is a nucleic acid, such as any described in Section II-D-1.
  • the nucleic acid contains a payload gene encoding a payload agent.
  • the exogenous agent is a protein, such as any described in Section II-D-2.
  • the protein is a payload agent.
  • the exogenous agent is a small molecule, such as any described in Section II-D-3.
  • the pay load agent encoded or contained by the exogenous agent is any described in Section III.
  • the exogenous agent is or encodes an engineered receptor, such as any described in Section III-A.
  • the exogenous agent is or encodes a gene-editing agent, such as any described in Section III-B.
  • the particle is a lipid particle.
  • the particle includes a bilayer of amphipathic lipids that encloses a lumen or cavity.
  • the particle has a lipid bilayer as the outermost surface.
  • the particle is spherical.
  • the particle is of elongated or irregular shape.
  • the lipid bilayer encloses a lumen.
  • the lumen is aqueous.
  • the lumen is in contact with the hydrophilic head groups on the interior of the lipid bilayer.
  • the lumen is a cytosol.
  • the cytosol contains cellular components present in a source cell.
  • the cytosol does not contain components present in a source cell.
  • the lumen is a cavity.
  • the cavity contains an aqueous environment. In some embodiments, the cavity does not contain an aqueous environment.
  • the particle comprises several different types of lipids.
  • the lipids are amphipathic lipids.
  • the amphipathic lipids are phospholipids.
  • the phospholipids comprise phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.
  • the lipids comprise phospholipids such as phosphocholines and phosphoinositols.
  • the lipids comprise DMPC, DOPC, and DSPC.
  • a particle or composition of particles can be assessed for one or more features related to their size, including diameter, average (e.g., mean or median) diameter, range of variation thereof above and below an average (e.g., mean or median) value of the diameter, coefficient of variation, polydispersity index, or other measure of size of particles.
  • Various methods for particle characterization can be used, including laser diffraction, dynamic light scattering (DLS; also known as photon correlation spectroscopy) or image analysis, such as microscopy or automated image analysis.
  • DLS dynamic light scattering
  • image analysis such as microscopy or automated image analysis.
  • the particle has a diameter of less than about 3 pm, less than about 2 pm, less than about 1 pm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 m, less than about 400 nm, less than about 300, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 50 nm, or less than about 20 nm. In some embodiments, the particle has a diameter of less than about 400 nm. In some embodiments, the particle has a diameter of less than about 150 nm.
  • the particle has a diameter of between at or about 2 pm and at or about 1 pm, between at or about 1 pm and at or about 900 nm, between at or about 900 nm and at or about 800 nm, between at or about 800 and at or about 700 nm, between at or about 700 nm and at or about 600 nm, between at or about 600 nm and at or about 500 nm, between at or about 500 nm and at or about 400 nm, between at or about 400 nm and at or about 300 nm, between at or about 300 nm and at or about 200 nm, between at or about 200 and at or about 100 nm, between at or about 100 and at or about 50 nm, or between at or about 20 nm and at or about 50 nm.
  • the particle has a diameter between at or about 10 nm and at or about 1000 nM, between at or about 25 nm and at or about 500 nm, between at or about 40 nm and at or about 300 nm, between at or about 50 nm and at or about 250 nm, between at or about 60 nm and at or about 225 nm, between at or about 70 nm and at or about 200 nm, between at or about 80 nm and at or about 175 nm, or between at or about 90 nm and at or about 150 nm.
  • the particle has a hydrodynamic radius, e.g., as determined by DLS, of about 100 nm to about two microns. In some embodiments, the particle has a hydrodynamic radius between at or about 2 pm and at or about 1 pm, between at or about 1 pm and at or about 900 nm, between at or about 900 nm and at or about 800 nm, between at or about 800 and at or about 700 nm, between at or about 700 nm and at or about 600 nm, between at or about 600 nm and at or about 500 nm, between at or about 500 nm and at or about 400 nm, between at or about 400 nm and at or about 300 nm, between at or about 300 nm and at or about 200 nm, between at or about 200 and at or about 100 nm, between at or about 100 and at or about 50 nm, or between at or about 20 nm and at or about 50 nm.
  • a hydrodynamic radius between at or about 2 pm and at
  • Non-limiting examples of particles include any as described in, or containing any features as described in, International Published PCT Application No. WO 2017/095946; WO 2017/095944; WO 2017/095940; WO 2019/157319; WO 2018/208728; WO 2019/113512; WO 2019/161281; WO 2020/102578; WO 2019/222403; WO 2020/014209; WO 2020/102485; WO 2020/102499; WO 2020/102503; WO 2013/148327; WO 2017/182585; WO 2011/058052; or WO 2017/068077, each of which are incorporated by reference in their entirety.
  • the particle is a viral-based particle, a viral particle, a virus-like particle (VLP), a nanoparticle, a vesicle, an exosome, a dendrimer, a lentiviral particle, an enucleated cell, a microvesicle, a membrane vesicle, an extracellular membrane vesicle, a plasma membrane vesicle, a giant plasma membrane vesicle, an apoptotic body, a mitoparticle, a pyrenocyte, a lysosome, another membrane enclosed vesicle, or a cell based particle
  • VLP virus-like particle
  • the lipid bilayer includes a synthetic lipid complex.
  • the synthetic lipid complex is a liposome.
  • the lipid bilayer is a vesicular structure characterized by a phospholipid bilayer membrane and an inner aqueous medium.
  • the lipid bilayer has multiple lipid layers separated by aqueous medium.
  • the lipid bilayer forms spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • the lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers
  • the lipid bilayer is derived from a source cell during a process to produce a lipid-containing particle. Exemplary methods for producing lipid-containing particles are described in Section IV.
  • the lipid bilayer includes membrane components of the host cell from which the lipid bilayer is derived, e.g., phospholipids, membrane proteins, etc.
  • the lipid bilayer includes a cytosol that includes components found in the cell from which the vehicle is derived, e.g., solutes, proteins, nucleic acids, etc., but not all of the components of a cell, e.g., lacking a nucleus.
  • the lipid bilayer is considered to be exosome-like.
  • the particle is virally derived.
  • the particle is a viral or virus-like particle, such as any described in Section II-A-1.
  • the particle can be a viral-based particle, such as a viral particle (e.g., lentiviral particle) or a virus-like particle (e.g., a lentiviral-like particle).
  • the provided particle is a targeted lentiviral particle.
  • the lipid bilayer is a viral envelope.
  • the viral envelope is obtained from a host cell.
  • the viral particle includes one or more viral structural proteins (e.g., capsid proteins).
  • the lipid bilayer is obtained from a membrane other than the plasma membrane of a host cell.
  • the viral envelope lipid bilayer is embedded with viral proteins, including viral glycoproteins.
  • the lipid particle is not virally derived.
  • the lipid particle can be a nanoparticle, a vesicle, an exosome, a dendrimer, an enucleated cell, a microvesicle, a membrane vesicle, an extracellular membrane vesicle, a plasma membrane vesicle, a giant plasma membrane vesicle, an apoptotic body, a mitoparticle, a pyrenocyte, a lysosome, another membrane enclosed vesicle, or a cell derived particle.
  • the provided particle is a cell-based particle, such as any described in Section II-A-3.
  • the lipid bilayer of the particle is or comprises the viral envelope.
  • the viral envelope may comprise a fusogen that is endogenous to the virus or is a pseudotyped fusogen.
  • the particle’s lumen or cavity comprises a viral nucleic acid, e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid.
  • the viral nucleic acid may be a viral genome.
  • the particle may further comprise one or more viral non-structural proteins, e.g., in its cavity or lumen.
  • the particles are lenti viral particles.
  • the lenti viral particle is Human Immunodeficiency Virus-1 (HIV-1).
  • the particle is limited in the number of polynucleotides that can be packaged.
  • nucleotides encoding polypeptides to be packaged can be modified such that they retain functional activity with fewer nucleotides in the coding region than that which encodes for the wild-type peptide. Such modifications can include truncations, or other deletions.
  • more than one polypeptide can be expressed from the same promoter, such that they are fusion polypeptides.
  • the insert size to be packaged (i.e., viral genome, or portions thereof; or heterologous polynucleotides as described) can be between 500-1000, 1000-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, or 7000-8000 nucleotides in length. In some embodiments, the insert can be over 8000 nucleotides, such as 9000, 10,000, or 11,000 nucleotides in length.
  • the particle such as retroviral particle, comprises one or more of gag polyprotein, polymerase (e.g., pol), integrase (e.g., a functional or non-functional variant), protease, and a fusogen.
  • the particle further comprises rev.
  • one or more of the aforesaid proteins are encoded in the retroviral genome (e.g., the insert as described above), and in some embodiments, one or more of the aforesaid proteins are provided in trans, e.g., by a helper cell, helper virus, or helper plasmid.
  • the nucleic acid (e.g., retroviral nucleic acid) comprises one or more of the following nucleic acid sequences: 5’ LTR (e.g., comprising U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT) Promoter operatively linked to a payload gene encoding a payload agent, payload gene (optionally comprising an intron before the open reading frame), Poly A tail sequence, WPRE, and 3’ LTR (e.g., comprising U5 and lacking a functional U3).
  • the nucleic acid further comprises a retroviral cisacting RNA packaging element and a cPPT/CTS element.
  • the nucleic acid further comprises one or more insulator element.
  • the recognition sites are situated between the poly A tail sequence and the WPRE.
  • the particle comprises supramolecular complexes formed by viral proteins that self-assemble into capsids.
  • the particle is a viral particle derived from viral capsids.
  • the particle is a viral particle derived from viral nucleocapsids.
  • the particle comprises nucleocapsid-derived proteins that retain the property of packaging nucleic acids.
  • the particle packages one or more viral nucleic acids (e.g., retroviral nucleic acids) during the expression process.
  • the nucleic acids do not encode any genes involved in virus replication.
  • the particle is replication defective.
  • the retroviral nucleic acid comprises one or more of (e.g., all of): a 5’ promoter (e.g., to control expression of the entire packaged RNA), a 5’ LTR (e.g., that includes R (poly adenylation tail signal) and/or U5 which includes a primer activation signal), a primer binding site, a psi packaging signal, a RRE element for nuclear export, a promoter directly upstream of the payload gene to control payload gene expression, a payload gene, a polypurine tract, and a 3’ LTR (e.g., that includes a mutated U3, a R, and U5).
  • the retroviral nucleic acid further comprises one or more of a cPPT, a WPRE, and/or an insulator element.
  • a retrovirus typically replicates by reverse transcription of its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome.
  • Illustrative retroviruses suitable for use in particular embodiments include Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spuma virus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentivirus.
  • M-MuLV Moloney murine leukemia virus
  • MoMSV Moloney murine sarcoma virus
  • Harvey murine sarcoma virus HaMuSV
  • murine mammary tumor virus MuMTV
  • GaLV
  • the retrovirus is a Gammretro virus. In some embodiments the retrovirus is an Epsilonretrovirus. In some embodiments the retrovirus is an Alpharetrovirus. In some embodiments the retrovirus is a Betaretro virus. In some embodiments the retrovirus is a Deltaretro virus. In some embodiments the retrovirus is a Lentivirus. In some embodiments the retrovirus is a Spumaretrovirus. In some embodiments the retrovirus is an endogenous retrovirus.
  • Illustrative lenti viruses include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
  • HIV based particle backbones e.g., HIV cis-acting sequence elements
  • a viral particle can comprise a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that typically facilitate transfer of a nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer.
  • Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acids.
  • Viral particles and transfer plasmids can comprise structural and/or functional genetic elements that are primarily derived from a virus.
  • a retroviral particle can comprise a viral particle or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus.
  • a lenti viral particle can comprise a viral particle or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus.
  • a lentiviral particle may comprise a lentiviral transfer plasmid (e.g., as naked DNA) or an infectious lentiviral particle.
  • a lentiviral transfer plasmid e.g., as naked DNA
  • infectious lentiviral particle e.g., infectious lentiviral particle.
  • elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is to be understood that the sequences of these elements can be present in RNA form in lentiviral particles and can be present in DNA form in DNA plasmids.
  • At least part of one or more protein coding regions that contribute to or are essential for replication may be absent compared to the corresponding wild- type virus. In some embodiments, this makes the viral particle replication-defective. In some embodiments, the particle is capable of transducing a target non-dividing host cell and/or integrating its genome into a host genome.
  • the structure of a wild-type retrovirus genome often comprises a 5' long terminal repeat (LTR) and a 3' LTR, between or within which are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome and gag, pol and env genes encoding the packaging components which promote the assembly of viral particles.
  • More complex retroviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell.
  • the viral genes are flanked at both ends by regions called long terminal repeats (LTRs).
  • LTRs are involved in pro viral integration and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes. Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome.
  • the LTRs themselves are typically similar (e.g., identical) sequences that can be divided into three elements, which are called U3, R and U5.
  • U3 is derived from the sequence unique to the 3' end of the RNA.
  • R is derived from a sequence repeated at both ends of the RNA and
  • U5 is derived from the sequence unique to the 5' end of the RNA.
  • the sizes of the three elements can vary considerably among different retroviruses.
  • the site of transcription initiation is typically at the boundary between U3 and R in one LTR and the site of poly (A) addition (termination) is at the boundary between R and U5 in the other LTR.
  • U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins.
  • Some retroviruses comprise any one or more of the following genes that code for proteins that are involved in the regulation of gene expression: tot, rev, tax and rex. With regard to the structural genes gag, pol and env themselves, gag encodes the internal structural protein of the virus.
  • Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid).
  • the pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome.
  • the env gene encodes the surface (SU) glycoprotein and the transmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. This interaction promotes infection, e.g., by fusion of the viral membrane with the cell membrane.
  • Retroviruses may also contain additional genes which code for proteins other than gag, pol and env.
  • additional genes include (in HIV), one or more of vif, vpr, vpx, vpu, tat, rev and nef.
  • El AV has (amongst others) the additional gene S2. Proteins encoded by additional genes serve various functions, some of which may be duplicative of a function provided by a cellular protein.
  • tat acts as a transcriptional activator of the viral LTR (Derse and Newbold 1993 Virology 194:530-6; Maury et al. 1994 Virology 200:632- 42).
  • TAR binds to a stable, stem-loop RNA secondary structure referred to as TAR.
  • RRE rev-response elements
  • Ttm an EIAV protein, Ttm, has been identified that is encoded by the first exon of tat spliced to the env coding sequence at the start of the transmembrane protein.
  • non-primate lentiviruses contain a fourth pol gene product which codes for a dUTPase. This may play a role in the ability of these lentiviruses to infect certain non-dividing or slowly dividing cell types.
  • a recombinant lenti viral particle is a particle with sufficient retroviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle capable of infecting a target cell. Infection of the target cell can comprise reverse transcription and integration into the target cell genome.
  • the RLV typically carries non-viral coding sequences which are to be delivered by the particle to the target cell, such as a nucleic acid encoding a payload agent as described herein.
  • an RLV is incapable of independent replication to produce infectious retroviral particles within the target cell.
  • the RLV lacks a functional gag-pol and/or env gene and/or other genes involved in replication.
  • the particle may be configured as a split-intron particle, e.g., as described in PCT patent application WO 99/15683, which is herein incorporated by reference in its entirety.
  • a minimal lentiviral genome may comprise, e.g., (5')R-U5-one or more first nucleotide sequences-U3-R(3').
  • the plasmid used to produce the lentiviral genome within a source cell can also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a source cell.
  • These regulatory sequences may comprise the natural sequences associated with the transcribed retroviral sequence, e.g., the 5' U3 region, or they may comprise a heterologous promoter such as another viral promoter, for example the CMV promoter.
  • Some lentiviral genomes comprise additional sequences to promote efficient virus production.
  • rev and RRE sequences may be included.
  • codon optimization may be used, e.g., the payload gene encoding the payload agent may be codon optimized, e.g., as described in WO 01/79518, which is herein incorporated by reference in its entirety.
  • Alternative sequences which perform a similar or the same function as the rev/RRE system may also be used.
  • a functional analogue of the rev/RRE system is found in the Mason Pfizer monkey virus. This is known as CTE and comprises an RRE-type sequence in the genome which is believed to interact with a factor in the infected cell. The cellular factor can be thought of as a rev analogue.
  • CTE may be used as an alternative to the rev/RRE system.
  • the Rex protein of HTLV-I can functionally replace the Rev protein of HIV-I. Rev and Rex have similar effects to IRE-BP.
  • a retroviral nucleic acid (e.g., a lentiviral nucleic acid, e.g., a primate or non-primate lentiviral nucleic acid) (1) comprises a deleted gag gene wherein the deletion in gag removes one or more nucleotides downstream of about nucleotide 350 or 354 of the gag coding sequence; (2) has one or more accessory genes absent from the retroviral nucleic acid; (3) lacks the tat gene but includes the leader sequence between the end of the 5' LTR and the ATG of gag; and (4) combinations of (1), (2) and (3).
  • the lentiviral particle comprises all of features (1) and (2) and (3). This strategy is described in more detail in WO 99/32646, which is herein incorporated by reference in its entirety.
  • a primate lentivirus minimal system requires none of the HIV/SIV additional genes vif, vpr, vpx, vpu, tat, rev and nef for either particle production or for transduction of dividing and non-dividing cells.
  • an EIAV minimal particle system does not require S2 for either particle production or for transduction of dividing and non-dividing cells.
  • additional genes may permit particles to be produced without the genes associated with disease in lentiviral (e.g. HIV) infections. In particular, tat is associated with disease. Secondly, the deletion of additional genes permits the particle to package more heterologous DNA. Thirdly, genes whose function is unknown, such as S2, may be omitted, thus reducing the risk of causing undesired effects. Examples of minimal lentiviral particles are disclosed in WO 99/32646 and in WO 98/17815.
  • the retroviral nucleic acid is devoid of at least tat and S2 (if it is an EIAV particle system), and possibly also vif, vpr, vpx, vpu and nef. In some embodiments, the retroviral nucleic acid is also devoid of rev, RRE, or both. [0308] In some embodiments the retroviral nucleic acid comprises vpx.
  • the Vpx polypeptide binds to and induces the degradation of the SAMHD1 restriction factor, which degrades free dNTPs in the cytoplasm.
  • the concentration of free dNTPs in the cytoplasm increases as Vpx degrades SAMHD1 and reverse transcription activity is increased, thus facilitating reverse transcription of the retroviral genome and integration into the target cell genome.
  • codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type.
  • viruses including HIV and other lentiviruses, use a large number of rare codons and by changing these to correspond to commonly used mammalian codons, increased expression of the packaging components in mammalian producer cells can be achieved.
  • codon optimization has a number of other advantages.
  • the nucleotide sequences encoding the packaging components may have RNA instability sequences (INS) reduced or eliminated from them.
  • INS RNA instability sequences
  • the amino acid sequence coding sequence for the packaging components is retained so that the viral components encoded by the sequences remain the same, or at least sufficiently similar that the function of the packaging components is not compromised.
  • codon optimization also overcomes the Rev/RRE requirement for export, rendering optimized sequences Rev independent.
  • codon optimization also reduces homologous recombination between different constructs within the particle system (for example between the regions of overlap in the gag-pol and env open reading frames).
  • codon optimization leads to an increase in viral titer and/or improved safety.
  • codons relating to INS are codon optimized.
  • sequences are codon optimized in their entirety, with the exception of the sequence encompassing the frameshift site of gag-pol.
  • the gag-pol gene comprises two overlapping reading frames encoding the gag-pol proteins.
  • the expression of both proteins depends on a frameshift during translation. This frameshift occurs as a result of ribosome "slippage" during translation. This slippage is thought to be caused at least in part by ribosome-stalling RNA secondary structures.
  • Such secondary structures exist downstream of the frameshift site in the gag-pol gene.
  • the region of overlap extends from nucleotide 1222 downstream of the beginning of gag (wherein nucleotide 1 is the A of the gag ATG) to the end of gag (nt 1503). Consequently, a 281 bp fragment spanning the frameshift site and the overlapping region of the two reading frames is preferably not codon optimized.
  • retaining this fragment will enable more efficient expression of the gag-pol proteins.
  • the beginning of the overlap is at nt 1262 (where nucleotide 1 is the A of the gag ATG).
  • the end of the overlap is at nt 1461.
  • the wild type sequence may be retained from nt 1156 to 1465.
  • derivations from optimal codon usage may be made, for example, in order to accommodate convenient restriction sites, and conservative amino acid changes may be introduced into the gag-pol proteins.
  • codon optimization is based on codons with poor codon usage in mammalian systems.
  • the third and sometimes the second and third base may be changed.
  • gag-pol sequences can be achieved by a skilled worker.
  • retroviral variants described which can be used as a starting point for generating a codon optimized gag-pol sequence. Lentiviral genomes can be quite variable. For example there are many quasi-species of HIV-I which are still functional. This is also the case for EIAV. These variants may be used to enhance particular parts of the transduction process. Examples of HIV-I variants may be found in the HIV databases maintained by Los Alamos National Laboratory. Details of EIAV clones may be found at the NCBI database maintained by the National Institutes of Health.
  • codon optimized sequences can be used in relation to any retrovirus, e.g., EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-I and HIV -2.
  • this method could be used to increase expression of genes from HTLV-I, HTLV-2, HFV, HSRV and human endogenous retroviruses (HERV), MLV and other retroviruses.
  • the retroviral particle comprises a packaging signal that comprises from 255 to 360 nucleotides of gag in particles that still retain env sequences, or about 40 nucleotides of gag in a particular combination of splice donor mutation, gag and env deletions.
  • the retroviral particle includes a gag sequence which comprises one or more deletions, e.g., the gag sequence comprises about 360 nucleotides derivable from the N-terminus.
  • the retroviral particle, helper cell, helper virus, or helper plasmid may comprise retroviral structural and accessory proteins, for example gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef proteins or other retroviral proteins.
  • the retroviral proteins are derived from the same retrovirus.
  • the retroviral proteins are derived from more than one retrovirus, e.g. 2, 3, 4, or more retroviruses.
  • the gag and pol coding sequences are generally organized as the Gag- Pol Precursor in native lentivirus.
  • the gag sequence codes for a 55-kD Gag precursor protein, also called p55.
  • the p55 is cleaved by the virally encoded protease (a product of the pol gene) during the process of maturation into four smaller proteins designated MA (matrix [pl7]), CA (capsid [p24]), NC (nucleocapsid [p9]) , and p6.
  • the pol precursor protein is cleaved away from Gag by a virally encoded protease, and further digested to separate the protease (plO), RT (p50), RNase H (pl5), and integrase (p31) activities.
  • the lentiviral particle is integration-deficient.
  • the pol is integrase deficient, such as by encoding due to mutations in the integrase gene.
  • the pol coding sequence can contain an inactivating mutation in the integrase, such as by mutation of one or more of amino acids involved in catalytic activity, i.e. mutation of one or more of aspartic 64, aspartic acid 116 and/or glutamic acid 152.
  • the integrase mutation is a D64V mutation.
  • the mutation in the integrase allows for packaging of viral RNA into a lentivirus.
  • the mutation in the integrase allows for packaging of viral proteins into a lentivirus. In some embodiments, the mutation in the integrase reduces the possibility of insertional mutagenesis. In some embodiments, the mutation in the integrase decreases the possibility of generating replication- competent recombinants (RCRs) (Wanisch et al. 2009. Mol Ther. 1798): 1316-1332).
  • RCRs replication- competent recombinants
  • native Gag-Pol sequences can be utilized in a helper particle (e.g., helper plasmid or helper virus), or modifications can be made.
  • These modifications include chimeric Gag-Pol, where the Gag and Pol sequences are obtained from different viruses (e.g., different species, subspecies, strains, clades, etc.), and/or where the sequences have been modified to improve transcription and/or translation, and/or reduce recombination.
  • viruses e.g., different species, subspecies, strains, clades, etc.
  • the retroviral nucleic acid includes a polynucleotide encoding a 150- 250 (e.g., 168) nucleotide portion of a gag protein that (i) includes a mutated INS1 inhibitory sequence that reduces restriction of nuclear export of RNA relative to wild-type INS1, (ii) contains two nucleotide insertion that results in frame shift and premature termination, and/or (iii) does not include INS2, INS3, and INS4 inhibitory sequences of gag.
  • a 150- 250 e.g., 168) nucleotide portion of a gag protein that (i) includes a mutated INS1 inhibitory sequence that reduces restriction of nuclear export of RNA relative to wild-type INS1, (ii) contains two nucleotide insertion that results in frame shift and premature termination, and/or (iii) does not include INS2, INS3, and INS4 inhibitory sequences of gag.
  • a particle described herein is a hybrid particle that comprises both retroviral (e.g., lentiviral) sequences and non-lentiviral viral sequences.
  • a hybrid particle comprises retroviral, e.g., lentiviral, sequences for reverse transcription, replication, integration and/or packaging.
  • most or all of the viral particle backbone sequences are derived from a lentivirus, e.g., HIV-1.
  • a lentivirus e.g., HIV-1.
  • retroviral and/or lentiviral sequences can be used, or combined and numerous substitutions and alterations in certain of the lentiviral sequences may be accommodated without impairing the ability of a transfer particle to perform the functions described herein.
  • a variety of lentiviral particles are described in Naldini et ah, (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos.
  • LTRs long terminal repeats
  • An LTR typically comprises a domain located at the ends of retroviral nucleic acid which, in their natural sequence context, are direct repeats and contain U3, R and U5 regions. LTRs generally promote the expression of retroviral genes (e.g., promotion, initiation and poly adenylation of gene transcripts) and viral replication.
  • the LTR can comprise numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences for replication and integration of the viral genome.
  • the viral LTR is typically divided into three regions called U3, R and U5.
  • the U3 region typically contains the enhancer and promoter elements.
  • the U5 region is typically the sequence between the primer binding site and the R region and can contain the polyadenylation sequence.
  • the R (repeat) region can be flanked by the U3 and U5 regions.
  • the LTR is typically composed of U3, R and U5 regions and can appear at both the 5' and 3' ends of the viral genome. In some embodiments, adjacent to the 5' LTR are sequences for reverse transcription of the genome (the tRNA primer binding site) and for efficient packaging of viral RNA into particles (the Psi site).
  • a packaging signal can comprise a sequence located within the retroviral genome which mediate insertion of the viral RNA into the viral capsid or particle, see e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.
  • Several retroviral particles use a minimal packaging signal (a psi [Y] sequence) for encapsidation of the viral genome.
  • retroviral nucleic acids comprise modified 5' LTR and/or 3' LTRs.
  • Either or both of the LTR may comprise one or more modifications including, but not limited to, one or more deletions, insertions, or substitutions.
  • Modifications of the 3' LTR are often made to improve the safety of lentiviral or retroviral systems by rendering viruses replication-defective, e.g., virus that is not capable of complete, effective replication such that infective virions are not produced (e.g., replicationdefective lentiviral progeny).
  • a particle is a self-inactivating (SIN) particle, e.g., replicationdefective particle, e.g., retroviral or lentiviral particle, in which the right (3') LTR enhancer- promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication.
  • SI self-inactivating
  • a replication incompetent also referred to herein as replication defective
  • the right (3') LTR U3 region can be used as a template for the left (5') LTR U3 region during viral replication and, thus, absence of the U3 enhancer-promoter inhibits viral replication.
  • the 3' LTR is modified such that the U5 region is removed, altered, or replaced, for example, with an exogenous poly(A) sequence
  • the 3' LTR, the 5' LTR, or both 3' and 5' LTRs may be modified LTRs.
  • Other modifications to the viral particle, i.e., retroviral or lentiviral particle, to render said particle replication incompetent are known in the art.
  • the U3 region of the 5' LTR is replaced with a heterologous promoter to drive transcription of the viral genome during production of viral particles.
  • heterologous promoters include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.
  • SV40 viral simian virus 40
  • CMV cytomegalovirus
  • MoMLV Moloney murine leukemia virus
  • RSV Rous sarcoma virus
  • HSV herpes simplex virus
  • promoters are able to drive high levels of transcription in a Tat- independent manner.
  • the heterologous promoter has additional advantages in controlling the manner in which the viral genome is transcribed.
  • the heterologous promoter can be inducible, such that transcription of all or part of the viral genome will occur only when the induction factors are present.
  • Induction factors include, but are not limited to, one or more chemical compounds or the physiological conditions such as temperature or pH, in which the host cells are cultured.
  • viral particles comprise a TAR (trans-activation response) element, e.g., located in the R region of lentiviral (e.g., HIV) LTRs.
  • This element interacts with the lentiviral trans-activator (tat) genetic element to enhance viral replication.
  • this element is not required, e.g., in embodiments wherein the U3 region of the 5' LTR is replaced by a heterologous promoter.
  • the R region e.g., the region within retroviral LTRs beginning at the start of the capping group (i.e., the start of transcription) and ending immediately prior to the start of the poly A tract can be flanked by the U3 and U5 regions.
  • the R region plays a role during reverse transcription in the transfer of nascent DNA from one end of the genome to the other.
  • the retroviral nucleic acid can also comprise a FLAP element, e.g., a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2.
  • a FLAP element e.g., a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2.
  • a retrovirus e.g., HIV-1 or HIV-2.
  • Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, et ah, 2000, Cell, 101:173, which are herein incorporated by reference in their entireties.
  • central initiation of the plus-strand DNA at the central polypurine tract (cPPT) and central termination at the central termination sequence (CTS) can lead to the formation of a three- stranded DNA
  • the retroviral or lentiviral particle backbones comprise one or more FLAP elements upstream or downstream of the payload gene encoding the payload agent.
  • a transfer plasmid includes a FLAP element, e.g., a FLAP element derived or isolated from HIV-L
  • a retroviral or lentiviral nucleic acid comprises one or more export elements, e.g., a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell.
  • export elements include the human immunodeficiency virus (HIV) rev response element (RRE) (see e.g., Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), and the hepatitis B virus post-transcriptional regulatory element (HPRE), which are herein incorporated by reference in their entireties.
  • the RNA export element is placed within the 3' UTR of a gene, and can be inserted as one or multiple copies.
  • expression of heterologous sequences e.g., nucleic acid encoding a payload agent
  • expression of heterologous sequences in viral particles is increased by incorporating one or more of, e.g., all of, posttranscriptional regulatory elements, polyadenylation sites, and transcription termination signals into the particles.
  • posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid at the protein, e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J.
  • a retroviral nucleic acid described herein comprises a posttranscriptional regulatory element such as a WPRE or HPRE
  • a retroviral nucleic acid described herein lacks or does not comprise a posttranscriptional regulatory element such as a WPRE or HPRE.
  • Elements directing the termination and polyadenylation of the heterologous nucleic acid transcripts may be included, e.g., to increase expression of the payload agent. Transcription termination signals may be found downstream of the polyadenylation signal.
  • particles comprise a polyadenylation sequence 3' of a polynucleotide encoding the payload agent.
  • a polyA site may comprise a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II.
  • Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency.
  • polyA signals that can be used in a retroviral nucleic acid, include AATAAA, ATT AAA, AGTAAA, a bovine growth hormone polyA sequence (BGHpA), a rabbit b- globin polyA sequence (rPgpA), or another suitable heterologous or endogenous polyA sequence.
  • BGHpA bovine growth hormone polyA sequence
  • rPgpA rabbit b- globin polyA sequence
  • a retroviral or lenti viral particle further comprises one or more insulator elements, e.g., an insulator element described herein.
  • the particles comprise a promoter operably linked to a polynucleotide encoding a payload agent.
  • the particles may have one or more LTRs, wherein either LTR comprises one or more modifications, such as one or more nucleotide substitutions, additions, or deletions.
  • the particles may further comprise one of more accessory elements to increase transduction efficiency (e.g., a cPPT/FLAP), viral packaging (e.g., a Psi packaging signal, RRE), and/or other elements that increase payload gene expression (e.g., poly (A) sequences), and may optionally comprise a WPRE or HPRE.
  • a lentiviral nucleic acid comprises one or more of, e.g., all of, e.g., from 5’ to 3’, a promoter (e.g., CMV), an R sequence (e.g., comprising TAR), a U5 sequence (e.g., for integration), a PBS sequence (e.g., for reverse transcription), a DIS sequence (e.g., for genome dimerization), a psi packaging signal, a partial gag sequence, an RRE sequence (e.g., for nuclear export), a cPPT sequence (e.g., for nuclear import), a promoter to drive expression of the payload agent, a payload gene encoding the payload agent, a WPRE sequence (e.g., for efficient transgene expression), a PPT sequence (e.g., for reverse transcription), an R sequence (e.g., for polyadenylation and termination), and a U5 signal (e.g.
  • a promoter e
  • the particle is a virus-like particle (VLP) that is derived from virus.
  • the viral envelope may comprise a fusogen, e.g., a fusogen that is endogenous to the virus or a pseudotyped fusogen.
  • the VLPS include those derived from retroviruses or lentiviruses. While VLPs mimic native virion structure, they lack the viral genomic information necessary for independent replication within a host cell. Therefore, in some aspects, VLPs are non-infectious. In particular embodiments, a VLP does not contain a viral genome.
  • the VLP’s bilayer of amphipathic lipids is or comprises the viral envelope.
  • the particle’s bilayer of amphipathic lipids is or comprises lipids derived from a cell.
  • a VLP contains at least one type of structural protein from a virus. In most cases this protein will form a proteinaceous capsid. In some cases the capsid will also be enveloped in a lipid bilayer originating from the cell from which the assembled VLP has been released (e.g., VLPs comprising a human immunodeficiency virus structural protein such as GAG).
  • the VLP further comprises a targeting moiety as an envelope protein within the lipid bilayer.
  • the particle comprises supramolecular complexes formed by viral proteins that self-assemble into capsids.
  • the particle is a virus-like particle derived from viral capsid proteins.
  • the particle is a virus-like particle derived from viral nucleocapsid proteins.
  • the particle comprises nucleocapsid-derived proteins that retain the property of packaging nucleic acids.
  • the particle comprises only viral structural glycoproteins among proteins from the viral genome. In some embodiments, the particle does not contain a viral genome.
  • the particle packages nucleic acids during the expression process, such as a nucleic acid encoding a payload agent.
  • the nucleic acids do not encode any genes involved in virus replication.
  • the particle is a virus-like particle, e.g., retrovirus-like particle, such as a lentivirus-like particle, that is replication defective.
  • the particle is a virus-like particle which comprises a sequence that is devoid of or lacking viral RNA, which may be the result of removing or eliminating the viral RNA from the sequence. In some embodiments, this may be achieved by using an endogenous packaging signal binding site on gag. In some embodiments, the endogenous packaging signal binding site is on pol. In some embodiments, the RNA which is to be delivered will contain a cognate packaging signal. In some embodiments, a heterologous binding domain (which is heterologous to gag) located on the RNA to be delivered, and a cognate binding site located on gag or pol, can be used to ensure packaging of the RNA to be delivered.
  • the heterologous sequence could be non-viral or it could be viral, in which case it may be derived from a different virus.
  • the particles could be used to deliver therapeutic RNA, in which case functional integrase and/or reverse transcriptase is not required.
  • the particles could also be used to deliver a therapeutic gene of interest, in which case pol is typically included.
  • the particle is a cell based particle that comprises a naturally derived membrane.
  • the naturally derived membrane comprises membrane vesicles prepared from cells or tissues.
  • the cell based particle comprises a vesicle that is obtainable from a cell.
  • the cell based particle comprises a microvesicle, an exosome, a membrane enclosed body, an apoptotic body (from apoptotic cells), a particle (which may be derived from e.g. platelets), an ectosome (derivable from, e.g., neutrophiles and monocytes in serum), a prostatosome (obtainable from prostate cancer cells), or a cardiosome (derivable from cardiac cells).
  • the source cell is an endothelial cell, a fibroblast, a blood cell (e.g., a macrophage, a neutrophil, a granulocyte, a leukocyte), a stem cell (e.g., a mesenchymal stem cell, an umbilical cord stem cell, bone marrow stem cell, a hematopoietic stem cell, an induced pluripotent stem cell e.g., an induced pluripotent stem cell derived from a subject’s cells), an embryonic stem cell (e.g., a stem cell from embryonic yolk sac, placenta, umbilical cord, fetal skin, adolescent skin, blood, bone marrow, adipose tissue, erythropoietic tissue, hematopoietic tissue), a myoblast, a parenchymal cell (e.g., hepatocyte), an alveolar cell, a neuron (e.g., a fibro
  • the cell based particle has a density of ⁇ 1, 1-1.1, 1.05-1.15, 1.1-1.2, 1.15-1.25, 1.2-1.3, 1.25-1.35, or >1.35 g/ml. In embodiments, the cell based particle has a size that is less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, of that of the source cell.
  • a composition containing a plurality of the cell based particle comprises less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or 10% source cells by protein mass or less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or 10% of cells having a functional nucleus.
  • the source cell used to make the cell based particle will not be available for testing after the particle is made.
  • the cell based particle is an extracellular vesicle, e.g., a cell based vesicle comprising a membrane that encloses an internal space and has a smaller diameter than the cell from which it is derived.
  • the extracellular vesicle has a diameter from 20 nm to 1000 nm.
  • the cell based particle is an apoptotic body, a fragment of a cell, a vesicle derived from a cell by direct or indirect manipulation, a vesiculated organelle, and a vesicle produced by a living cell (e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane).
  • the extracellular vesicle is derived from a living or dead organism, explanted tissues or organs, or cultured cells.
  • the cell based particle is a nanovesicle, e.g., a cell-derived small (e.g., between 20-250 nm in diameter, or 30-150 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from said cell by direct or indirect manipulation.
  • the production of nanovesicles can, in some instances, result in the destruction of the source cell.
  • the nanovesicle may comprise a lipid or fatty acid and polypeptide.
  • the cell based particle is an exosome.
  • the exosome is a cell-derived small (e.g., between 20-300 nm in diameter, or 40-200 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from said cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane.
  • production of exosomes does not result in the destruction of the source cell.
  • the exosome comprises lipid or fatty acid and polypeptide.
  • the cell based particle is a microvesicle.
  • the micro vesicle has a diameter of about 100 nm to about 2000 nm.
  • the cell based particle is a cell ghost.
  • a vesicle is a plasma membrane vesicle, e.g. a giant plasma membrane vesicle.
  • the cell based particle is derived from a source cell with a genetic modification which results in increased expression of an immunomodulatory agent, such as an immunosuppressive agent.
  • an immunomodulatory agent such as an immunosuppressive agent.
  • the immunosuppressive agent is on an exterior surface of the cell.
  • the immunosuppressive agent is incorporated into the exterior surface of the particle.
  • the particle comprises an immunomodulatory agent attached to the surface of the solid particle by a covalent or non-covalent bond
  • cell based particles are generated by inducing budding of an exosome, microvesicle, membrane vesicle, extracellular membrane vesicle, plasma membrane vesicle, giant plasma membrane vesicle, apoptotic body, mitoparticle, pyrenocyte, lysosome, or other membrane enclosed vesicle.
  • cell based particles are generated by inducing cell enucleation.
  • Enucleation may be performed using assays such as genetic, chemical (e.g., using Actinomycin D, see Bayona-Bafaluyet al., “A chemical enucleation method for the transfer of mitochondrial DNA to p° cells” Nucleic Acids Res. 2003 Aug 15; 31(16): e98), mechanical methods (e.g., squeezing or aspiration, see Lee et al., “A comparative study on the efficiency of two enucleation methods in pig somatic cell nuclear transfer: effects of the squeezing and the aspiration methods.” Anim Biotechnol. 2008;19(2):71- 9), or combinations thereof.
  • assays such as genetic, chemical (e.g., using Actinomycin D, see Bayona-Bafaluyet al., “A chemical enucleation method for the transfer of mitochondrial DNA to p° cells” Nucleic Acids Res. 2003 Aug 15; 31(16): e98
  • the cell based particles are generated by inducing cell fragmentation.
  • cell fragmentation can be performed using the following methods, including, but not limited to: chemical methods, mechanical methods (e.g., centrifugation (e.g., ultracentrifugation, or density centrifugation), freeze-thaw, or sonication), or combinations thereof.
  • a characteristic of a cell based particle is described by comparison to a reference cell.
  • the reference cell is the source cell.
  • the reference cell is a HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080, or BJ cell.
  • a characteristic of a composition of cell based particles is described by comparison to a population of reference cells, e.g., a population of source cells, or a population of HeLa, HEK293, HFF- 1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080, or BJ cells.
  • the particle has a fusogen. In some embodiments, the particle has more than one fusogen. In some embodiments, the fusogen contains a mammalian protein, such as any described in Section II-B-1. In some embodiments, the fusogen contains a viral protein, such as any described in Section II-B-2.
  • the fusogen promotes mixing between lipids in the particle and lipids in the target cell. In some embodiments, the fusogen facilitates the fusion of the particle to a membrane of the target cell. In some embodiments, the particle integrates into the membrane of the target cell. In some embodiments, the membrane is the plasma membrane of the target cell. In some embodiments, the fusogen promotes formation of one or more pores between the interior of the particle and the cytosol of the target cell.
  • the fusogen is endogenous to the particle.
  • the particle is pseudotyped with the fusogen, such as when the particle is a viral particle or virus-like particle.
  • the fusogen is exposed on the surface of the particle. In some embodiments, the fusogen is exposed on the surface of the lipid bilayer of the particle. In some embodiments, a portion of the fusogen is embedded in the lipid bilayer of the particle. [0361] In some embodiments, the particle is a viral particle or a virus-like particle. In some embodiments, the fusogen is exposed on the surface of the viral envelope of the particle. In some embodiments, a portion of the fusogen is embedded in the viral envelope of the particle.
  • the fusogen is present at a copy number of at least, or no more than, 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the fusogen comprised by the particle is disposed in the cell membrane. In embodiments, the particle also comprises fusogen internally, e.g., in the cytoplasm or an organelle.
  • the fusogen comprises (or is identified as comprising) about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, or more, or about 1-30%, 5- 20%, 10- 15%, 12-15%, 13-14%, or 13.6% of the total protein in a particle, e.g., as determined by a mass spectrometry assay.
  • the fusogen comprises (or is identified as comprising) about 13.6% of the total protein in the particle.
  • the fusogen is (or is identified as being) more or less abundant than one or more additional proteins of interest.
  • the fusogen has (or is identified as having) a ratio to EGFP of about 140, 145, 150, 151, 152, 153, 154, 155, 156, 157 (e.g., 156.9), 158, 159, 160, 165, or 170.
  • the fusogen has (or is identified as having) a ratio to GAPDH of about 50, 55, 60, 65, 70 (e.g., 69), 75, 80, or 85, or about 1-30%, 5-20%, 10-15%, 12-15%, 13-14%, or 13.6%.
  • the fusogen has (or is identified as having) a ratio to CNX of about 500, 510, 520, 530, 540, 550, 560 (e.g., 558.4), 570, 580, 590, or 600, or about 300-800, 400-700, 500-600, 520-590, 530-580, 540-570, 550-560, or 558.4, e.g., by a mass spectrometry assay.
  • the fusogen is a protein fusogen.
  • the fusogen is a native protein or a derivative of a native protein.
  • the fusogen is a synthetic protein.
  • the fusogen is a mammalian protein or a homologue of a mammalian protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the mammalian protein), such as any described in Section II-B-1.
  • the fusogen is a non-mammalian protein.
  • the fusogen is a viral protein or a homologue of a viral protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the viral protein), such as any described in Section II-B-2.
  • the fusogen is a fragment of any of the foregoing.
  • the fusogen is a variant of any of the foregoing.
  • the fusogen is a protein fusion containing one or more proteins or fragments thereof.
  • the fusogen is a protein fusion containing one or more of any of the foregoing.
  • the fusogen is mutated to reduce binding for the native binding partner of the fusogen. In some embodiments the fusogen is randomly mutated. In some embodiments the fusogen is rationally mutated. In some embodiments the fusogen is subjected to directed evolution. In some embodiments the fusogen is truncated and only a subset of the peptide is used in the particle.
  • the fusogen is or contains a mammalian protein.
  • exemplary mammalian fusogens include a SNARE family protein such as vSNAREs or tSNAREs, a syncytin protein such as Syncytin-1 (DOI: 10.1128/JVI.76.13.6442-6452.2002) and Syncytin-2, myomaker (biorxiv.org/content/early/2017/04/02/123158, doi.org/10.1101/123158, doi: 10.1096/fj.201600945R, doi: 10.1038/nature 12343), myomixer (www.nature.com/nature/journal/v499/n7458/full/naturel2343.html, doi: 10.1038/nature 12343), myomerger (science.sciencemag.org/content/early/2017/04/05/science.aam9361, DOI: 10.1126/science.aam9361), FGFRL1 (fibroblast
  • the fusogen is encoded by a human endogenous retroviral element (hERV) found in the human genome. Additional exemplary fusogens are disclosed in US 6,099,857A and US 2007/0224176, the entire contents of each of which are hereby incorporated by reference.
  • hERV human endogenous retroviral element
  • a viral fusogen is a Class I viral membrane fusion protein, a Class II viral membrane protein, a Class III viral membrane fusion protein, a viral membrane glycoprotein, or other viral fusion proteins, or a homologue thereof, a fragment thereof, a variant thereof, or a protein fusion containing one or more proteins or fragments thereof, such as one or more of any of the foregoing.
  • Class I viral membrane fusion proteins include Baculovirus F protein, e.g., F proteins of the nucleopolyhedrovirus (NPV) genera, e.g., Spodoptera exigua MNPV (SeMNPV) F protein and Lymantria dispar MNPV (LdMNPV), and paramyxovirus F proteins.
  • NPV nucleopolyhedrovirus
  • SeMNPV Spodoptera exigua MNPV
  • LdMNPV Lymantria dispar MNPV
  • Class II viral membrane proteins include tick bone encephalitis E (TBEV E) and Semliki Forest Virus E1/E2.
  • Class III viral membrane fusion proteins include rhabdovirus G (e.g., fusogenic protein G of the Vesicular Stomatatis Virus (VSV-G), Cocal virus G protein), herpesvirus glycoprotein B (e.g., Herpes Simplex virus 1 (HSV-1) gB), Epstein Barr Virus glycoprotein B (EBV gB), thogotovirus G, baculovirus gp64 (e.g., Autographa California multiple NPV (AcMNPV) gp64), and Borna disease virus (BDV) glycoprotein (BDV G).
  • rhabdovirus G e.g., fusogenic protein G of the Vesicular Stomatatis Virus (VSV-G), Cocal virus G protein
  • herpesvirus glycoprotein B e.g., Herpes Simplex virus 1 (HSV-1) gB
  • thogotovirus G
  • viral fusogens e.g., membrane glycoproteins and viral fusion proteins
  • viral syncytia proteins such as influenza hemagglutinin (HA) or mutants, or fusion proteins thereof; human immunodeficiency virus type 1 envelope protein (HIV-1 ENV), gpl20 from HIV binding LFA-1 to form lymphocyte syncytium, HIV gp41, HIV gpl60, or HIV Trans- Activator of Transcription (TAT); viral glycoprotein VSV-G, viral glycoprotein from vesicular stomatitis virus of the Rhabdoviridae family; glycoproteins gB and gH-gL of the varicella-zoster virus (VZV); murine leukaemia virus (MLV)-lOAl; Gibbon Ape Leukemia Virus glycoprotein (GaLV); type G glycoproteins in Rabies, Mokola, vesicular stomatitis virus and Togavirus
  • Non-mammalian fusogens include viral fusogens, homologues thereof, fragments thereof, and fusion proteins comprising one or more proteins or fragments thereof, such as one or more of any of the foregoing.
  • Viral fusogens include class I fusogens, class II fusogens, class III fusogens, and class IV fusogens.
  • class I fusogens such as human immunodeficiency virus (HIV) gp41 have a characteristic postfusion conformation with a signature trimer of a-helical hairpins with a central coiled-coil structure.
  • Class I viral fusion proteins include proteins having a central postfusion six-helix bundle.
  • Class I viral fusion proteins include influenza HA, parainfluenza F, HIV Env, Ebola GP, hemagglutinins from orthomyxoviruses, F proteins from paramyxoviruses (e.g. Measles, (Katoh et al. BMC Biotechnology 2010, 10:37)), ENV proteins from retroviruses, and fusogens of filoviruses and coronaviruses.
  • class II viral fusogens such as dengue E glycoprotein, have a structural signature of P- sheets forming an elongated ectodomain that refolds to result in a trimer of hairpins.
  • the class II viral fusogen lacks the central coiled coil.
  • Class II viral fusogen can be found in alphaviruses (e.g., El protein) and flaviviruses (e.g., E glycoproteins).
  • Class II viral fusogens include fusogens from Semliki Forest virus, Sinbis, rubella virus, and dengue virus.
  • class III viral fusogens such as the vesicular stomatitis virus G glycoprotein, combine structural signatures found in classes I and II.
  • a class III viral fusogen comprises a helices (e.g., forming a six-helix bundle to fold back the protein as with class I viral fusogens), and sheets with an amphiphilic fusion peptide at its end, reminiscent of class II viral fusogens.
  • Class III viral fusogens can be found in rhabdo viruses and herpesviruses.
  • class IV viral fusogens are fusion-associated small transmembrane (FAST) proteins (doi:10.1038/sj.emboj.7600767, Nesbitt, Rae L., "Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins” (2012). Electronic Thesis and Dissertation Repository. Paper 388), which are encoded by nonenveloped reoviruses.
  • the class IV viral fusogens are sufficiently small that they do not form hairpins (doi: 10.1146/annurev-cellbio- 101512-122422, doi:10.1016/j.devcel.2007.12.008).
  • the fusogen is any of the fusogenic moieties described in WO2017/182585; WO2022/164935; WO2021/076788; Hamilton et al. bioRxiv 2022.08.24.505004; Nikolic et al. Nat Commun 9, 1029 (2018); Dobson et al. Nat. Methods. 19, 449-460 (2022); and Yu et al. bioRxiv 2021.12.13.472464, for instance any of the VSV or variant VSV glycoproteins described therein, such as VSV glycoproteins that have reduced binding to native receptors.
  • the fusogen is a Baboon Endogenous Retrovirus (BaEV) envelope glycoprotein.
  • BoEV envelope glycoproteins and variants thereof are described in PCT/US2022/031459; US9249426; Aguila et al. Journal of Virology 2003 77(2):1281-1291; Bernadin et al. Blood Advances 2019 3(3):461-475; Colamartino et al. Frontiers in Immunology 2019 10:2873; Girard-Gagnepain et al. Blood 2014 124(8): 1221-1231; and Levy et al. Journal of Thrombosis and Haemostasis 2016 14:2478-2492.
  • Wild-type BaEV envelope glycoproteins are retroviral envelope proteins containing a C- terminal cytoplasmic tail (e.g., corresponding to amino acids 512-545 of SEQ ID NO:252), a transmembrane domain (e.g., corresponding to amino acids 489-511 of SEQ ID NO:252), and an extracellular domain (e.g., corresponding to amino acids 1-488 of SEQ ID NO:252).
  • Maturation of the precursor protein in the Golgi which requires the minimal sequence [KR]-X-[KR]-R (wherein X is any amino acid), results in two subunits, the surface unit protein or gp70, and the transmembrane protein p20E.
  • the surface unit protein or gp70 (e.g., corresponding to amino acids 1-358 of SEQ ID NO:252) and the transmembrane protein p20E (e.g., corresponding to amino acids 359-545 of SEQ ID NO:252) remain associated in a labile interaction that may include a disulfide bond.
  • fusogenicity is controlled by a short, 17 amino acid sequence termed a fusion inhibitory R peptide (e.g., set forth in SEQ ID NO:253), which is localized on the C-terminal of the cytoplasmic tail domain.
  • the fusion inhibitory R peptide harbors the tyrosine endocytosis signal YXXL, and its cleavage by the viral protease is thought to potentiate fusogenic activation through molecular rearrangements in the membrane-spanning domain and the extracellular region of the envelope glycoprotein (Salamango et al (2015) Journal of virology 89(24): 12492-12500).
  • the gp70 mediates receptor binding to the ASCT-2 and ASCT-1 receptors on host cells.
  • the glycoprotein 70 (g70) subunit or a biologically active portion thereof binds the ASCT-2 and ASCT-1 receptors.
  • the p20E acts as a class I viral fusion protein.
  • the interaction of the gp70 subunit with a host cell membrane triggers refolding of the p20E and is believed to activate the fusogenic potential by unmasking the fusion peptide.
  • the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
  • BaEV baboon endogenous virus
  • the fusogen is a truncated BaEV envelope glycoprotein.
  • Exemplary BaEV envelope glycoproteins and truncates thereof are described in PCT/US2022/031459.
  • the truncated BaEV envelope glycoprotein comprises a cytoplasmic tail with a partial fusion inhibitory R peptide relative to a wild-type BaEV envelope glycoprotein, wherein the R peptide contains a contiguous portion of the inhibitory R peptide but lacks the full length R peptide of wild-type BaEV envelope glycoprotein.
  • the truncated BaEV envelope glycoprotein has a cytoplasmic tail that is composed of a partial inhibitory R peptide with at least one, at least two, or at least three contiguous amino-terminal amino acids of the inhibitory R peptide but less than the full-length R peptide relative to wild-type BaEV envelope glycoprotein.
  • the truncated BaEV envelope glycoprotein has a cytoplasmic tail that has a partial inhibitory R peptide composed of 1 to 16 contiguous amino-terminal amino acids of the inhibitory R peptide of the wild- type BaEV envelope glycoprotein, such as is composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 12, 14, 15 or 16 amino-terminal amino acids of the inhibitory R peptide of the wild-type BaEV envelope glycoprotein.
  • the truncated BaEV envelope glycoprotein is set forth in any of SEQ ID NO:254-260. In som embodiments, the truncated BaEV envelope glycoprotein s BaEV R+8 set fort hin SEQ ID NO:259.
  • the fusogen is a modified BaEV envelope glycoprotein.
  • the cytoplasmic tail domain of the BaEV envelope glycoprotein is devoid of the fusion inhibitory R peptide.
  • the expression “fusion inhibitory R peptide” refers to the C-terminal portion of the cytoplasmic tail domain of the envelope glycoprotein which harbours a tyrosine endocytosis signal — YXXL — and which is cleaved by viral protease during virion maturation, thus enhancing membrane fusion of the envelope glycoprotein.
  • the fusion inhibitory R peptide of the BaEV envelope glycoprotein is typically located between amino acids 547 and 564 of the wild-type BaEV envelope glycoprotein.
  • the modified BaEV envelope glycoprotein is set forth in SEQ ID NO: 261 (BaEVRLess).
  • the cytoplasmic tail domain of the BaEV envelope glycoprotein is replaced by the cytoplasmic tail domain of a murine leukemia virus (MLV) envelope glycoprotein.
  • MLV murine leukemia virus
  • the Murine Leukemia Virus envelope glycoprotein is notably described in Ott et al. (1990) J. Virol. 64:757- 766.
  • the Murine Leukemia Virus envelope glycoprotein is that of strain 4070A.
  • MLV envelope glycoprotein refers to the wild-type form of the MLV envelope glycoprotein or to a mutant of said wild-type MLV envelope glycoprotein which is at least 80%, preferably at least 85%, still preferably at least 90%, more preferably at least 95%, still more preferably at least 99% identical to said wild-type MLV envelope glycoprotein, provided that said mutant glycoprotein retains the capacity of the wild-type envelope glycoprotein of interacting with viral core proteins, in particular with lentiviral core proteins.
  • the cytoplasmic tail domain of the MLV envelope glycoprotein is located between amino acids 622 and 654 of the wild-type MLV envelope glycoprotein.
  • the fusogen is BaEVTR.
  • the modified BaEV envelope glycoprotein is set forth in SEQ ID NO: 262 (BaEVTR).
  • the fusogen is able to target the CD34+ progenitor subpopulations indicated in FIG. 7 for BaEVTR.
  • the fusogen is able to target HSCs, MPPs, CMPs, MLPs, or ETPs.
  • the fusogen is able to target HSCs, MPPs, CMPs, MLPs, and ETPs.
  • the target cells are HSCs, MPPs, CMPs, MLPs, or ETPs.
  • the target cells are MPPs, CMPs, MLPs, and ETPs.
  • a VLP includes a BaEVTR glycoprotein fusogen (e.g., a fusogen comprising a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof or a truncated BaEV envelope glycoprotein or a functional variant thereof)
  • the VLP also comprises a gag protein (e.g. an MLV-gag protein).
  • the MLV-gag protein is part of a fusion protein, where the fusion protein is a cleavable fusion protein between (i) a viral structural protein (e.g. GAG (e.g.
  • VLP includes a BaEVTR glycoprotein fusogen and a gag protein (e.g. an MLV-gag protein)
  • production of such a VLP includes varying the ratio of polynucleotide encoding the BaEVTR glycoprotein fusogen and the polynucleotide encoding the gag protein.
  • the ratio of polynucleotide encoding the BaEVTR glycoprotein fusogen to the polynucleotide encoding the gag protein includes a 1:1, 1:2, 1:3, 1:4, or 1:5 ratio.
  • the fusogen is or contains a G or H protein.
  • the G or H protein is a Paramyxovirus (e.g., Morbillivirus or Henipavirus) G or H protein or a biologically active portion thereof.
  • the Henipavirus G protein is a Hendr a (HeV) virus G protein, a Nipah (NiV) virus G-protein (NiV-G), a Cedar (CedPV) virus G-protein, a Mojiang virus G- protein, a bat Paramyxovirus G-protein, a Kumasi virus G-protein, a Langya virus G-protein, or a biologically active portion thereof.
  • Table 1 A non-limited list of exemplary G proteins is shown in Table 1.
  • the Henipavirus attachment G proteins are type II transmembrane glycoproteins containing an N-terminal cytoplasmic tail (e.g., corresponding to amino acids 1-49 of SEQ ID NO:1), a transmembrane domain (e.g., corresponding to amino acids 50-70 of SEQ ID NO:1, and an extracellular domain containing an extracellular stalk (e.g., corresponding to amino acids 71-187 of SEQ ID NO:1), and a globular head (corresponding to amino acids 188-602 of SEQ ID NO:1).
  • the N-terminal cytoplasmic domain is within the inner lumen of the lipid bilayer and the C-terminal portion is the extracellular domain that is exposed on the outside of the lipid bilayer.
  • Regions of the stalk in the C- terminal region have been shown to be involved in interactions with F protein and triggering of F protein fusion (Liu et al. 2015 J of Virology 89:1838).
  • the globular head mediates receptor binding to henipavirus entry receptors ephrin B2 and ephrin B3, but is dispensable for membrane fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13)e00577-19).
  • tropism of the G protein is modified. Binding of the G protein to a binding partner can trigger fusion mediated by a compatible F protein or biologically active portion thereof.
  • G protein sequences disclosed herein are predominantly disclosed as expressed sequences including an N-terminal methionine required for start of translation. As such N-terminal methionines are commonly cleaved co- or post-translationally, the mature protein sequences for all G protein sequences disclosed herein are also contemplated as lacking the N-terminal methionine.
  • G glycoproteins are highly conserved between henipavirus species.
  • the G protein of NiV and HeV viruses share 79% amino acids identity.
  • Studies have shown a high degree of compatibility among G proteins with F proteins of different species as demonstrated by heterotypic fusion activation (Brandel-Tretheway et al. Journal of Virology. 2019).
  • a retargeted lipid particle can contain heterologous proteins from different species.
  • the G protein has a sequence set forth in any of SEQ ID NOs: 1-11 or is a functionally active variant or biologically active portion thereof that has a sequence that is at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% identical to any one of SEQ ID NOs:l, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.
  • the G protein has a sequence set forth in SEQ ID NO:1 or is a functionally active variant or biologically active portion thereof that has a sequence that is at least at or about 80%, at least at or about 90%, at least at or about 95%, or at least at or about 99% identical to SEQ ID NO:1.
  • the G protein has a sequence set forth in SEQ ID N0:4 or is a functionally active variant or biologically active portion thereof that has a sequence that is at least at or about 80%, at least at or about 90%, at least at or about 95%, or at least at or about 99% identical to SEQ ID NO:4.
  • the G protein has a sequence set forth in SEQ ID NO:5 or is a functionally active variant or biologically active portion thereof that has a sequence that is at least at or about 80%, at least at or about 90%, at least at or about 95%, or at least at or about 99% identical to SEQ ID NO:5.
  • the G protein or functionally active variant or biologically active portion is a protein that retains fusogenic activity in conjunction with a Henipa virus F protein, e.g. NiV-F or HeV-F.
  • Fusogenic activity includes the activity of the G protein in conjunction with a Henipavirus F protein to promote or facilitate fusion of two membrane lumens, such as the lumen of the targeted lipid particle having embedded in its lipid bilayer a henipavirus F and G protein, and a cytoplasm of a target cell, e.g. a cell that contains a surface receptor or molecule that is recognized or bound by the targeted envelope protein.
  • the F protein and G protein are from the same Henipavirus species (e.g. NiV-G and NiV-F). In some embodiments, the F protein and G protein are from different Henipavirus species (e.g. NiV-G and HeV-F).
  • the G protein has the sequence of amino acids set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10 or SEQ ID NO: 11 or is a functionally active variant thereof or a biologically active portion thereof that retains fusogenic activity.
  • the biologically active portion has an amino acid sequence having at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:1, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10 or SEQ ID NO: 11 and retains fusogenic activity in conjunction with a Henipavirus F protein (e.g., NiV-F or HeV-F).
  • a Henipavirus F protein e.g., NiV-F or HeV-F
  • Reference to retaining fusogenic activity includes activity (in conjunction with a Henipavirus F protein) that is between at or about 10% and at or about 150% or more of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO: 1, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10 or SEQ ID NO: 11 such as at least or at least about 10% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 15% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 20% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 25% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or
  • the G protein is a mutant G protein that is a functionally active variant or biologically active portion containing one or more amino acid mutations, such as one or more amino acid insertions, deletions, substitutions or truncations.
  • the mutations described herein relate to amino acid insertions, deletions, substitutions or truncations of amino acids compared to a reference G protein sequence.
  • the reference G protein sequence is the wild- type sequence of a G protein or a biologically active portion thereof.
  • the functionally active variant or the biologically active portion thereof is a mutant of a wild-type Hendra (HeV) virus G protein, a wild-type Nipah (NiV) virus G-protein (NiV-G), a wild-type Cedar (CedPV) virus G-protein, a wild-type Mojiang virus G-protein, a wild-type bat Paramyxovirus G-protein, a Kumasi virus G-protein, a Langya virus G-protein, or biologically active portion thereof.
  • the wild-type G protein has the sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:7, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10 or SEQ ID NO: 11.
  • the G protein is a mutant G protein that is a biologically active portion that is an N-terminally and/or C-terminally truncated fragment of a wild- type Hendra (HeV) virus G protein, a wild-type Nipah (NiV) virus G-protein (NiV-G), a wild-type Cedar (CedPV) virus G- protein, a wild-type Mojiang virus G-protein, a wild-type bat Paramyxovirus G-protein, a Kumasi virus G-protein, or a Langya virus G-protein.
  • the truncation is an N-terminal truncation of all or a portion of the cytoplasmic domain.
  • the mutant G protein is a biologically active portion that is truncated and lacks up to 49 contiguous amino acid residues at or near the N-terminus of the wild-type G protein, such as a wild-type G protein set forth in any one of SEQ ID NO:1, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:7, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.
  • the mutant F protein is truncated and lacks up to 49 contiguous amino acids, such as up to 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 30, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 contiguous amino acids at the N-terminus of the wild-type G protein.
  • the G protein is a wild-type Nipah virus G (NiV-G) protein or a Hendra virus G protein, or is a functionally active variant or biologically active portion thereof.
  • the G protein is a NiV-G protein that has the sequence set forth in SEQ ID NO:1, SEQ ID NOG or SEQ ID NOG, or is a functional variant or a biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or or
  • the G protein is a NiV-G protein that has the sequence set forth in SEQ ID NO:1, or is a functional variant or a biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99% sequence identity to SEQ ID NO:1.
  • the G protein is a NiV-G protein that has the sequence set forth in SEQ ID NO:1.
  • the G protein is a NiV-G protein that has the sequence set forth in SEQ ID N0:4, or is a functional variant or a biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99% sequence identity to SEQ ID NO:4.
  • the G protein is a NiV-G protein that has the sequence set forth in SEQ ID NO:4. In some embodiments, the G protein is a NiV-G protein that has the sequence set forth in SEQ ID NO:5, or is a functional variant or a biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99% sequence identity to SEQ ID NO:5. In some embodiments, the G protein is a
  • the G protein is a mutant NiV-G protein that is a biologically active portion of a wild-type NiV-G.
  • the biologically active portion is an N- terminally truncated fragment.
  • the mutant NiV-G protein is truncated and lacks up to 5 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 6 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 7 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 8 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID
  • the mutant NiV-G protein is truncated and lacks 5 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5).
  • the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the mutant NiV-G protein is truncated and lacks 10 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5).
  • the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:44. In some embodiments, the mutant NiV-G protein is truncated and lacks 15 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:45. In some embodiments, the mutant NiV-G protein is truncated and lacks 20 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5).
  • the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the mutant NiV-G protein is truncated and lacks 25 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the mutant NiV-G protein is truncated and lacks 30 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5).
  • the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the mutant NiV-G protein is truncated and lacks 34 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:42.
  • the NiV-G protein is a biologically active portion that does not contain a cytoplasmic domain.
  • the NiV-G protein without the cytoplasmic domain is encoded by SEQ ID NO:22.
  • the mutant NiV-G protein comprises a sequence set forth in any of SEQ ID NOS: 12-14, 17, 18 and 22, or 42-45 or is a functional variant thereof that has an amino acid sequence having at least at or 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%,
  • the mutant NiV-G protein has a 5 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO: 12 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 12 or such as set forth
  • the mutant NiV-G protein has a 10 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO:44 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:44.
  • the mutant NiV-G protein has a 20 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO: 13 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 13.
  • the mutant NiV-G protein has a 25 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO: 14 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 14.
  • the mutant NiV-G protein has a 33 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO: 17 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 17.
  • the mutant NiV-G protein has a 34 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO: 18 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:18.
  • the mutant NiV-G protein has a 48 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO:22 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:22.
  • the mutant NiV-G protein has a 15 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), such as set forth in SEQ ID NO:45 or a functional variant thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:
  • the mutant NiV-G protein has a 20 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), such as set forth in SEQ ID NO: 13 or a functional variant thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:
  • the mutant NiV-G protein has a 25 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), such as set forth in SEQ ID NO: 14 or a functional variant thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:
  • the mutant NiV-G protein has a 30 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), such as set forth in SEQ ID NO:43 or a functional variant thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:
  • the mutant NiV-G protein has a 34 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:, SEQ ID NO:4, or SEQ ID NO:5), such as set forth in SEQ ID NO:42 or a functional variant thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:
  • the mutant NiV-G protein has a 48 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), such as set forth in SEQ ID NO:22 or a functional variant thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:
  • the G protein is a mutant HeV-G protein that is a biologically active portion of a wild- type HeV-G.
  • the biologically active portion is an N-terminally truncated fragment.
  • the G protein is a wild-type HeV-G protein that has the sequence set forth in SEQ ID NO:23 or 24, or is a functional variant or biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at or about 85%, at least at or about 86%, at least at or about 87%, at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:23or 24.
  • the G protein is a mutant HeV-G protein that is a biologically active portion of a wild-type HeV-G (SEQ ID NO:23 or SEQ ID NO:24).
  • the biologically active portion is an N-terminally truncated fragment.
  • the mutant HeV-G protein is truncated and lacks up to 5 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 6 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 7 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24) or up to 8 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 9 contiguous amino acid residues at or near the N- terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 10 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 11 contig
  • the HeV-G protein is a biologically active portion that does not contain a cytoplasmic domain.
  • the mutant HeV-G protein lacks the N-terminal cytoplasmic domain of the wild-type HeV-G protein (SEQ ID NO:23 or 24), such as set forth in SEQ ID NO:25 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity
  • the mutant HeV-G protein lacks the N-terminal cytoplasmic domain of the wild-type HeV-G protein (SEQ ID NO:23 or 24), such as set forth in SEQ ID NO:26 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:26.
  • SEQ ID NO:23 or 24 such as set forth in SEQ ID NO:26 or a functional variant thereof having at least
  • the G protein or the functionally active variant or biologically active portion thereof binds to Ephrin B2 or Ephrin B3.
  • the G protein has the sequence of amino acids set forth in any one of SEQ ID NO:24, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or is a functionally active variant thereof or a biologically active portion thereof that is able to bind to Ephrin B2 or Ephrin B3.
  • the functionally active variant or biologically active portion has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at or about 86%, at least at or about 87%, at least at or about 88%, or at least at or about 89% , at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to any of SEQ ID NO:24, SEQ ID NO:23, SEQ ID N0:4, SEQ ID N0:6, SEQ ID N0:5, SEQ ID N0:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, and retains binding to Ephrin
  • the functionally active variant or biologically active portion has an amino acid sequence having at least about 80%, at least about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, and retains binding to Ephrin B2 or B3.
  • Reference to retaining binding to Ephrin B2 or B3 includes binding that is at least or at least about 5% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, 10% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, 15% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ
  • the G protein is NiV-G or a functionally active variant or biologically active portion thereof and binds to Ephrin B2 or Ephrin B3.
  • the NiV-G has the sequence of amino acids set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, or is a functionally active variant thereof or a biologically active portion thereof that is able to bind to Ephrin B2 or Ephrin B3.
  • the functionally active variant or biologically active portion has an amino acid sequence having at least about 80%, at least about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27 and retains binding to Ephrin B2 or B3.
  • Exemplary biologically active portions include N-terminally truncated variants lacking all or a portion of the cytoplasmic domain, e.g.
  • Reference to retaining binding to Ephrin B2 or B3 includes binding that is at least or at least about 5% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 10% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 15% of the level or degree of binding of the corresponding wildtype NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 20% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 25% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:
  • the G protein or the biologically thereof is a mutant G protein that exhibits reduced binding for the native binding partner of a wild-type G protein.
  • the mutant G protein or the biologically active portion thereof is a mutant of wild-type Niv-G and exhibits reduced binding to one or both of the native binding partners Ephrin B2 or Ephrin B3.
  • the mutant G-protein or the biologically active portion, such as a mutant NiV-G protein exhibits reduced binding to the native binding partner.
  • the reduced binding to Ephrin B2 or Ephrin B3 is reduced by greater than at or about 5%, at or about 10%, at or about 15%, at or about 20%, at or about 25%, at or about 30%, at or about 40%, at or about 50%, at or about 60%, at or about 70%, at or about 80%, at or about 90%, or at or about 100%.
  • the mutations described herein can improve transduction efficiency. In some embodiments, the mutations described herein allow for specific targeting of other desired cell types that are not Ephrin B2 or Ephrin B3. In some embodiments, the mutations described herein result in at least the partial inability to bind at least one natural receptor, such has reduce the binding to at least one of Ephrin B2 or Ephrin B3. In some embodiments, the mutations described herein interfere with natural receptor recognition.
  • the G protein is HeV-G or a functionally active variant or biologically active portion thereof and binds to Ephrin B2 or Ephrin B3.
  • the HeV- G has the sequence of amino acids set forth in SEQ ID NO:23 or 24, or is a functionally active variant thereof or a biologically active portion thereof that is able to bind to Ephrin B2 or Ephrin B3.
  • the functionally active variant or biologically active portion has an amino acid sequence having at least about 80%, at least about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:23 or 24 and retains binding to Ephrin B2 or B3.
  • Exemplary biologically active portions include N- terminally truncated variants lacking all or a portion of the cytoplasmic domain, e.g.
  • Reference to retaining binding to Ephrin B2 or B3 includes binding that is at least or at least about 5% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 10% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 15% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 20% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 25% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 30% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 35% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO
  • the G protein or the biologically thereof is a mutant G protein that exhibits reduced binding for the native binding partner of a wild-type G protein.
  • the mutant G protein or the biologically active portion thereof is a mutant of wildtype Niv-G and exhibits reduced binding to one or both of the native binding partners Ephrin B2 or Ephrin B3.
  • the mutant G-protein or the biologically active portion, such as a mutant NiV-G protein exhibits reduced binding to the native binding partner.
  • the G protein contains one or more amino acid substitutions in a residue that is involved in the interaction with one or both of Ephrin B2 and Ephrin B3.
  • the amino acid substitutions correspond to mutations E501 A, W504A, Q530A and E533A with reference to numbering set forth in SEQ ID NO:4.
  • the G protein is a mutant G protein.
  • the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A and E533A with reference to numbering set forth in SEQ ID NO:4.
  • the G protein is a mutant G protein that contains one or more amino acid substitutions elected from the group consisting of E501A, W504A, Q530A and E533A with reference to SEQ ID NO:4 and is a biologically active portion thereof containing an N-terminal truncation.
  • the mutant NiV-G protein or the biologically active portion thereof is truncated and lacks up to 5 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 6 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:4), 7 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 8 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 9 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), up to 10 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 11 contiguous amino acid residues at or near the N-terminus of the wild-type NiV
  • the mutant NiV-G protein has the amino acid sequence set forth in SEQ ID NO: 17 or 18 or an amino acid sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 17 or 18.
  • the G protein has the sequence of amino acids set forth in SEQ ID NO: 17 or 18.
  • the mutant NiV-G protein has the amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 17.
  • the G protein has the sequence of amino acids set forth in SEQ ID NO 17.
  • the mutant NiV-G protein has the amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 18.
  • the G protein has the sequence of amino acids set forth in SEQ ID NO 18.
  • the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A and E533A with reference to numbering set forth in SEQ ID NO:4.
  • the G protein is a mutant G protein that contains one or more amino acid substitutions elected from the group consisting of E501 A, W504A, Q530A and E533A with reference to SEQ ID NO:4 and is a biologically active portion thereof containing an N-terminal truncation.
  • mutant Paramyxovirus G/H glycoproteins comprising one or more amino acid mutations that result in decreased glycosylation of the protein.
  • the one or more amino acid mutations also called deglycosylation mutations, can be one or more amino acid substitutions (also referred to as mutations).
  • the mutant Paramyxovirus G/H glycoprotein comprises an amino acid substitution at one or more amino acid positions that reduce glycosylation of the G/H glycoprotein.
  • the one or more amino acid substitutions disrupts an N-linked glycosylation site.
  • the one or more amino acid substitutions disrupts an O-linked glycosylation site.
  • the mutant Paramyxovirus G/H glycoprotein is derived from Morbillivirus (e.g., measles virus (MeV), canine distemper virus, Cetacean morbilli virus, Peste-des-driven-ruminants virus, Phocine distemper virus, Rinderpest virus), Henipavirus (e.g., Hendra (HeV) virus, Nipah (NiV) virus, a Cedar (CedPV) virus, Mojiang virus, a Langya virus or bat Paramyxovirus).
  • Morbillivirus e.g., measles virus (MeV), canine distemper virus, Cetacean morbilli virus, Peste-des-driven-ruminants virus, Phocine distemper virus, Rinderpest virus
  • Henipavirus e.g., Hendra (HeV) virus, Nipah (NiV) virus, a Cedar (CedPV) virus, Mojiang virus
  • the mutant Paramyxovirus G/H glycoprotein is a mutant of a Paramyxovirus G/H glycoprotein derived from Nipah virus or Measles virus.
  • the mutant Paramyxovirus G/H protein is selected from the group consisting of SEQ ID NOs: 2-11, 375, and 376, or a modified Paramyxovirus G/H glycoprotein derived from any one of 2-11, 375, and 376 containing an altered cytoplasmic tail .
  • the mutant Paramyxovirus G/H protein has a sequence of amino acids that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% to any one of SEQ ID NOs: 2-11, 375, and 376 and contains the acid substitution at one or more amino acid positions that reduce glycosylation of the G/H glycoprotein as provided herein.
  • N-glycosylation sites can be determined using the sequence of a protein. For example, N-glycosylation often occurs at sites with the sequence N-X-S/T in which “X” is any amino acid except P.
  • Various algorithms and tools are available for prediction of both N- and O- linked glycosylation, including SprintGly (http://sparks-lab.org/server/sprint-gly/), NetNGlyc (https://services.healtlttech.dtu.dk/service.php7NetNGlyc-l.0), NetOGlyc (https://services.healtlttech.dtu.dk/service.php?NetOGlyc-4.0), and GlycoMine struct
  • the Paramyxovirus G/H glycoprotein to which the deglycosylation mutation is made is a NiV-G set forth in SEQ ID NO: 4 or a modified Nipah G glycoprotein (NiV-G) that has an altered cytoplasmic tail compared to native NiV-G (e.g., SEQ ID NO: 4).
  • the variant Paramyxovirus G/H protein has a sequence of amino acids that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% to SEQ ID NO: 4 and contains the acid substitution at one or more amino acid positions that reduce glycosylation of the G/H glycoprotein as provided herein
  • Exemplary modified NiV- G proteins with altered cytoplasmic tails to which the one or more amino acid substitutions for reducing glycosylation can be incorporated are described in Section II.B.
  • amino acid positions for substitutions are described herein with positions “corresponding to” positions of a reference sequence. It is understood that the amino acid substitutions are not limited to being made in only the reference sequence but also can be made in similar sequences by identification of residues that align or correspond with the reference positions. For instance, positions “corresponding to” to positions of a protein in a reference sequence can be identified upon alignment of a similar sequence with the referenced sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.
  • amino acid positions for mutations are described herein with reference to the exemplary truncated NiV-G sequence set forth in SEQ ID NO:42; however, similar amino acid positions for mutations as described can be made in other modified NiV-G sequences, such as any as described in Section II.B, by sequence alignment and identification of the corresponding residues.
  • the one or more amino acid mutations are at positions corresponding to positions 39, 126, 128, 273, 345, 384, 448, and 496 of SEQ ID NO:42.
  • the variant Paramyxovirus G/H glycoprotein comprises an amino acid mutation at any one of positions 39, 126, 128, 273, 345, 384, 448, and 496 of SEQ ID NO:42.
  • the variant Paramyxovirus G/H glycoprotein comprises two or more amino acid mutations at any of positions corresponding to positions 39, 126, 128, 273, 345, 384, 448, and 496 of SEQ ID NO:42., such as mutations at 2, 3, 4, 5, 7, or 8 of the positions.
  • the one or more amino acid mutations is at a position corresponding to position 39 of SEQ ID NO:42. In some embodiments, the one or more amino acid mutations is at a position corresponding to position 126 of SEQ ID NO:42. In some embodiments, the one or more amino acid mutations is at a position corresponding to position 128 of SEQ ID NO:42. In some embodiments, the one or more amino acid mutations is at a position corresponding to position 273 of SEQ ID NO: 42. In some embodiments, the one or more amino acid mutations is at a position corresponding to position 345 of SEQ ID NO:42.
  • the one or more amino acid mutations is at a position corresponding to position 384 of SEQ ID NO:42. In some embodiments, the one or more amino acid mutations is at a position corresponding to position 448 of SEQ ID NO:42. In some embodiments, the one or more amino acid mutations is at a position corresponding to position 496 of SEQ ID NO:42.
  • the native amino acid at the position comprising the amino acid mutation is asparagine or serine.
  • the amino acid mutation is an amino acid substitution.
  • the mutation is an asparagine to glutamine substitution.
  • the mutation is a serine to alanine substitution.
  • the mutation is an asparagine to glutamine substitution at a position corresponding to position 39 (N39Q) of SEQ ID NO:42. In some embodiments, the mutation is an asparagine to glutamine substitution at a position corresponding to position 126 (N126Q) of SEQ ID NO: 42. In some embodiments, the mutation is an asparagine to glutamine substitution at a position corresponding to position 273 (N273Q) of SEQ ID NO: 42. In some embodiments, the mutation is an asparagine to glutamine substitution at a position corresponding to position 345 (N345Q) of SEQ ID NO: 42.
  • the mutation is an asparagine to glutamine substitution at a position corresponding to position 384 (N384Q) of SEQ ID NO: 42. In some embodiments, the mutation is an asparagine to glutamine substitution at a position corresponding to position 448 (N448Q) of SEQ ID NO: 42. In some embodiments, the mutation is an asparagine to glutamine substitution at a position corresponding to position 496 (N496Q) of SEQ ID NO: 42.
  • the mutation is a serine to alanine substitution at a position corresponding to position 128 (S128A) of SEQ ID NO: 42.
  • the G/H glycoprotein is derived from Nipah virus G protein and the one or more amino acid substitutions are at positions corresponding to positions selected from the group consisting of 39, 126, 128, 273, 345, 384, 448, and 496 of SEQ ID NO: 42.
  • the one or more amino acid substitutions are selected from N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q or any combination thereof.
  • the G/H glycoprotein is a mutant NiV-G containing one amino acid substitution from any one of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G/H glycoprotein is a mutant NiV-G containing two amino acid substitutions from any two of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q.
  • the G/H glycoprotein is a mutant NiV-G containing three amino acid substitutions from any three of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G/H glycoprotein is a mutant NiV-G containing four amino acid substitutions from any one of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q.
  • the G/H glycoprotein is a mutant NiV-G containing five amino acid substitutions from any one of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G/H glycoprotein is a mutant NiV-G containing six amino acid substitutions from any one of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q.
  • the G/H glycoprotein is a mutant NiV-G containing seven amino acid substitutions from any one of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G/H glycoprotein is a mutant NiV-G containing eight amino acid substitutions from any one of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q.
  • the one or more amino acid substitutions are in the SEQ ID NO: 147 or a or a modified Nipah G glycoprotein (NiV-G) that has an altered cytoplasmic tail compared to native NiV-G (e.g., SEQ ID NO:42).
  • the amino acid substitutions are in a modified NiV-G protein described in Section II.B.
  • the amino acid substitutions are in the NiV-G set forth in SEQ ID NO:42.
  • the variant Nipah-G protein comprises at least three amino acid substitutions.
  • the amino acid substitutions are at positions 273, 384, and 496 of SEQ ID NO:42.
  • the amino acid substitutions are at positions 273, 345, and 496 of SEQ ID NO:42.
  • the amino acid substitutions are at positions 39, 126, and 128 of SEQ ID NO:42.
  • the amino acid substitutions are at positions 39, 273, and 345 of SEQ ID NO:42.
  • the amino acid substitutions are at positions 39, 384, and 448 of SEQ ID NO:42.
  • the amino acid substitutions are at positions 39, 448, and 496 of SEQ ID NO:42. In some embodiments, the amino acid substitutions are at positions 39, 128, and 273 of SEQ ID NO:42. In some embodiments, the amino acid substitutions are at positions 39, 345, and 384 of SEQ ID NO:42. In some embodiments, the amino acid substitutions are at positions 39, 384, and 448 of SEQ ID NO:42.
  • the variant Nipah-G protein comprises at least two amino acid substitutions.
  • the amino acid substitutions are at positions 273, and 496 of SEQ ID NO:42.
  • the amino acid substitutions are at positions 345, and 496 of SEQ ID NO:42.
  • the amino acid substitutions are at positions 39 and 128 of SEQ ID NO:
  • amino acid substitutions are at positions 39, and 345 of SEQ ID NO:42.
  • amino acid substitutions are at positions 39, and 448 of SEQ ID NO:42.
  • amino acid substitutions are at positions 39 and 496 of SEQ ID NO:42.
  • amino acid substitutions are at positions 39 and 273 of SEQ ID NO:42.
  • amino acid substitutions are at positions 39 and 384 of SEQ ID NO:42.
  • amino acid substitutions are at positions 384 and 448 of SEQ ID NO:42.
  • the amino acid substitution is at position 39 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 126 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 128 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 273 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 345 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 384 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 448 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 496 of SEQ ID NO:42.
  • the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 39 of SEQ ID NO:42. In some embodiments, the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 126 of SEQ ID NO:42. In some embodiments, the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 273 of SEQ ID NO:42. In some embodiments, the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 345 of SEQ ID NO:42. In some embodiments, the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 384 of SEQ ID NO:42.
  • the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 448 of SEQ ID NO:42. In some embodiments, the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 496 of SEQ ID NO:42. In some embodiments, the mutant Nipah-G protein comprises a serine to alanine substitution at position 128 of SEQ ID NO:42.
  • the mutant Nipah-G protein comprises the sequence selected from the group consisting of any one of SEQ ID NOs: 377-503, such as any exemplary mutant Nipah-G proteins set forth in Table 2 A below.
  • the mutant Nipah-G protein comprises the sequence of SEQ ID NO: 379.
  • the variant Nipah-G protein comprises the sequence of SEQ ID NO: 400.
  • the variant Nipah-G protein comprises the sequence of SEQ ID NO: 404.
  • the Paramyxovirus G/H glycoprotein to which the deglycosylation mutations is made is a Measles virus H (Mev-H) protein or a modified MeV-H protein that has an altered cytoplasmic tail compared to native MeV-H (e.g., SEQ ID NO:375).
  • Mev-H Measles virus H
  • SEQ ID NO:375 modified MeV-H protein that has an altered cytoplasmic tail compared to native MeV-H
  • the mutant Paramyxovirus G/H protein has a sequence of amino acids that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% to SEQ ID NO: 375 and contains the acid substitution at one or more amino acid positions that reduce glycosylation of the G/H glycoprotein as provided herein.
  • the G/H glycoprotein is derived from Measles virus H (Mev-H) protein and the one or more amino acid substitutions are at positions corresponding to positions selected from the group consisting of 168, 187, 200, 215, 238 of SEQ ID NO: 375.
  • the mutant Mev-H protein comprises at least two amino acid substitutions, such as 2, 3, 4, or 5 substitutions at positions 168, 187, 200, 215, 238 of SEQ ID NO: 375.
  • the Paramyxovirus G/H glycoprotein to which the deglycosylation mutations is made is a Canine distemper virus H (CDV-H) protein or a modified CDV-H protein that has an altered cytoplasmic tail compared to native CDV-H (e.g., SEQ ID NO:376).
  • CDV-H Canine distemper virus H
  • SEQ ID NO:376 a modified CDV-H protein that has an altered cytoplasmic tail compared to native CDV-H
  • the mutant Paramyxovirus G/H protein has a sequence of amino acids that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% to SEQ ID NO: 376 and contains the acid substitution at one or more amino acid positions that reduce glycosylation of the G/H glycoprotein as provided herein.
  • the G/H glycoprotein is derived from Canine distemper virus H (CDV-H) protein and the one or more amino acid substitutions are at positions corresponding to positions selected from the group consisting of 19, 149, 422 of SEQ ID NO: 376.
  • the variant CDV-H protein comprises at least two amino acid substitutions, such as 2 or 3 substitutions at positions 19, 149, 422 of SEQ ID NO: 376.
  • the fusogen contains a protein with a hydrophobic fusion peptide domain. In some embodiments, the fusogen is or contains a F protein. In some embodiments, the fusogen contains a henipavirus F protein molecule or biologically active portion thereof.
  • the Henipavirus F protein is a Hendra (Hev) virus F protein, a Nipah (NiV) virus F-protein, a Cedar (CedPV) virus F protein, a Mojiang virus F protein, a bat Paramyxovirus F protein, a Kumasi virus F protein, a Eangya virus F protein, or a biologically active portion thereof.
  • F proteins of henipaviruses are encoded as Fo precursors containing a signal peptide (e.g. corresponding to amino acid residues 1-26 of SEQ ID NO:28). Following cleavage of the signal peptide, the mature Fo (e.g. SEQ ID NO:29) is transported to the cell surface, then endocytosed and cleaved by cathepsin L into the mature fusogenic subunits Fl and F2.
  • a signal peptide e.g. corresponding to amino acid residues 1-26 of SEQ ID NO:28
  • the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 38.
  • the Fo comprises the amino acid sequence of SEQ ID NO:41.
  • the Fl subunit comprises the sequence amino acid sequence set forth in SEQ ID NO:46.
  • the F2 subunit comprises the sequence amino acid sequence set forth in SEQ ID NO:39.
  • the Fl and F2 subunits are associated by a disulfide bond and recycled back to the cell surface.
  • the Fl subunit contains the fusion peptide domain located at the N terminus of the Fl subunit, where it is able to insert into a cell membrane to drive fusion. In some aspects, fusion is blocked by association of the F protein with G protein, until the G protein engages with a target molecule resulting in its disassociation from F and exposure of the fusion peptide to mediate membrane fusion.
  • the sequence and activity of the F protein is highly conserved.
  • the F protein of NiV and HeV viruses share 89% amino acid sequence identity.
  • the henipavirus F proteins exhibit compatibility with G proteins from other species to trigger fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13):e00577-19).
  • the F protein is heterologous to the G protein, i.e. the F and G protein or biologically active portions are from different henipavirus species.
  • the F protein is from Hendra virus and the G protein is from Nipah virus.
  • the F protein can be a chimeric F protein containing regions of F proteins from different species of Henipavirus. In some embodiments, switching a region of amino acid residues of the F protein from one species of Henipavirus to another can result in fusion to the G protein of the species comprising the amino acid insertion. (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13):e00577-19). In some cases, the chimeric F protein contains an extracellular domain from one henipavirus species and a transmembrane and/or cytoplasmic domain from a different henipavirus species.
  • the F protein contains an extracellular domain of Hendra virus and a transmembrane/cytoplasmic domain of Nipah virus.
  • F protein sequences disclosed herein are predominantly disclosed as expressed sequences including an N-terminal signal sequence.
  • N-terminal signal sequences are commonly cleaved co- or post-translationally, the mature protein sequences for all F protein sequences disclosed herein are also contemplated as lacking the N-terminal signal sequence.
  • the F protein is encoded by a nucleotide sequence that encodes the sequence set forth by any one of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or is a functionally active variant or a biologically active portion thereof that has a sequence that is at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% identical to any one of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO
  • the F protein is encoded by a nucleotide sequence that encodes the sequence set forth by any one of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • the F protein or the functionally active variant or biologically active portion thereof retains fusogenic activity in conjunction with a Henipavirus G protein, such as a G protein set forth above (e.g. NiV-G or HeV-G).
  • Fusogenic activity includes the activity of the F protein in conjunction with a G protein to promote or facilitate fusion of two membrane lumens, such as the lumen of the targeted lipid particle having embedded in its lipid bilayer a henipavirus F and G protein, and a cytoplasm of a target cell, e.g. a cell that contains a surface receptor or molecule that is recognized or bound by the targeted envelope protein.
  • the F protein and G protein are from the same Henipavirus species (e.g. NiV-G and NiV-F). In some embodiments, the F protein and G protein are from different Henipavirus species (e.g. NiV-G and HeV-F). In particular embodiments, the F protein of the functionally active variant or biologically active portion retains the cleavage site cleaved by cathepsin L (e.g. corresponding to the cleavage site between amino acids 109-110 of SEQ ID NO: 30).
  • the F protein has the sequence of amino acids set forth in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or is a functionally active variant thereof or a biologically active portion thereof that retains fusogenic activity.
  • the functionally active variant comprises an amino acid sequence having at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, and retains fusogenic activity in conjunction with a Henipavirus G protein (e.g., NiV-G or HeV-G).
  • a Henipavirus G protein e.g., NiV-G or HeV-G
  • the biologically active portion has an amino acid sequence having at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • Reference to retaining fusogenic activity includes activity (in conjunction with a Henipavirus G protein) that between at or about 10% and at or about 150% or more of the level or degree of binding of the corresponding wild-type F protein, such as set forth in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, such as at least or at least about 10% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 15% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 20% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 25% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as
  • the F protein is a mutant F protein that is a functionally active fragment or a biologically active portion containing one or more amino acid mutations, such as one or more amino acid insertions, deletions, substitutions or truncations.
  • the mutations described herein relate to amino acid insertions, deletions, substitutions or truncations of amino acids compared to a reference F protein sequence.
  • the reference F protein sequence is the wild- type sequence of an F protein or a biologically active portion thereof.
  • the mutant F protein or the biologically active portion thereof is a mutant of a wild-type Hendra (Hev) virus F protein, a Nipah (NiV) virus F-protein, a Cedar (CedPV) virus F protein, a Mojiang virus F protein, a bat Paramyxovirus F protein, a Kumasi virus F protein, or a Langya virus F protein.
  • the wild-type F protein is encoded by a sequence of nucleotides that encodes any one of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • the mutant F protein is a biologically active portion of a wild- type F protein that is an N-terminally and/or C-terminally truncated fragment.
  • the mutant F protein or the biologically active portion of a wild-type F protein thereof comprises one or more amino acid substitutions.
  • the mutations described herein can improve transduction efficiency.
  • the mutations described herein can increase fusogenic capacity. Exemplary mutations include any as described, see e.g. Khetawat and Broder 2010 Virology Journal 7:312; Witting et al. 2013 Gene Therapy 20:997-1005; published international; patent application No. WO/2013/148327.
  • the mutant F protein is a biologically active portion that is truncated and lacks up to 20 contiguous amino acid residues at or near the C-terminus of the wild-type F protein, such as a wild-type F protein encoded by a sequence of nucleotides encoding the F protein set forth in any one of SEQ ID NOS: 28-37.
  • the mutant F protein is truncated and lacks up to 20 contiguous amino acids, such as up to 19, 18 , 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 contiguous amino acids at the C-terminus of the wild-type F protein.
  • the mutant F protein comprises the sequence set forth in SEQ ID NO: 15.
  • the mutant F protein comprises the sequence set forth in SEQ ID NO:20. In some embodiments, the mutant F protein is truncated and lacks up to 19 contiguous amino acids, such as up to 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 contiguous amino acids at the C-terminus of the wild-type F protein.
  • the F protein or the functionally active variant or biologically active portion thereof comprises an Fl subunit or a fusogenic portion thereof.
  • the Fl subunit is a proteolytically cleaved portion of the Fo precursor.
  • the Fo precursor is inactive.
  • the cleavage of the Fo precursor forms a disulfide-linked F1+F2 heterodimer.
  • the cleavage exposes the fusion peptide and produces a mature F protein.
  • the cleavage occurs at or around a single basic residue.
  • the cleavage occurs at Arginine 109 of NiV-F protein.
  • cleavage occurs at Lysine 109 of the Hendr a virus F protein.
  • the F protein is a wild-type Nipah virus F (NiV-F) protein or is a functionally active variant or biologically active portion thereof.
  • the Fo precursor is encoded by a sequence of nucleotides encoding the sequence set forth in SEQ ID NO:20.
  • the encoding nucleic acid can encode a signal peptide sequence that has the sequence MVVILDKRCY CNLLILILMI SECSVG (SEQ ID NO:38).
  • the F protein is cleaved into an Fl subunit comprising the sequence set forth in SEQ ID NO:46 and an F2 subunit comprising the sequence set forth in SEQ ID NO: 39.
  • the F protein is a NiV-F protein that is encoded by a sequence of nucleotides encoding the sequence set forth in SEQ ID NO:30, or is a functionally active variant or biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at or about 86%, at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:30.
  • the F protein is a NiV-F protein that is encoded by a sequence of nucleotides encoding the sequence set forth in SEQ ID NO:30.
  • the NiV-F-protein has the sequence of set forth in 30, or is a functionally active variant or a biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at or about 86%, at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to 30.
  • the NiV-F-protein has the sequence of
  • the F protein or the functionally active variant or the biologically active portion thereof includes an Fl subunit that has the sequence set forth in SEQ ID NO:46, or an amino acid sequence having, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:46.
  • the F protein or the functionally active variant or biologically active portion thereof includes an F2 subunit that has the sequence set forth in SEQ ID NO:39, or an amino acid sequence having, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:39.
  • the F protein or the functionally active variant or the biologically active portion thereof includes an Fl subunit that has the sequence set forth in SEQ ID NO:46, or an amino acid sequence having, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at or about 86%, at least at or about 87%, at least at or about 88%, or at least at or about 89% at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 46.
  • the F protein or the functionally active variant or biologically active portion thereof includes an F2 subunit that has the sequence set forth in SEQ ID NO:39, or an amino acid sequence having, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at or about 86%, at least at or about 87%, at least at or about 88%, or at least at or about 89% at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:39.
  • the F protein is a mutant NiV-F protein that is a biologically active portion thereof that is truncated and lacks up to 20 contiguous amino acid residues at or near the C- terminus of the wild-type NiV-F protein (e.g. set forth SEQ ID NO:40).
  • the mutant NiV-F protein comprises an amino acid sequence set forth in SEQ ID NO:20.
  • the mutant NiV-F protein has a sequence that has at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:20.
  • the mutant F protein contains an Fl protein that has the sequence set forth in SEQ ID NO:46.
  • the mutant F protein has a sequence that has at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:46.
  • the F protein is a mutant NiV-F protein that is a biologically active portion thereof that comprises a 20 amino acid truncation at or near the C-terminus of the wild-type NiV- F protein (SEQ ID NO:40); and a point mutation on an N-linked glycosylation site.
  • the mutant NiV-F protein comprises an amino acid sequence set forth in SEQ ID NO: 15.
  • the mutant NiV-F protein has a sequence that has at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 15.
  • the F protein is a mutant NiV-F protein that is a biologically active portion thereof that comprises a 25 amino acid truncation at or near the C-terminus of the wild-type NiV- F protein (SEQ ID NO:40). In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof that comprises a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO:40). In some embodiments, the NiV-F protein is encoded by a nucleotide sequence that encodes the sequence set forth in SEQ ID NO:20.
  • the NiV-F proteins is encoded by a nucleotide sequence that encodes sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:20.
  • the F protein is a mutant NiV-F protein that is a biologically active portion thereof that comprises a 22 amino acid truncation at or near the C-terminus of the wild-type NiV- F protein (SEQ ID NO:40).
  • the NiV-F protein comprises the amino acid sequence set forth in SEQ ID NO:21, or an amino acid sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:21.
  • the NiV-F protein is encoded by a nucleotide sequence that encodes the sequence set forth in SEQ ID NO:21.
  • the NiV-F proteins is encoded by a nucleotide sequence that encodes sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:21.
  • a VLP includes a fusogen that contains a G or H protein and an F protein (e.g., a G or H protein and F protein from a Paramyxovirus (e.g., Morbillivirus or Henipavirus) G or H protein or a biologically active portion thereof), the VLP also comprises a gag protein (e.g. an MLV-gag protein).
  • the MLV-gag protein is part of a fusion protein, where the the fusion protein is a cleavable fusion protein between (i) a viral structural protein (e.g. GAG (e.g.
  • production of such a VLP includes varying the ratio of polynucleotide encoding the G or H protein and an F protein and the polynucleotide encoding the gag protein.
  • the ratio of polynucleotide encoding the G or H protein and an F protein to the polynucleotide encoding the gag protein includes a 1:1, 1:2, 1:3, 1:4, or 1:5 ratio.
  • the particle includes a targeting agent that binds to a target molecule.
  • the target molecule is expressed on a target cell.
  • the targeting agent targets the particle to the target cell.
  • the particle preferentially targets a target cell compared to a non-target cell.
  • the particle preferentially fuses with a target cell compared to a non-target cell.
  • the fusogen is fused to the targeting agent of the particle. In some aspects, the fusogen is retargeted by the targeting agent to display altered tropism.
  • the targeting agent is linked to an exposed domain of the fusogen.
  • the exposed domain is the N-terminus.
  • the exposed domain is the C-terminus.
  • the targeting agent is fused to the fusogen directly.
  • the targeting agent is fused to the fusogen via a linker.
  • the linker is a peptide linker.
  • the targeting agent and fusogen are fused together, directly or indirectly, such as via the linker, in a fusion protein.
  • the targeting agent is fused to a Paramyxovirus attachment protein comprised by the fusogen. In some embodiments, the targeting agent is fused to the C-terminus of the Paramyxovirus attachment protein. In some embodiments, the targeting agent is fused to the Paramyxovirus attachment protein directly. In some embodiments, the targeting agent is fused to the Paramyxovirus attachment protein via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the targeting agent and Paramyxovirus attachment protein are fused together, directly or indirectly, such as via the linker, in a fusion protein.
  • the Paramyxovirus attachment protein is a Paramyxovirus G or H protein. In some embodiments, the Paramyxovirus attachment protein is a Paramyxovirus G protein. In some embodiments, the Paramyxovirus attachment protein is a Paramyxovirus H protein. In some embodiments, the Paramyxovirus attachment protein is any described in Section II-B-2-b. In some embodiments, the Paramyxovirus attachment protein is a mutated variant having reduced binding to its native receptor, such as any described in Section II-B-2-b.
  • the targeting agent is fused to a separate transmembrane domain incorporated into the lipid bilayer, not the fusogen.
  • the transmembrane domain may be any naturally- occurring or non-naturally occurring transmembrane domain.
  • the transmembrane domain may be a transmembrane domain of a receptor, a transmembrane protein, preferably a viral transmembrane protein, a fragment of a transmembrane protein, a transmembrane peptide or a variant thereof, such as a genetically modified transmembrane domain of a receptor, a genetically modified transmembrane protein, a genetically modified fragment of a transmembrane protein, or a genetically modified transmembrane peptide.
  • transmembrane domains are the transmembrane domain (TMD) of the platelet-derived growth factor receptor (PDGFR), the transmembrane domain of CD34, or the VSVG glycoprotein transmembrane domain.
  • the C-terminus of the transmembrane domain is fused, directly or indirectly (for example via a linker, such as a peptide linker), to the N-terminus of the targeting agent.
  • the linker is a flexible peptide linker.
  • the linker is 1-20 amino acids, such as 1-20 amino acids predominantly composed of glycine.
  • the linker is 1-20 amino acids, such as 1-20 amino acids predominantly composed of glycine and serine.
  • the linker is a flexible peptide linker containing amino acids Glycine and Serine, referred to as GS -linkers.
  • the peptide linker includes the sequences GS, GGS, GGGGS (SEQ ID NO:263), GGGGGS (SEQ ID NO:264), or combinations thereof.
  • the polypeptide linker has the sequence (GGS)n (SEQ IDNO:268), wherein n is 1 to 10. In some embodiments, the polypeptide linker has the sequence (GGGGS)n, (SEQ ID NO:265) wherein n is 1 to 10. In some embodiments, the polypeptide linker has the sequence (GGGGGS)n (SEQ ID NO:266), wherein n is 1 to 6. In some embodiments, the peptide linker is (GmS)n (SEQ ID NO:267), wherein each of m and n is an integer between 1 to 4, inclusive.
  • a “target cell” refers to a cell of a type that is specifically targeted by the particle.
  • the target molecule is expressed on the target cell.
  • the target cell is a hematopoietic cell.
  • the target cell is CD34+.
  • the target cell is a CD34+ progenitor cell.
  • a “non-target cell” refers to a cell of a type to which targeting of the particle is not desired.
  • the target molecule is expressed at higher levels on target cells than on non-target cells.
  • the target molecule is not expressed on the non-target cell.
  • a non-target cell is a non-hematopoietic cell.
  • the non-target cell is CD34-.
  • the non-target cell is not a CD34+ progenitor cell.
  • the target cell is a hematopoietic lineage cell.
  • hematopoietic cell includes blood cells, both from the myeloid and the lymphoid lineage.
  • hematopoietic cell includes both undifferentiated or poorly differentiated cells, such as hematopoietic stem cells and progenitor cells, and differentiated cells such as T lymphocytes, B lymphocytes, or dendritic cells.
  • the hematopoietic cells are hematopoietic stem cells (HSCs), CD34+ progenitor cells, in particular peripheral blood CD34+ cells, very early progenitor CD34+ cells, B-cell CD19+ progenitors, myeloid progenitor CD13+ cells, T lymphocytes, B lymphocytes, monocytes, dendritic cells, cancer B cells in particular B-cell chronic lymphocytic leukemia (BCLL) cells and marginal zone lymphoma (MZL) B cells, or thymocytes.
  • HSCs hematopoietic stem cells
  • CD34+ progenitor cells in particular peripheral blood CD34+ cells, very early progenitor CD34+ cells, B-cell CD19+ progenitors, myeloid progenitor CD13+ cells, T lymphocytes, B lymphocytes, monocytes, dendritic cells, cancer B cells in particular B-cell chronic lymphocytic leukemia (BCLL) cells and marginal zone lymph
  • hematopoietic cells are produced from bone marrow hematopoietic stem cells.
  • a hematopoietic cell is a hematopoietic stem cell (HSC), which are cells able to replenish all blood cell types and to self-renew.
  • HSC hematopoietic stem cell
  • Hematopoietic stem cells may be in particular defined as cells that keep the levels of myeloid, T cells, and B cells at robustly detectable levels (typically more than 1 % of peripheral blood cells) for 16 weeks when injected into the circulation of a recipient mouse with a depleted hematopoietic system (Schroeder (2010) Cell Stem Cell 6:203-207).
  • the hematopoietic cell is a "CD34+ progenitor cell,” which is a heterogeneous cell population that include a subpopulation of HSCs, pluripotent stem cells and cells in the early stages of lineage commitment.
  • CD34+ progenitor cells continuously migrate to and from the bone marrow in normal adult animals. They can differentiate to produce all hematopoietic cell lineages found in the circulation.
  • the hematopoietic cell is a very early progenitor CD34+ cell which is a subgroup of CD34+ progenitor cells enriched from HSCs.
  • the hematopoietic cell is a "peripheral blood CD34+ cell”, which is a CD34+ cell present in the blood.
  • the hematopoietic cell is a B cell CD 19+ progenitor, which is a population of B-lineage cells that express cell surface CD10, CD34, and CD19.
  • the hematopoietic cell is a myeloid progenitor CD 13+ cells, which is a population of myeloid lineage cells that express cell surface CD34 and CD13, and in some cases, also CD33.
  • the target cell is selected from the group consisting of myeloid- lymphoid balanced hematopoietic lineage cells, myeloid-biased hematopoietic lineage cells, lymphoid- biased hematopoietic lineage cells, a platelet-biased hematopoietic lineage cells, a platelet-myeloid- biased hematopoietic lineage cells, a long-term repopulating hematopoietic lineage cells, an intermediateterm repopulating hematopoietic lineage cells, or a short-term repopulating hematopoietic lineage cells.
  • the target cell is selected from monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes and platelets. In some embodiments, the target cell is selected from T cells, B cells, natural killer (NK) cells and innate lymphoid cells.
  • the target cell is an effector cell, e.g., a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions.
  • a target cell may include one or more of a monocyte, macrophage, neutrophil, dendritic cell, eosinophil, mast cell, platelet, large granular lymphocyte, Langerhans' cell, natural killer (NK) cell, T lymphocyte (e.g., T cell), a Gamma delta T cell, B lymphocyte (e.g., B cell) and may be from any organism including humans, mice, rats, rabbits, and monkeys.
  • the hematopoietic cell is a T cell.
  • the T cell is a naive T cell.
  • the T cell is a memory T cell.
  • the hematopoietic cell is a B cell.
  • the target cell is a resting B cell, such as a naive or a memory B cell.
  • the target cell is a cancer B cell, such as a B-cell chronic lymphocytic leukemia (BCLL) cell or a marginal zone lymphoma (MZL) B cell.
  • BCLL B-cell chronic lymphocytic leukemia
  • MZL marginal zone lymphoma
  • the target cell is a thymocyte.
  • the target cell is a natural killer (NK) cell.
  • the thymocyte expresses CD4 or CD8.
  • the thymocyte does not express CD4 or CD8.
  • the natural killer (NK) cell is a cell that expresses CD56.
  • the target molecule is expressed on CD34+ progenitor cells.
  • the target cell is a CD34+ progenitor cell.
  • the target molecule is expressed on all CD34+ progenitor cells.
  • the target molecule is expressed on at least a subset of CD34+ progenitor cells.
  • the target molecule is expressed on HSCs. In some embodiments, the target molecule is expressed on MPPs. In some embodiments, the target molecule is expressed on MLPs. In some embodiments, the target molecule is expressed on ETPs. In some embodiments, the target molecule is expressed on MEPs. In some embodiments, the target molecule is expressed on CMPs. In some embodiments, the target molecule is expressed on GMPs. In some embodiments, the target molecule is expressed on any combination of the foregoing CD34+ progenitor subpopulations. In some embodiments, the target molecule is expressed on HSCs and MPPs. In some embodiments, the target molecule is expressed on myeloid progenitors.
  • the target molecule is expressed on lymphoid progenitors. In some embodiments, the cell surface molecule is expressed on myeloid progenitors. In some embodiments, the cell surface molecule is expressed on HSCs, MPPs, MEPs, CMPs, and GMPs.
  • the target cells are HSCs. In some embodiments, the target cells are MPPs. In some embodiments, the target cells are MLPs. In some embodiments, the target cells are ETPs. In some embodiments, the target cells are MEPs. In some embodiments, the target cells are CMPs. In some embodiments, the target cells are GMPs. In some embodiments, the target cells are any combination of the foregoing CD34+ progenitor subpopulations. In some embodiments, the target cells are HSCs and MPPs. In some embodiments, the target cells are myeloid progenitors. In some embodiments, the target cells are lymphoid progenitors. In some embodiments, the target cells are myeloid progenitors. In some embodiments, the target cells are any one or more, e.g., each of, HSCs, MPPs, MEPs, CMPs, and GMPs.
  • the target molecule is ASCT1.
  • the target molecule is ASCT2.
  • the target cell is ASCT2+.
  • the target cells are the CD34+ progenitor subpopulations indicated in FIG. 7 for ASCT2.
  • the target cells are lymphoid progenitors.
  • the target cells are MLPs or ETPs. In some embodiments, the target cells are MLPs and ETPs.
  • the target molecule is CD 105. In some embodiments, the target cell is CD105+.
  • the target molecule is CD110. In some embodiments, the target cell is CD110+.
  • the target molecule is CD 117.
  • the target cell is CD117+.
  • the target cells are the CD34+ progenitor subpopulations indicated in FIG. 7 for CD117.
  • the target cells are myeloid progenitors.
  • the target cells are MPPs, MEPs, CMPs, or GMPs.
  • the target cells are MPPs, MEPs, CMPs, and GMPs.
  • the target molecule is CD 133. In some embodiments, the target cell is CD133+.
  • the target molecule is CD 146. In some embodiments, the target cell is CD146+.
  • the target molecule is CD 164. In some embodiments, the target cell is CD164+.
  • the target molecule is CD34. In some embodiments, the target cell is CD34+.
  • the target molecule is CD46. In some embodiments, the target cell is CD46+.
  • the target molecule is CD49f. In some embodiments, the target cell is CD49f+. [0498] In some embodiments, the target molecule is CD90. In some embodiments, the target cell is CD90+.
  • the target molecule is EPCR. In some embodiments, the target cell is EPCR+.
  • the target molecule is ITGA3. In some embodiments, the target cell is ITGA3+.
  • the targeting agent can include any targeting protein able to confer specific binding to the target molecule expressed on the target cell.
  • a targeting protein can also include an antibody or an antigen-binding fragment thereof (e.g., Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VE or VH), nanobodies, or camelid VHH domains), an antigen-binding fibronectin type III (Fn3) scaffold such as a fibronectin polypeptide minibody, a ligand, a cytokine, a chemokine, or a T cell receptor (TCRs).
  • the targeting agent is an antibody or antigen binding fragment thereof.
  • the targeting agent contains a humanized antibody molecule, intact IgA, IgG, IgE or IgM antibody; bi- or multi- specific antibody (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies®; minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®;
  • the targeting agent is a single chain molecule. In some embodiments, the targeting agent is a single domain antibody. In some embodiments, the targeting agent is a single chain variable fragment. In particular embodiments, the targeting agent contains an antibody variable sequence (s) that is human or humanized.
  • the single domain antibody is naturally occurring. In some embodiments, the single domain antibody is synthetic.
  • the single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide.
  • the single domain antibody is a heavy chain only antibody variable domain. In some embodiments, the single domain antibody does not include light chains.
  • the heavy chain antibody devoid of light chains is referred to as a VHH.
  • the single domain antibodies have a molecular weight of 12-15 kDa.
  • the single domain antibody antibodies include camelid antibodies or shark antibodies.
  • the single domain antibody is derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca, vicuna and guanaco.
  • the single domain antibody is referred to as immunoglobulin new antigen receptors (IgNARs) and is derived from cartilaginous fishes.
  • the single domain antibody is generated by splitting dimeric variable domains of human or mouse IgG into monomers and camelizing critical residues.
  • the single domain antibody can be generated from phage display libraries.
  • the phage display libraries are generated from a VHH repertoire of camelids immunized with various antigens, as described in Arbabi et al., FEBS Letters, 414, 521-526 (1997); Lauwereys et al., EMBO J., 17, 3512-3520 (1998); Decanniere et al., Structure, 7, 361-370 (1999).
  • the phage display library is generated comprising antibody fragments of a non-immunized camelid.
  • single domain antibodies a library of human single domain antibodies is synthetically generated by introducing diversity into one or more scaffolds.
  • the targeting agent can be engineered to bind the Fc region of an antibody that targets an antigen on a target cell, redirecting fusion activity towards cells that display the antibody’s target (DOI: 10.1128/JVI.75.17.8016-8020.2001, doi:10.1038/nml l92).
  • altered and non-altered fusogens may be displayed on the same retroviral particle or VLP (doi: 10.1016/j.biomaterials.2014.01.051).
  • a single-chain variable fragment can be used as the targeting agent to redirect fusion activity towards target cells that display the scFv binding target (doi:10.1038/nbtl060, DOI 10.1182/blood-2012-l 1-468579, doi:10.1038/nmeth,1514, doi:10.1006/mthe.2002.0550, HUMAN GENE THERAPY 11:817- 826, doi:10.1038/nbt942, doi:10.1371/journal.pone.0026381, DOI 10.1186/sl2896-015-0142-z).
  • DARPin designed ankyrin repeat proteins
  • a single domain antibody e.g., a VHH
  • VHH single domain antibody
  • receptor ligands and antigens can be used as the targeting agent to redirect fusion activity towards target cells that display the target receptor (DOI: 10.1089/hgtb.2012.054, DOI: 10.1128/JVI.76.7.3558-3563.2002).
  • the target molecule is CD 133.
  • the target cell is CD133+.
  • the targeting agent is an anti-CD133 antibody.
  • Exemplary anti-CD133 antibodies include CART133, AC133, 293C3-SDIE, CMab-43, RW03, 293C3H9 (293C3), and W6B3H10 (W6B3); and anti-CD133 antibodies disclosed in US Patent Nos. US8722858, US9249225, US9624303, US10106623, US10711068, US11098109, US11214628, US11352435, and US11220551; US Patent Application Nos. US20130224202; PCT Application Nos.
  • the targeting agent is a CD 133 binding agent that is an antibody or antigen-binding fragment.
  • the CD 133 targeting agent is a single chain antibody, such as a single chain variable fragment (scFv).
  • the CD133 binding agent contains (a) a variable heavy (VH) chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 271, 272, 273, respectively, and a variable light (VL) chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 280, 281, 282, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 284, 285, and 286, respectively; (c) a VH chain comprising a CDR-H1, a C
  • the CD 133 binding agent contains a VH and a VL, e.g. containined in an scFv, selected from (a) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 314, 315, and 273, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 316, 317, and 282, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 284, 285,
  • the CD 133 binding agent is a CD 133 binding agent comprising a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 270, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 274, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • VH heavy chain variable
  • VL light chain variable
  • the CD 133 binding agent is a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 279, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 283, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD 133 binding agent is a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 297, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 301, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD 133 binding agent is a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 306, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 310, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD133 binding agent is a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 288, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 292, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD 133 binding agent is a CD 133 binding agent comprising a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 270 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 274.
  • the CD 133 binding agent is a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 279 and a VL region comprising the amino acid sequence of SEQ ID NO: 283.
  • the CD133 binding agent is a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 297 and a VL region comprising the amino acid sequence of SEQ ID NO: 301. In some embodiments, the CD133 binding agent is a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 306 and a VL region comprising the amino acid sequence of SEQ ID NO: 310. In some embodiments, the CD133 binding agent is a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 288 and a VL region comprising the amino acid sequence of SEQ ID NO: 292.
  • the CD 133 binding agent is an scFv comprising the amino acid sequence set forth in SEQ ID NO: 269, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD133 binding agent is an scFv comprising the amino acid sequence set forth in SEQ ID NO: 278, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD 133 binding agent is an scFv comprising the amino acid sequence set forth in SEQ ID NO: 287, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD133 binding agent is an scFv comprising the amino acid sequence set forth in SEQ ID NO: 296, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD 133 binding agent is an scFv comprising the amino acid sequence set forth in SEQ ID NO: 305, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the target molecule is CD 105.
  • the target cell is CD105+.
  • the targeting agent is an anti-CD105 antibody.
  • Exemplary anti-CD105 antibodies include carotuximab, TRC105, huRH105, and TCR205; and anti-CD105 antibodies disclosed in US Patent Nos. US8221753, US8609094, US9150652, US95181212, US9926375, US9944714, US 10155820, and US 10336831; US Patent Application Nos. US20100098692, US20100196398, US20170007714, and US20220233591; PCT Application Nos.
  • the target molecule is CD 117.
  • the target cell is CD117+.
  • the targeting agent is an anti-CD 117 antibody.
  • the targeting agent is a CD117 binding agent that is an antibody or antigen-binding fragment.
  • the CD117 binding agent is a single domain antibody, such as a VHH.
  • the CD117 binding agent comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising a CDR-H1, a CDR- H2, and a CDR-H3 contained within an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374, wherein the target molecule is CD117.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 324, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 325, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 326, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 327, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 328, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 329, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 330, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 331, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 332, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 333, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 334, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 335, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 336, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 337, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 338, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 339, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 340, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 341, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 342, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 343, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 344, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 345, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 346, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 347, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 348, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 349, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 350, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 351, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 352, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 353, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 354, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 355, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 356, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 357, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 358, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 360, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 361, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 362, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 363, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 364, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 365, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 366, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 367, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 368, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 369, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 370, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 371, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 372, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 373, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 374, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 324. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 325. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 326. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 327. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 328. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 329.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 330. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 331. In some embodiments, the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 332. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 333. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 334. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 335.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 336. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 337. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 338. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 339. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 340. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 341.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 342. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 343. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 344. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 345. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 346. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 347.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 348. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 349. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 350. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 351. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 352. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 353.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 354. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 355. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 356. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 357. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 358. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 359.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 360. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 361. In some embodiments, the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 362. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 363. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 364. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 365.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 366. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 367. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 368. In some embodiments, the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 369. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 370. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 371.
  • the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 372. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 373. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 374.
  • the target molecule is EPCR. In some embodiments, the target cell is EPCR+. In some embodiments, the targeting agent is an anti-EPCR antibody. Exemplary anti-EPCR antibodies include JRK1494, JRK1535; and anti-EPCR antibodies disclosed in US Patent Application Nos. US20210355231 and US20220127374; and PCT Application Nos. W02020051277 and WO2020161478.
  • the target molecule is CD34.
  • the target cell is CD34+.
  • the targeting agent is an anti-CD34 antibody.
  • Exemplary anti-CD34 antibodies include h4C8, 9C5; and anti-CD34 antibodies disclosed in US Patent Nos. US8399249, US8927696, and US10106623; US Patent Application Nos. US20090221003, US20130143238, US20100311955, US20130172533, US20170320966, US20170298148, US20180169177, US20190135945; and PCT Application Nos. W02009079922 and WO2015121383.
  • the target molecule is ASCT1.
  • the target molecule is ASCT2.
  • the target cell is ASCT2+.
  • the targeting agent is an anti-ASCT2 antibody.
  • Exemplary anti-ASCT2 antibodies include idactamab, MEDI7247, KM4008, KM4012, KM4018; and anti-ASCT2 antibodies disclosed in US Patent Nos. US8268592, US8501180, US8945870, US8673592, and US10829554; US Patent Application Nos. US20180273617, US20190367605, US 20210024629; and PCT Application Nos. WO2017083451, WO2018089393.
  • the target molecule is CD90. In some embodiments, the target cell is CD90+. In some embodiments, the targeting agent is an anti-CD90 antibody.
  • Exemplary anti-CD90 antibodies include EPR3133, CL1028, CL1040, AF-9, JF10-09, 5E10, 7E1B11; and anti-CD90 antibodies disclosed in US Patent Application No. US20210054068; and PCT Application No.
  • the target molecule is CD 164.
  • the target cell is CD164+.
  • the targeting agent is an anti-CD164 antibody.
  • Exemplary anti-CD164 antibodies include 67D2, H-4, 32G1, EML2058, 5C5, N6B6, 4B4, and 15-11-14; and anti-CD164 antibodies disclosed in PCT Application No. W02006002438; and German Patent Nos. DE19727813C1 and DE19727815C1.
  • the target molecule is CD49f.
  • the target cell is CD49f+.
  • the targeting agent is an anti-CD49f antibody.
  • Exemplary anti-CD49f antibodies include CL6957, GoH3, SR45-00, and MP4F10; and anti-CD49f antibodies disclosed in US Patent Nos. US5538725, US10030071; US Patent Application Nos. US20110301227, US20160194400, US20160280789; and PCT Application Nos. WG2015034052 and WO2018127655.
  • the target molecule is CD 146.
  • the target cell is CD146+.
  • the targeting agent is an anti-CD146 antibody.
  • Exemplary anti-CD146 antibodies include imaprelimab, PRX003, ABX-MA1, huAA98, M2H-1, M2J-1, and JM1-24-3; and anti- CD146 antibodies disclosed in US Patent Nos. US6924360, US7067131, US709844, US9447190, US9782500, US10407506, US10414825, US10407507, US10584177, US10905771, US11427648; US Patent Application Nos.
  • targeting agent can be any described in the referenced associated documents that bind to the associated target molecule.
  • the particle contains an exogenous agent.
  • the exogenous agent is in the lumen of the particle.
  • the exogenous agent is a nucleic acid, such as any described in Section II-D-1.
  • the nucleic acid contains a payload gene encoding a payload agent.
  • the exogenous agent encodes a protein, such as any described in Section II-D-2.
  • the exogenous agent is a protein, such as any described in Section II-D-2.
  • the protein is a payload agent.
  • the exogenous agent is a small molecule, such as any described in Section II-D-3.
  • the pay load agent encoded or contained by the exogenous agent is any described in Section III.
  • the exogenous agent is or encodes an engineered receptor, such as any described in Section III-A.
  • the exogenous agent is or encodes a gene-editing agent, such as any described in Section III-B.
  • the exogenous agent is a protein or a nucleic acid (e.g., a DNA, a chromosome (e.g. a human artificial chromosome), or an RNA, e.g., an mRNA or miRNA).
  • the exogenous agent is a protein.
  • the exogenous agent is a nucleic acid (e.g., a DNA, a chromosome (e.g. a human artificial chromosome), or an RNA, e.g., an mRNA or miRNA).
  • the exogenous agent comprises or encodes a membrane protein.
  • the exogenous agent comprises or encodes a therapeutic agent.
  • the therapeutic agent is chosen from one or more of a protein, e.g., an enzyme, a transmembrane protein, a receptor, or an antibody; a nucleic acid, e.g., DNA, a chromosome (e.g. a human artificial chromosome), or an RNA, e.g., mRNA, siRNA, or miRNA; or a small molecule.
  • the particle delivers to a target cell at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the exogenous agent (e.g., an exogenous agent comprising or encoding a therapeutic agent) comprised by the particle.
  • the exogenous agent e.g., an exogenous agent comprising or encoding a therapeutic agent
  • the particles that contact, e.g., fuse, with the target cell or cells delivers to the target cell or cells an average of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the exogenous agent (e.g., an exogenous agent comprising or encoding a therapeutic agent) comprised by the particles that contact, e.g., fuse, with the target cell or cells.
  • the exogenous agent e.g., an exogenous agent comprising or encoding a therapeutic agent
  • a composition containing a plurality of the particle delivers to a target tissue at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the exogenous agent (e.g., an exogenous agent comprising or encoding a therapeutic agent) comprised by the composition.
  • the exogenous agent e.g., an exogenous agent comprising or encoding a therapeutic agent
  • the exogenous agent is not expressed naturally in the cell from which the particle is derived. In some embodiments, the exogenous agent is expressed naturally in the cell from which the particle is derived. In some embodiments, the exogenous agent is loaded into the particle via expression in the cell from which the particle is derived (e.g. expression from DNA or mRNA introduced via transfection, transduction, or electroporation). In some embodiments, the exogenous agent is expressed from DNA integrated into the genome or maintained episomally. In some embodiments, expression of the exogenous agent is constitutive. In some embodiments, expression of the exogenous agent is induced. In some embodiments, expression of the exogenous agent is induced immediately prior to generating the particle. In some embodiments, expression of the exogenous agent is induced at the same time as expression of the fusogen of the particle.
  • the exogenous agent is loaded into the particle via expression in the cell from which the particle is derived (e.g. expression from DNA or mRNA introduced via transfection, transduction, or electropor
  • the exogenous agent is loaded into the particle via electroporation into the particle itself or into the cell from which the particle is derived. In some embodiments, the exogenous agent is loaded into the particle via transfection (e.g., of a DNA or mRNA encoding the exogenous agent) into the particle itself or into the cell from which the particle is derived.
  • transfection e.g., of a DNA or mRNA encoding the exogenous agent
  • the exogenous agent includes one or more nucleic acid sequences, one or more polypeptides, a combination of nucleic acid sequences and/or polypeptides, one or more organelles, or any combination of the foregoing.
  • the exogenous agent includes one or more cellular components.
  • the exogenous agent includes one or more cytosolic and/or nuclear components.
  • the exogenous agent includes a mixture of proteins, nucleic acids, or metabolites, e.g., multiple polypeptides, multiple nucleic acids, multiple small molecules; combinations of nucleic acids, polypeptides, and small molecules; ribonucleoprotein complexes (e.g. Cas9-gRNA complex); multiple transcription factors, multiple epigenetic factors, reprogramming factors (e.g. Oct4, Sox2, cMyc, and Klf4); multiple regulatory RNAs; and any combination of the foregoing.
  • proteins, nucleic acids, or metabolites e.g., multiple polypeptides, multiple nucleic acids, multiple small molecules; combinations of nucleic acids, polypeptides, and small molecules; ribonucleoprotein complexes (e.g. Cas9-gRNA complex); multiple transcription factors, multiple epigenetic factors, reprogramming factors (e.g. Oct4, Sox2, cMyc, and Klf4); multiple regulatory RNAs; and any combination of
  • the exogenous agent includes a nucleic acid.
  • the exogenous agent may comprise RNA to enhance expression of an endogenous protein, or a siRNA or miRNA that inhibits protein expression of an endogenous protein.
  • the endogenous protein may modulate structure or function in the target cells.
  • the exogenous agent may include a nucleic acid encoding an engineered protein that modulates structure or function in the target cells.
  • the exogenous agent is a nucleic acid that targets a transcriptional activator that modulate structure or function in the target cells
  • the nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, the nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.
  • the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.
  • the nucleic acid is partly or wholly single stranded; in some embodiments, the nucleic acid is partly or wholly double stranded.
  • the nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.
  • the nucleic acid may include variants, e.g., having an overall sequence identity with a reference nucleic acid of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
  • a variant nucleic acid does not share at least one characteristic sequence element with a reference nucleic acid.
  • a variant nucleic acid shares one or more of the biological activities of the reference nucleic acid.
  • a nucleic acid variant has a nucleic acid sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. In some embodiments, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue as compared to a reference.
  • a variant nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues that participate in a particular biological activity relative to the reference.
  • a variant nucleic acid comprises not more than about 15, about 12, about 9, about 3, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference.
  • a variant nucleic acid comprises fewer than about 27, about 24, about 21, about 18, about 15, about 12, about 9, about 6, about 3, or fewer than about 9, about 6, about 3, or about 2 additions or deletions as compared to the reference.
  • the exogenous agent includes a nucleic acid, e.g., DNA, nDNA (nuclear DNA), mtDNA (mitochondrial DNA), protein coding DNA, gene, operon, chromosome, genome, transposon, retrotransposon, viral genome, intron, exon, modified DNA, mRNA (messenger RNA), tRNA (transfer RNA), modified RNA, microRNA, siRNA (small interfering RNA), tmRNA (transfer messenger RNA), rRNA (ribosomal RNA), mtRNA (mitochondrial RNA), snRNA (small nuclear RNA), small nucleolar RNA (snoRNA), SmY RNA (mRNA trans-splicing RNA), gRNA (guide RNA), TERC (telomerase RNA component), aRNA (antisense RNA), cis-NAT (Cis-natural antisense transcript), CRISPR RNA (crRNA), IncRNA (a nucleic acid, e
  • the nucleic acid encodes one or more (e.g. two or more) inhibitory RNA molecules directed against one or more RNA targets.
  • An inhibitory RNA molecule can be, e.g., a miRNA or an shRNA.
  • the inhibitory molecule can be a precursor of a miRNA, such as for example, a Pri-miRNA or a Pre-miRNA, or a precursor of an shRNA.
  • the inhibitory molecule can be an artificially derived miRNA or shRNA.
  • the inhibitory RNA molecule can be a dsRNA (either transcribed or artificially introduced) that is processed into an siRNA or the siRNA itself.
  • the inhibitory RNA molecule can be a miRNA or shRNA that has a sequence that is not found in nature, or has at least one functional segment that is not found in nature, or has a combination of functional segments that are not found in nature.
  • at least one or all of the inhibitory RNA molecules are miR-155.
  • a retroviral vector described herein encodes two or more inhibitory RNA molecules directed against one or more RNA targets. Two or more inhibitory RNA molecules, in some embodiments, can be directed against different targets. In other embodiments, the two or more inhibitory RNA molecules are directed against the same target.
  • the exogenous agent comprises a shRNA.
  • a shRNA can comprise a double-stranded structure that is formed by a single self complementary RNA strand.
  • shRNA constructs can comprise a nucleotide sequence identical to a portion, of either coding or non-coding sequence, of a target gene. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence can also be used. Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene can be used.
  • the length of the duplex-forming portion of an shRNA is at least 20, 2 1 or 22 nucleotides in length, e.g., corresponding in size to RNA products produced by Dicer-dependent cleavage.
  • the shRNA construct is at least 25, 50, 100, 200, 300 or 400 bases in length. In certain embodiments, the shRNA construct is 400-800 bases in length.
  • shRNA constructs are highly tolerant of variation in loop sequence and loop size.
  • a retroviral vector that encodes an siRNA, an miRNA, an shRNA, or a ribozyme comprises one or more regulatory sequences, such as, for example, a strong constitutive pol III, e.g., human U6 snRNA promoter, the mouse U6 snRNA promoter, the human and mouse H 1 RNA promoter and the human tRNA-val promoter, or a strong constitutive pol II promoter.
  • a strong constitutive pol III e.g., human U6 snRNA promoter, the mouse U6 snRNA promoter, the human and mouse H 1 RNA promoter and the human tRNA-val promoter, or a strong constitutive pol II promoter.
  • the nucleic acid is operatively linked to a “positive target cell-specific regulatory element” (or positive TCSRE).
  • the positive TCSRE is a functional nucleic acid sequence.
  • the positive TCSRE contains a promoter or enhancer.
  • the TCSRE is a nucleic acid sequence that increases the level of an exogenous agent in a target cell.
  • the nucleic acid is operatively linked to a “negative target cellspecific regulatory element” (or negative TCSRE).
  • the negative TCSRE is a functional nucleic acid sequence.
  • the negative TCSRE is a miRNA recognition site that causes degradation of inhibition of the particle in a non-target cell.
  • the nucleic acid is operatively linked to a “non-target cell-specific regulatory element” (or NTCSRE).
  • the NTCSRE comprises a nucleic acid sequence that decreases the level of an exogenous agent in a non-target cell compared to in a target cell.
  • the NTCSRE comprises a non-target cell-specific miRNA recognition sequence, non-target cell-specific protease recognition site, non-target cell-specific ubiquitin ligase site, non-target cell-specific transcriptional repression site, or non-target cell-specific epigenetic repression site.
  • the NTCSRE comprises a tissue-specific miRNA recognition sequence, tissue-specific protease recognition site, tissuespecific ubiquitin ligase site, tissue-specific transcriptional repression site, or tissue-specific epigenetic repression site.
  • the NTCSRE comprises a non-target cell-specific miRNA recognition sequence, non-target cell-specific protease recognition site, non-target cell-specific ubiquitin ligase site, non-target cell-specific transcriptional repression site, or non-target cell-specific epigenetic repression site.
  • the NTCSRE comprises a non-target cell-specific miRNA recognition sequence and the miRNA recognition sequence is able to be bound by one or more of miR3 1, miR363, or miR29c.
  • the NTCSRE is situated or encoded within a transcribed region encoding the exogenous agent, optionally wherein an RNA produced by the transcribed region comprises the miRNA recognition sequence within a UTR or coding region.
  • the exogenous agent is or encodes a protein.
  • the protein includes moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified.
  • the protein includes more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
  • the protein contains L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs. In some embodiments, the protein contains natural amino acids, non-natural amino acids, synthetic amino acids, or combinations of any of the foregoing. In some embodiments, the protein is selected from antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
  • the protein is a wild-type protein. In some embodiments, the protein is a mutant protein. In some embodiments, the protein includes a polypeptide or its variants, e.g., having an overall sequence identity with a reference polypeptide of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, a variant polypeptide shares one or more of the biological activities of the reference polypeptide.
  • a polypeptide variant has an amino acid sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. In some embodiments, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue as compared to a reference.
  • a variant polypeptide comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional that participate in a particular biological activity relative to the reference. In some embodiments, a variant polypeptide comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference.
  • the protein includes a polypeptide, e.g., enzymes, structural polypeptides, signaling polypeptides, regulatory polypeptides, transport polypeptides, sensory polypeptides, motor polypeptides, defense polypeptides, storage polypeptides, transcription factors, antibodies, cytokines, hormones, catabolic polypeptides, anabolic polypeptides, proteolytic polypeptides, metabolic polypeptides, kinases, transferases, hydrolases, lyases, isomerases, ligases, enzyme modulator polypeptides, protein binding polypeptides, lipid binding polypeptides, membrane fusion polypeptides, cell differentiation polypeptides, epigenetic polypeptides, cell death polypeptides, nuclear transport polypeptides, nucleic acid binding polypeptides, reprogramming polypeptides, DNA editing polypeptides, DNA repair polypeptides, DNA recombination polypeptides, transposase
  • a polypeptide
  • Zinc -finger nucleases transcription- activator-like nucleases (TALENs), cas9 and homologs thereof), recombinases, transposases, DNA polymerases, RNA polymerases, reverse transciptases, and any combination of the foregoing.
  • TALENs transcription- activator-like nucleases
  • cas9 and homologs thereof
  • recombinases recombinases
  • transposases DNA polymerases
  • RNA polymerases reverse transciptases, and any combination of the foregoing.
  • the protein targets a target protein in the cell for degradation. In some embodiments, the protein targets a target protein in the cell for degradation by localizing the protein to the proteasome. In some embodiments, the target protein is a wild-type protein. In some embodiments, the target protein is a mutant protein.
  • the exogenous agent includes a small molecule, e.g., ions (e.g. Ca 2+ , C1-, Fe 2+ ), carbohydrates, lipids, reactive oxygen species, reactive nitrogen species, isoprenoids, signaling molecules, heme, polypeptide cofactors, electron accepting compounds, electron donating compounds, metabolites, ligands, and any combination of the foregoing.
  • the small molecule is a pharmaceutical that interacts with a target in the target cell.
  • the small molecule targets a protein in the target cell for degradation.
  • the small molecule targets a protein in the target cell for degradation by localizing the protein to the proteasome.
  • the small molecule is a proteolysis targeting chimera molecule (PROTAC).
  • the exogenous agent is a nucleic acid, such as any described in Section II-D-1.
  • the nucleic acid contains a payload gene encoding a payload agent, for instance a protein, such as any described in Section II-D-2.
  • the exogenous agent is a protein, such as any described in Section II-D-2.
  • the protein is a payload agent.
  • the payload agent comprises a cytosolic protein, e.g., a protein that is produced in the recipient cell and localizes to the recipient cell cytoplasm.
  • the payload agent comprises a secreted protein, e.g., a protein that is produced and secreted by the recipient cell.
  • the payload agent comprises a nuclear protein, e.g., a protein that is produced in the recipient cell and is imported to the nucleus of the recipient cell.
  • the payload agent comprises an organellar protein (e.g., a mitochondrial protein), e.g., a protein that is produced in the recipient cell and is imported into an organelle (e.g., a mitochondrial) of the recipient cell.
  • organellar protein e.g., a mitochondrial protein
  • the protein is a wild-type protein or a mutant protein.
  • the protein is a fusion or chimeric protein.
  • the payload agent comprises a membrane protein.
  • the membrane protein comprises a chimeric antigen receptor (CAR), a T cell receptor, an integrin, an ion channel, a pore forming protein, a Toll-Like Receptor, an interleukin receptor, a cell adhesion protein, or a transport protein.
  • the payload agent is an engineered receptor, such as antigen receptors including chimeric antigen receptors (CARs), and other antigen-binding receptors such as transgenic T cell receptors (TCRs).
  • antigen receptors including chimeric antigen receptors (CARs), and other antigen-binding receptors such as transgenic T cell receptors (TCRs).
  • CARs chimeric antigen receptors
  • TCRs transgenic T cell receptors
  • the pay load agent is a chimeric antigen receptor (CAR) comprising an antigen binding domain.
  • the CAR is or comprises a first generation CAR comprising an antigen binding domain, a transmembrane domain, and signaling domain (e.g., one, two or three signaling domains).
  • the CAR comprises a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains.
  • a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain which upon successful signaling of the CAR induces expression of a cytokine gene.
  • the antigen binding domain is or comprises an scFv or Fab.
  • the antigen binding domain targets an antigen characteristic of a cell type. In some embodiments, the antigen binding domain targets an antigen characteristic of a neoplastic cell. In some embodiments, the antigen characteristic of a neoplastic cell is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor, Epidermal Growth Factor Receptors (EGFR) (including ErbBl/EGFR, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), Fibroblast Growth Factor Receptors (FGFR) (including FGF1, FGF2, FGF3, FGF4, FGF5, FGF1, FGF2, FGF3,
  • EphB3, EphB4, and EphB6) CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CIC- Kb, Bestrophins, TMEM16A, GABA receptor, glycin receptor, ABC transporters, NAV1.1, NAVI.2, NAVI.3, NAVI.4, NAV1.5, NAVI.6, NAV1.7, NAVI.8, NAV1.9, sphingosin-1 -phosphate receptor (S1P1R), NMDA channel, transmembrane protein, multispan transmembrane protein, T-cell receptor motifs; T-cell alpha chains; T-cell P chains; T-cell y chains; T-cell 5 chains; CCR7; CD3; CD4; CD5; CD7; CD8; CDllb; CDllc;
  • the antigen binding domain targets an antigen characteristic of a T cell.
  • the antigen characteristic of a T cell is selected from a cell surface receptor, a membrane transport protein (e.g., an active or passive transport protein such as, for example, an ion channel protein, a pore-forming protein, etc.), a transmembrane receptor, a membrane enzyme, and/or a cell adhesion protein characteristic of a T cell.
  • an antigen characteristic of a T cell may be a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor, AKT1; AKT2; AKT3; ATF2; BCL10; CALM1; CD3D (CD35); CD3E (CD3a); CD3G (CD3y); CD4; CD8; CD28; CD45; CD80 (B7-1); CD86 (B7-2); CD247 (CD3Q; CTLA4 (CD152); ELK1; ERK1 (MAPK3); ERK2; FOS; FYN; GRAP2 (GADS); GRB2; HLA-DRA; HLA- DRB1; HLA-DRB3; HLA-DRB4; HLA-
  • the antigen binding domain targets an antigen characteristic of a disorder. In some embodiments, the antigen binding domain targets an antigen characteristic of an autoimmune or inflammatory disorder. In some embodiments, the autoimmune or inflammatory disorder is selected from chronic graft-vs-host disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, goodpasture, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, cold agglutinin disease, Pemphigus vulgaris, Grave's disease, autoimmune hemolytic anemia, Hemophilia A, Primary Sjogren's Syndrome, thrombotic thrombocytopenia purrpura, neuromyelits optica, Evan's syndrome, IgM mediated neuropathy, cyroglobulinemia, dermatomyositis, idiopathic thrombocytopenia, ankylosing spondylitis, bull
  • GVHD chronic graf
  • the antigen characteristic of an an autoimmune or inflammatory disorder is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, or histidine kinase associated receptor.
  • a CAR antigen binding domain binds to a ligand expressed on B cells, plasma cells, plasmablasts, CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, CD28, TNF, interferon receptors, GM-CSF, ZAP-70, LFA-1, CD3 gamma, CD5 or CD2.
  • a ligand expressed on B cells, plasma cells, plasmablasts, CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, CD28, TNF, interferon receptors, GM-CSF, ZAP-70, LFA-1, CD3 gamma, CD5 or CD2.
  • the antigen binding domain targets an antigen characteristic of senescent cells, e.g., urokinase-type plasminogen activator receptor (uPAR).
  • uPAR urokinase-type plasminogen activator receptor
  • the CAR may be used for treatment or prophylaxis of disorders characterized by the aberrant accumulation of senescent cells, e.g., liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis.
  • the antigen binding domain targets an antigen characteristic of an infectious disease.
  • infectious disease is selected from HIV, hepatitis B virus, hepatitis C virus, Human herpes virus, Human herpes virus 8 (HHV-8, Kaposi sarcoma- associated herpes virus (KSHV)), Human T-lymphotrophic virus-1 (HTLV-1), Merkel cell polyomavirus (MCV), Simian virus 40 (SV40), Eptstein-Barr virus, CMV, human papillomavirus.
  • the antigen characteristic of an infectious disease is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor, HIV Env, gpl20, or CD4-induced epitope on HIV-1 Env.
  • the CAR transmembrane domain comprises at least a transmembrane region of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or functional variant thereof.
  • the transmembrane domain comprises at least a transmembrane region(s) of CD8a, CD8P, 4-1BB/CD137, CD28, CD34, CD4, FcaRIy, CD16, OX40/CD134, CD3 ⁇ , CD3a, CD3y, CD35, TCRa, TCRp, TCR ⁇ , CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, and FGFR2B, or functional variant thereof.
  • the CAR comprises at least one signaling domain selected from one or more of B7-1/CD80; B7-2/CD86; B7-H1/PD-L1; B7-H2; B7-H3; B7-H4; B7-H6; B7-H7;
  • the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof.
  • the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; and (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof.
  • the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof.
  • the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof, and/or (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof.
  • the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; (iii) a 4- IBB domain, or a CD 134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the CAR further comprises one or more spacers, e.g., wherein the spacer is a first spacer between the antigen binding domain and the transmembrane domain.
  • the first spacer includes at least a portion of an immunoglobulin constant region or variant or modified version thereof.
  • the spacer is a second spacer between the transmembrane domain and a signaling domain.
  • the second spacer is an oligopeptide, e.g., wherein the oligopeptide comprises glycine-serine doublets.
  • the payload agent is or comprises a CAR, e.g., a first generation CAR or a nucleic acid encoding a first generation CAR.
  • a first generation CAR comprises an antigen binding domain, a transmembrane domain, and signaling domain.
  • a signaling domain mediates downstream signaling during T cell activation.
  • the payload agent is or comprises a second generation CAR or a nucleic acid encoding a second generation CAR.
  • a second generation CAR comprises an antigen binding domain, a transmembrane domain, and two signaling domains.
  • a signaling domain mediates downstream signaling during T cell activation.
  • a signaling domain is a costimulatory domain.
  • a costimulatory domain enhances cytokine production, CAR T cell proliferation, and or CAR T cell persistence during T cell activation.
  • the payload agent is or comprises a third generation CAR or a nucleic acid encoding a third generation CAR.
  • a third generation CAR comprises an antigen binding domain, a transmembrane domain, and at least three signaling domains.
  • a signaling domain mediates downstream signaling during T cell activation.
  • a signaling domain is a costimulatory domain.
  • a costimulatory domain enhances cytokine production, CAR T cell proliferation, and or CAR T cell persistence during T cell activation.
  • a third generation CAR comprises at least two costimulatory domains. In some embodiments, the at least two costimulatory domains are not the same.
  • the payload agent is or comprises a fourth generation CAR or a nucleic acid encoding a fourth generation CAR.
  • a fourth generation CAR comprises an antigen binding domain, a transmembrane domain, and at least two, three, or four signaling domains.
  • a signaling domain mediates downstream signaling during T cell activation.
  • a signaling domain is a costimulatory domain.
  • a costimulatory domain enhances cytokine production, CAR T cell proliferation, and or CAR T cell persistence during T cell activation.
  • a first, second, third, or fourth generation CAR further comprises a domain which upon successful signaling of the CAR induces expression of a cytokine gene.
  • a cytokine gene is endogenous or exogenous to a target cell comprising a CAR which comprises a domain which upon successful signaling of the CAR induces expression of a cytokine gene.
  • a cytokine gene encodes a pro-inflammatory cytokine.
  • a cytokine gene encodes IL-1, IL-2, IL-9, IL-12, IL-18, TNF, or IFN-gamma, or functional fragment thereof.
  • a domain which upon successful signaling of the CAR induces expression of a cytokine gene is or comprises a transcription factor or functional domain or fragment thereof. In some embodiments a domain which upon successful signaling of the CAR induces expression of a cytokine gene is or comprises a transcription factor or functional domain or fragment thereof. In some embodiments a transcription factor or functional domain or fragment thereof is or comprises a nuclear factor of activated T cells (NF AT), an NF-kB, or functional domain or fragment thereof. See, e.g., Zhang. C. et al., Engineering CAR-T cells. Biomarker Research. 5:22 (2017); WO 2016126608; Sha, H. et al. Chimaeric antigen receptor T-cell therapy for tumour immunotherapy. Bioscience Reports Jan 27, 2017, 37 (1).
  • NF AT nuclear factor of activated T cells
  • a CAR antigen binding domain is or comprises an antibody or antigen-binding portion thereof. In some embodiments, a CAR antigen binding domain is or comprises an scFv or Fab. In some embodiments a CAR antigen binding domain comprises an scFv or Fab fragment of a T-cell alpha chain antibody; T-cell P chain antibody; T-cell y chain antibody; T-cell 5 chain antibody; CCR7 antibody; CD3 antibody; CD4 antibody; CD5 antibody; CD7 antibody; CD8 antibody; CDl lb antibody; CDl lc antibody; CD16 antibody; CD19 antibody; CD20 antibody; CD21 antibody; CD22 antibody; CD25 antibody; CD28 antibody; CD34 antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45RO antibody; CD52 antibody; CD56 antibody; CD62E antibody; CD68 antibody; CD80 antibody; CD95 antibody; CD117 antibody; CD127 antibody; CD133 antibody; CD137 (4-1 BB) antibody; CD 163 antibody; F4
  • an antigen binding domain binds to a cell surface antigen of a cell.
  • a cell surface antigen is characteristic of one type of cell. In some embodiments, a cell surface antigen is characteristic of more than one type of cell.
  • a CAR antigen binding domain binds a cell surface antigen characteristic of a T cell.
  • an antigen characteristic of a T cell may be a cell surface receptor, a membrane transport protein (e.g., an active or passive transport protein such as, for example, an ion channel protein, a pore-forming protein, etc.), a transmembrane receptor, a membrane enzyme, and/or a cell adhesion protein characteristic of a T cell.
  • an antigen characteristic of a T cell may be a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, or histidine kinase associated receptor.
  • a CAR comprises a signaling domain which is a costimulatory domain. In some embodiments a CAR comprises a second costimulatory domain. In some embodiments a CAR comprises at least two costimulatory domains. In some embodiments a CAR comprises at least three costimulatory domains. In some embodiments a CAR comprises a costimulatory domain selected from one or more of CD27, CD28, 4-1BB, CD134/OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.
  • LFA-1 lymphocyte function-associated antigen-1
  • the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (IT AM), or functional variant thereof.
  • the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (IT AM), or functional variant thereof; and (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof.
  • the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (IT AM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof.
  • the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (IT AM), or functional variant thereof; (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof, and/or (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof.
  • the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (IT AM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; (iii) a 4- IBB domain, or a CD 134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.
  • IT AM immunoreceptor tyrosine-based activation motif
  • the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain.
  • the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.
  • the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion.
  • exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
  • the intracellular signaling domain includes intracellular components of a 4-1BB signaling domain and a CD3-zeta signaling domain. In some embodiments, the intracellular signaling domain includes intracellular components of a CD28 signaling domain and a CD3zeta signaling domain.
  • the CAR comprises an extracellular antigen binding domain (e.g., antibody or antibody fragment, such as an scFv) that binds to an antigen (e.g. tumor antigen), a spacer (e.g. containing a hinge domain, such as any as described herein), a transmembrane domain (e.g. any as described herein), and an intracellular signaling domain (e.g. any intracellular signaling domain, such as a primary signaling domain or costimulatory signaling domain as described herein).
  • the intracellular signaling domain is or includes a primary cytoplasmic signaling domain.
  • the intracellular signaling domain additionally includes an intracellular signaling domain of a costimulatory molecule (e.g., a costimulatory domain).
  • the CAR contains one or more domains that combine an antigen- or ligand-binding domain (e.g. antibody or antibody fragment) that provides specificity for a desired antigen (e.g., tumor antigen) with intracellular signaling domains.
  • the intracellular signaling domain is a stimulating or an activating intracellular domain portion, such as a T cell stimulating or activating domain, providing a primary activation signal or a primary signal.
  • the intracellular signaling domain contains or additionally contains a costimulatory signaling domain to facilitate effector functions.
  • chimeric receptors when genetically engineered into immune cells can modulate T cell activity, and, in some cases, can modulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells with improved longevity, survival and/or persistence in vivo, such as for use in adoptive cell therapy methods.
  • Exemplary antigen receptors including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, W02013/123061, U.S. patent app. Pub. Nos. US2002131960, US2013287748, US20130149337, U.S. Patent Nos. 6,451,995,
  • the antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in WO/2014055668.
  • CARs examples include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US 7,446,190, US 8,389,282, Kochenderfer et al., (2013) Nature Reviews Clinical Oncology, 10, 267-276; Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US 7,446,190, and US 8, 389, 282.
  • the recombinant receptors such as CARs, generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, generally a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.
  • the antigen binding domain of the CAR molecule comprises an antibody, an antibody fragment, an scFv, a Fv, a Fab, a (Fab')2, a single domain antibody (SdAb), a VH or VL domain, or a camelid VHH domain.
  • a CAR antigen binding domain is or comprises an antibody or antigen-binding portion thereof. In some embodiments, a CAR antigen binding domain is or comprises an scFv or Fab. In some embodiments, a CAR antigen binding domain comprises an scFv or Fab fragment of a CD19 antibody; CD22 antibody; T-cell alpha chain antibody; T-cell chain antibody; T- cell y chain antibody; T-cell 5 chain antibody; CCR7 antibody; CD3 antibody; CD4 antibody; CD5 antibody; CD7 antibody; CD8 antibody; CDl lb antibody; CDl lc antibody; CD16 antibody; CD20 antibody; CD21 antibody; CD25 antibody; CD28 antibody; CD34 antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45RO antibody; CD52 antibody; CD56 antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD95 antibody; CD117 antibody; CD127 antibody; CD133 antibody; CD 137 (4-1 BB) antibody; CD 163 antibody; F
  • a CAR comprises a signaling domain which is a costimulatory domain. In some embodiments, a CAR comprises a second costimulatory domain. In some embodiments, a CAR comprises at least two costimulatory domains. In some embodiments, a CAR comprises at least three costimulatory domains. In some embodiments, a CAR comprises a costimulatory domain selected from one or more of CD27, CD28, 4-1BB, CD134/OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.
  • LFA-1 lymphocyte function-associated antigen-1
  • a CAR comprises two or more costimulatory domains, two costimulatory domains are different. In some embodiments, if a CAR comprises two or more costimulatory domains, two costimulatory domains are the same.
  • the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on the engineered cells.
  • the antigen targeted by the receptor includes antigens associated with a B cell malignancy, such as any of a number of known B cell markers.
  • the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD47, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.
  • the CAR binds to CD 19. In some embodiments, the CAR binds to CD22. In some embodiments, the CAR binds to CD 19 and CD22. In some embodiments, the CAR is selected from the group consisting of a first generation CAR, a second generation CAR, a third generation CAR, and a fourth generation CAR. In some embodiments, the CAR includes a single binding domain that binds to a single target antigen. In some embodiments, the CAR includes a single binding domain that binds to more than one target antigen, e.g., 2, 3, or more target antigens.
  • the CAR includes two binding domains such that each binding domain binds to a different target antigens. In some embodiments, the CAR includes two binding domains such that each binding domain binds to the same target antigen.
  • CD 19- specific, CD22-specific and CD19/CD22-bispecific CARs can be found in W02012/079000, WO2016/149578 and W02020/014482, the disclosures including the sequence listings and figures are incorporated herein by reference in their entirety.
  • the chimeric antigen receptor includes an extracellular portion containing an antibody or antibody fragment. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv.
  • the antigen targeted by the antigen-binding domain is CD 19.
  • the antigen-binding domain of the recombinant receptor, e.g., CAR, and the antigen-binding domain binds, such as specifically binds or specifically recognizes, a CD19, such as a human CD19.
  • the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD 19.
  • the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1.
  • the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US 2016/0152723.
  • the antigen is CD 19.
  • the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD 19.
  • the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1.
  • the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US 2016/0152723.
  • the scFv is derived from FMC63.
  • FMC63 generally refers to a mouse monoclonal IgGl antibody raised against Naim-1 and -16 cells expressing CD19 of human origin (Fing, N. R., et al. (1987). Leucocyte typing III. 302).
  • the antibody portion of the recombinant receptor e.g., CAR
  • the spacer includes at least a portion of an immunoglobulin constant region, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region.
  • the constant region or portion is of a human IgG, such as IgG4 or IgGl.
  • the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain.
  • the spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer.
  • Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, WO2014031687, U.S. Patent No. 8,822,647 or published app. No. US 2014/0271635.
  • the constant region or portion is of a human IgG, such as IgG4 or IgGl.
  • the antigen receptor comprises an intracellular domain linked directly or indirectly to the extracellular domain.
  • the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain.
  • the intracellular signaling domain comprises an IT AM.
  • the antigen recognition domain e.g. extracellular domain
  • the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g. scFv) and intracellular signaling domain.
  • the antigen-binding component e.g., antibody
  • the antigen-binding component is linked to one or more transmembrane and intracellular signaling domains.
  • a transmembrane domain that naturally is associated with one of the domains in the receptor e.g., CAR
  • the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • the CAR transmembrane domain comprises at least a transmembrane region of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or functional variant thereof.
  • the transmembrane domain comprises at least a transmembrane region(s) of CD8a, CD8P, 4-1BB/CD137, CD28, CD34, CD4, FcaRIy, CD16, OX40/CD134, CD3 ⁇ , CD3a, CD3y, CD35, TCRa, TCRp, TCR ⁇ , CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, and FGFR2B, or functional variant thereof.
  • the transmembrane domain in some embodiments is derived either from a natural or from a synthetic source.
  • the domain in some aspects is derived from any membrane-bound or transmembrane protein.
  • Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, CD 154.
  • the transmembrane domain in some embodiments is synthetic.
  • the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • the linkage is by linkers, spacers, and/or transmembrane domain(s).
  • the transmembrane domain contains a transmembrane portion of CD28.
  • the extracellular domain and transmembrane domain can be linked directly or indirectly.
  • the extracellular domain and transmembrane are linked by a spacer, such as any described herein.
  • the receptor contains extracellular portion of the molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion.
  • intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone.
  • a short oligo- or polypeptide linker for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
  • T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).
  • primary cytoplasmic signaling sequences those that initiate antigen-dependent primary activation through the TCR
  • secondary cytoplasmic signaling sequences those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal.
  • the CAR includes one or both of such signaling components.
  • the receptor e.g., the CAR
  • the CAR generally includes at least one intracellular signaling component or components.
  • the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine -based activation motifs or IT AMs.
  • IT AM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon.
  • cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
  • the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain.
  • the antigen-binding portion is linked to one or more cell signaling modules.
  • cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains.
  • the intracellular component is or includes a CD3-zeta intracellular signaling domain.
  • the intracellular component is or includes a signaling domain from Fc receptor gamma chain.
  • the receptor e.g., CAR
  • the receptor includes the intracellular signaling domain and further includes a portion, such as a transmembrane domain and/or hinge portion, of one or more additional molecules such as CD8, CD4, CD25, or CD 16.
  • the CAR or other chimeric receptor is a chimeric molecule of CD3-zeta (CD3-z) or Fc receptor and a portion of one of CD8, CD4, CD25 or CD16.
  • the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR.
  • the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors.
  • a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal.
  • the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement.
  • TCR T cell receptor
  • co-receptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement.
  • full activation generally requires not only signaling through the TCR, but also a costimulatory signal.
  • a component for generating secondary or co-stimulatory signal is also included in the CAR.
  • the CAR does not include a component for generating a costimulatory signal.
  • an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.
  • the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule.
  • the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, 0X40, DAP10, and ICOS.
  • the same CAR includes both the activating and costimulatory components.
  • the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between the transmembrane domain and intracellular signaling domain.
  • the T cell costimulatory molecule is CD28 or 41BB.
  • the T cell costimulatory molecule is 41BB.
  • the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen.
  • the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668).
  • the cells include one or more stimulatory or activating CAR and/or a costimulatory CAR.
  • the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl.
  • the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain.
  • the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.
  • the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion.
  • exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
  • the intracellular signaling domain includes intracellular components of a 4-1BB signaling domain and a CD3-zeta signaling domain. In some embodiments, the intracellular signaling domain includes intracellular components of a CD28 signaling domain and a CD3zeta signaling domain.
  • a CD19 specific CAR includes an anti-CD19 single-chain antibody fragment (scFv), a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) costimulatory signaling domain, and a CD3 ⁇ signaling domain.
  • a CD22 specific CAR includes an anti-CD22 scFv, a transmembrane domain such as one derived from human CD8a, a 4- 1BB (CD137) co-stimulatory signaling domain, and a CD3 ⁇ signaling domain.
  • a CD19/CD22-bispecific CAR includes an anti-CD19 scFv, an anti-CD22 scFv, a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co-stimulatory signaling domain, and a CD3 ⁇ signaling domain.
  • the CAR comprises a commercial CAR construct carried by a T cell.
  • CAR-T cell based therapies include brexucabtagene autoleucel (TECARTUS®), axicabtagene ciloleucel (YESCARTA®), idecabtagene vicleucel (ABECMA®), lisocabtagene maraleucel (BREYANZI®), tisagenlecleucel (KYMRIAH®), Descartes-08 and Descartes- 11 from Cartesian Therapeutics, CTL110 from Novartis, P-BMCA-101 from Poseida Therapeutics, AUTO4 from Autolus Limited, UCARTCS from Cellectis, PBCAR19B and PBCAR269A from Precision Biosciences, FT819 from Fate Therapeutics, and CYAD-211 from Clyad Oncology.
  • TECARTUS® brexucabtagene autoleucel
  • YESCARTA® axicabtagene ciloleucel
  • the antigen binding domain targets an antigen characteristic of an autoimmune or inflammatory disorder.
  • the ABD binds an antigen associated with an autoimmune or inflammatory disorder.
  • the antigen is expressed by a cell associated with an autoimmune or inflammatory disorder.
  • the autoimmune or inflammatory disorder is selected from chronic graft-vs-host disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, goodpasture syndrome, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, cold agglutinin disease, Pemphigus vulgaris, Grave's disease, autoimmune hemolytic anemia, Hemophilia A, Primary Sjogren's Syndrome, thrombotic thrombocytopenia purrpura, neuromyelits optica, Evan's syndrome, IgM mediated neuropathy, cryoglobulinemia, dermatomyositis, idiopathic thrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquired angioedema, chronic urticarial, antiphospholipid demyelinating polyneuropathy, and autoimmune thrombocytopenia or neutropenia or
  • the antigen characteristic of an autoimmune or inflammatory disorder is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, or histidine kinase associated receptor.
  • an antigen binding domain of a CAR binds to a ligand expressed on B cells, plasma cells, or plasmablasts. In some embodiments, an antigen binding domain of a CAR binds to CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, CD28, TNF, interferon receptors, GM-CSF, ZAP-70, LFA-1, CD3 gamma, CD5 or CD2. See, e.g., US 2003/0077249; WO 2017/058753; WO 2017/058850, the contents of which are herein incorporated by reference.
  • the antigen binding domain targets an antigen characteristic of senescent cells, e.g., urokinase-type plasminogen activator receptor (uPAR).
  • uPAR urokinase-type plasminogen activator receptor
  • the ABD binds an antigen associated with a senescent cell.
  • the antigen is expressed by a senescent cell.
  • the CAR may be used for treatment or prophylaxis of disorders characterized by the aberrant accumulation of senescent cells, e.g., liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis.
  • the antigen binding domain targets an antigen characteristic of an infectious disease.
  • the ABD binds an antigen associated with an infectious disease.
  • the antigen is expressed by a cell affected by an infectious disease.
  • the infectious disease is selected from HIV, hepatitis B virus, hepatitis C virus, Human herpes virus, Human herpes virus 8 (HHV-8, Kaposi sarcoma-associated herpes virus (KSHV)), Human T-lymphotrophic virus-1 (HTLV-1), Merkel cell polyomavirus (MCV), Simian virus 40 (SV40), Epstein-Barr virus, CMV, human papillomavirus.
  • the antigen characteristic of an infectious disease is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme- linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor, HIV Env, gpl20, or CD4-induced epitope on HIV-1 Env.
  • an antigen binding domain binds to a cell surface antigen of a cell.
  • a cell surface antigen is characteristic of (e.g., expressed by) a particular or specific cell type. In some embodiments, a cell surface antigen is characteristic of more than one type of cell.
  • a CAR antigen binding domain binds a cell surface antigen characteristic of a T cell, such as a cell surface antigen on a T cell.
  • an antigen characteristic of a T cell may be a cell surface receptor, a membrane transport protein (e.g., an active or passive transport protein such as, for example, an ion channel protein, a pore-forming protein, etc.), a transmembrane receptor, a membrane enzyme, and/or a cell adhesion protein characteristic of a T cell.
  • an antigen characteristic of a T cell may be a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, or histidine kinase associated receptor.
  • the CAR comprises an extracellular antigen binding domain (e.g., antibody or antibody fragment, such as an scFv) that binds to an antigen (e.g. tumor antigen), a spacer (e.g.
  • the intracellular signaling domain is or includes a primary cytoplasmic signaling domain.
  • the intracellular signaling domain additionally includes an intracellular signaling domain of a costimulatory molecule (e.g., a costimulatory domain). Examples of exemplary components of a CAR are described in Table 5. In provided aspects, the sequences of each component in a CAR can include any combination listed in Table 5.
  • the antigen receptor further includes a marker and/or cells expressing the CAR or other antigen receptor further includes a surrogate marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor.
  • a surrogate marker such as a cell surface marker
  • the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor, such as truncated version of such a cell surface receptor (e.g., tEGFR).
  • the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A.
  • a linker sequence such as a cleavable linker sequence, e.g., T2A.
  • a marker, and optionally a linker sequence can be any as disclosed in published patent application No. WO2014031687.
  • the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence.
  • the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.
  • the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self’ by the immune system of the host into which the cells will be adoptively transferred.
  • the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered.
  • the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.
  • CARs are referred to as first, second, and/or third generation CARs.
  • a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding;
  • a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD 137;
  • a third generation CAR is one that includes multiple costimulatory domains of different costimulatory receptors.
  • the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof.
  • the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4- IBB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof.
  • the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge -only spacer.
  • an Ig molecule such as a human Ig molecule
  • an Ig hinge e.g. an IgG4 hinge, such as a hinge -only spacer.
  • the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgGlIn other embodiments, the spacer is or contains an Ig hinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only. In some embodiments, the spacer is or comprises a glycineserine rich sequence or other flexible linker such as known flexible linkers.
  • the CAR includes an antibody such as an antibody fragment, including scFvs, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28 -derived intracellular signaling domain, and a CD3 zeta signaling domain.
  • an antibody such as an antibody fragment, including scFvs
  • a spacer such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28 -derived intracellular signal
  • the CAR includes an antibody or fragment, such as scFv, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-lBB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.
  • the recombinant receptors, such as CARs, expressed by the cells administered to the subject generally recognize or specifically bind to a molecule that is expressed in, associated with, and/or specific for the disease or condition or cells thereof being treated.
  • the receptor Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an ITAM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition.
  • the cells express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition.
  • the payload agent is a T cell receptor (TCR) or antigen-binding portion thereof that recognizes a peptide epitope or T cell epitope of a target polypeptide, such as an antigen of a tumor, viral or autoimmune protein.
  • TCR T cell receptor
  • a “T cell receptor” or “TCR” is a molecule that contains a variable a and b chains (also known as TCRalpha and TCRbeta, respectively) or a variable g and d chains (also known as TCRalpha and TCRbeta, respectively), or antigen-binding portions thereof, and which is capable of specifically binding to a peptide bound to an MHC molecule.
  • the TCR is in the ab form.
  • TCRs that exist in alpha-beta and gamma-delta forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions.
  • a TCR can be found on the surface of a cell or in soluble form.
  • a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the term “TCR” should be understood to encompass full TCRs as well as antigen-binding portions or antigen-binding fragments thereof.
  • the TCR is an intact or full-length TCR, including TCRs in the ab form or gd form.
  • the TCR is an antigen-binding portion that is less than a full-length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC -peptide complex.
  • an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC-peptide complex, to which the full TCR binds.
  • an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable b chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex.
  • the variable chains of a TCR contain complementarity determining regions involved in recognition of the peptide, MHC and/or MHC-peptide complex.
  • the engineered receptor is a chimeric autoantibody receptor (CAAR).
  • CAAR binds, e.g., specifically binds, or recognizes, an autoantibody.
  • a cell expressing the CAAR such as a T cell engineered to express a CAAR, can be used to bind to and kill autoantibody-expressing cells, but not normal antibody expressing cells.
  • CAAR-expressing cells can be used to treat an autoimmune disease associated with expression of self-antigens, such as autoimmune diseases.
  • CAAR-expressing cells can target B cells that ultimately produce the autoantibodies and display the autoantibodies on their cell surfaces, mark these B cells as disease-specific targets for therapeutic intervention.
  • CAAR-expressing cells can be used to efficiently targeting and killing the pathogenic B cells in autoimmune diseases by targeting the disease-causing B cells using an antigen-specific chimeric autoantibody receptor.
  • the recombinant receptor is a CAAR, such as any described in U.S. Patent Application Pub. No. US 2017/0051035.
  • the CAAR comprises an autoantibody binding domain, a transmembrane domain, and one or more intracellular signaling region or domain (also interchangeably called a cytoplasmic signaling domain or region).
  • the intracellular signaling region comprises an intracellular signaling domain.
  • the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain that is capable of stimulating and/or inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component (e.g. an intracellular signaling domain or region of a CD3-zeta) chain or a functional variant or signaling portion thereof), and/or a signaling domain comprising an immunoreceptor tyrosine- based activation motif (IT AM).
  • TCR T cell receptor
  • the autoantibody binding domain comprises an autoantigen or a fragment thereof.
  • the choice of autoantigen can depend upon the type of autoantibody being targeted.
  • the autoantigen may be chosen because it recognizes an autoantibody on a target cell, such as a B cell, associated with a particular disease state, e.g. an autoimmune disease, such as an autoantibody-mediated autoimmune disease.
  • the autoimmune disease includes pemphigus vulgaris (PV).
  • Exemplary autoantigens include desmoglein 1 (Dsgl) and Dsg3.
  • the pay load agent is a CAR.
  • CARs also known as chimeric immunoreceptors, chimeric T cell receptors, or artificial T cell receptors
  • CARs are receptor proteins that have been engineered to give host cells (e.g., T cells) the new ability to target a specific protein.
  • the receptors are chimeric because they combine both antigen-binding and T cell activating functions into a single receptor.
  • the provided particles may be used to express one or more CARs in a host cell (e.g., a T cell) for use in cell-based therapies against various target antigens.
  • the CAR may comprise an extracellular binding domain (also referred to as a “binder”) that specifically binds a target antigen, a transmembrane domain, and an intracellular signaling domain.
  • the CAR may further comprise one or more additional elements, including one or more signal peptides, one or more extracellular hinge domains, and/or one or more intracellular costimulatory domains. Domains may be directly adjacent to one another, or there may be one or more amino acids linking the domains.
  • the nucleotide sequence encoding a CAR may be derived from a mammalian sequence, for example, a mouse sequence, a primate sequence, a human sequence, or combinations thereof.
  • the sequence of the CAR may be humanized.
  • the nucleotide sequence encoding a CAR may also be codon-optimized for expression in a mammalian cell, for example, a human cell.
  • the nucleotide sequence encoding a CAR may be at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the nucleotide sequences disclosed herein.
  • the sequence variations may be due to codon-optimalization, humanization, restriction enzyme-based cloning scars, and/or additional amino acid residues linking the functional domains, etc.
  • the CAR may comprise a signal peptide at the N-terminus.
  • signal peptides include CD8a signal peptide, IgK signal peptide, and granulocytemacrophage colony-stimulating factor receptor subunit alpha (GMCSFR-a, also known as colony stimulating factor 2 receptor subunit alpha (CSF2RA)) signal peptide, and variants thereof, the amino acid sequences of which are provided in Table 6 below.
  • the extracellular binding domain of the CAR may comprise one or more antibodies specific to one target antigen or multiple target antigens.
  • the antibody may be an antibody fragment, for example, an scFv, or a single-domain antibody fragment, for example, a VHH.
  • the scFv may comprise a heavy chain variable region (Vu) and a light chain variable region (VL) of an antibody connected by a linker.
  • the VH and the VL may be connected in either order, i.e., Vn-linkcr-Vi. or Vi.-linkcr-Vn.
  • Non-limiting examples of linkers include Whitlow linker, (G4S) n (n can be a positive integer, e.g., 1, 2, 3, 4, 5, 6, etc.) linker, and variants thereof.
  • the antigen may be an antigen that is exclusively or preferentially expressed on tumor cells, or an antigen that is characteristic of an autoimmune or inflammatory disease.
  • target antigens include, but are not limited to, CD5, CD19, CD20, CD22, CD23, CD30, CD70, Kappa, Lambda, and B cell maturation agent (BCMA), G-protein coupled receptor family C group 5 member D (GPRC5D) (associated with leukemias); CS1/SLAMF7, CD38, CD138, GPRC5D, TACI, and BCMA (associated with myelomas); GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRa, IL-13Ra, Mesothelin, MUC1, MUC16, and ROR1 (associated with solid tumors).
  • the extracellular binding domain of the CAR can be codon- optimized for expression in a host cell or have variant sequences to increase functions of the extracellular binding domain.
  • the CAR may comprise a hinge domain, also referred to as a spacer.
  • hinge domains include CD8a hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge-CH2-CH3 domain, and variants thereof, the amino acid sequences of which are provided in Table
  • the transmembrane domain of the CAR may comprise a transmembrane region of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3s.
  • the transmembrane domain may comprise a transmembrane region of CD8a, CD8P, 4- 1BB/CD137, CD28, CD34, CD4, FcaRIy, CD16, OX40/CD134, CD3 ⁇ , CD3a, CD3y, CD35, TCRa, TCRP, TCR ⁇ , CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, and FGFR2B, or a functional variant thereof, including the human versions of each of these sequences.
  • Table 8 provides the amino acid sequences of a few exemplary transmembrane domains.
  • the intracellular signaling domain and/or intracellular costimulatory domain of the CAR may comprise one or more signaling domains selected from B7-1/CD80, B7- 2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, PDCD6, 4-1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNF
  • the intracellular signaling domain and/or intracellular costimulatory domain comprises one or more signaling domains selected from a CD3 ⁇ domain, an ITAM, a CD28 domain, 4-1BB domain, or a functional variant thereof.
  • Table 7 provides the amino acid sequences of a few exemplary intracellular costimulatory and/or signaling domains.
  • the CD3 ⁇ signaling domain of SEQ ID NO:99 may have a mutation, e.g., a glutamine (Q) to lysine (K) mutation, at amino acid position 14 (see SEQ ID NO:62).
  • the CAR is a CD19 CAR (“CD19-CAR”).
  • the CD19 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD19, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and/or an intracellular signaling domain in tandem.
  • the signal peptide of the CD 19 CAR comprises a CD 8 a signal peptide.
  • the CD8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:47 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 47.
  • the signal peptide comprises an IgK signal peptide.
  • the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:48 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:48.
  • the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide.
  • the GMCSFR-a or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:49 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 49.
  • the extracellular binding domain of the CD 19 CAR is specific to CD 19, for example, human CD 19.
  • the extracellular binding domain of the CD 19 CAR can be codon- optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain.
  • the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.
  • the extracellular binding domain of the CD19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (Vu) and the light chain variable region (VL) of FMC63 connected by a linker.
  • FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun. 34(16-17): 1157-1165 (1997) and PCT Application Publication No. WO2018/213337, the entire contents of each of which are incorporated by reference herein.
  • the amino acid sequences of the entire FMC63-derived scFv (also referred to as FMC63 scFv) and its different portions are provided in Table 10 below.
  • the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:63, 64, or 69, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 163, 64, or 69.
  • the CD19- specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 65-67 and 70-72. In some embodiments, the CD19-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 65-67. In some embodiments, the CD19-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 70-72.
  • the CD19-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified.
  • the extracellular binding domain of the CD 19 CAR comprises or consists of the one or more CDRs as described herein.
  • the linker linking the VH and the VL portions of the scFv is a Whitlow linker having an amino acid sequence set forth in SEQ ID NO: 68.
  • the Whitlow linker may be replaced by a different linker, for example, a 3xG4S linker having an amino acid sequence set forth in SEQ ID NO: 143, which gives rise to a different FMC63-derived scFv having an amino acid sequence set forth in SEQ ID NO:73.
  • the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 73 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:73.
  • the extracellular binding domain of the CD 19 CAR is derived from an antibody specific to CD19, including, for example, SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol. 138(9):2793-2799 (1987)), 4G7 (Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418-427 (1987)), B4 HB12b (Kansas & Tedder, J.
  • SJ25C1 Bejcek et al., Cancer Res. 55:2346-2351 (1995)
  • HD37 Pezutto et al., J. Immunol. 138(9):2793-2799 (1987)
  • 4G7 (Meeker
  • the extracellular binding domain of the CD19 CAR can comprise or consist of the Vu, the VL, and/or one or more CDRs of any of the antibodies.
  • the hinge domain of the CD19 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain.
  • the CD8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:50 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:50.
  • the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain.
  • the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:51 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:51.
  • the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain.
  • the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:53 or SEQ ID NO:54, or an amino acid sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:53 or SEQ ID NO:54.
  • the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain.
  • the IgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:55 or an amino acid sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:55.
  • the transmembrane domain of the CD 19 CAR comprises a CD 8 a transmembrane domain, for example, a human CD8a transmembrane domain.
  • the CD 8 a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:56 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:56.
  • the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain.
  • the CD28 transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:57 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:57.
  • the intracellular costimulatory domain of the CD 19 CAR comprises a 4-1BB costimulatory domain.
  • 4-1BB also known as CD137, transmits a potent costimulatory signal to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes.
  • the 4-1BB costimulatory domain is human.
  • the 4-1BB costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:59 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:59.
  • the intracellular costimulatory domain comprises a CD28 costimulatory domain.
  • CD28 is another co-stimulatory molecule on T cells.
  • the CD28 costimulatory domain is human.
  • the CD28 costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:60 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:60.
  • the intracellular costimulatory domain of the CD19 CAR comprises a 4- IBB costimulatory domain and a CD28 costimulatory domain as described.
  • the intracellular signaling domain of the CD19 CAR comprises a CD3 zeta (Q signaling domain.
  • CD3 zeta associates with T cell receptors (TCRs) to produce a signal and contains immunoreceptor tyrosine-based activation motifs (ITAMs).
  • TCRs T cell receptors
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • the CD3 zeta signaling domain refers to amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation.
  • the CD3 zeta signaling domain is human.
  • the CD3 zeta signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 61 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:61.
  • the payload agent is a CD19 CAR, including, for example, a CD19 CAR comprising the CD19-specific scFv having sequences set forth in SEQ ID NO:63 or SEQ ID NO:73, the CD8a hinge domain of SEQ ID NO:50, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3 ⁇ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
  • the CD 19 CAR may additionally comprise a signal peptide (e.g., a CD 8 a signal peptide) as described.
  • the payload agent is a CD19 CAR, including, for example, a CD19 CAR comprising the CD19-specific scFv having sequences set forth in SEQ ID NO:63 or SEQ ID NO:73, the IgG4 hinge domain of SEQ ID NO:53 or SEQ ID NO:54, the CD28 transmembrane domain of SEQ ID NO:57, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3 ⁇ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
  • the CD19 CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide) as described.
  • the payload agent is a CD19 CAR, including, for example, a CD19 CAR comprising the CD19-specific scFv having sequences set forth in SEQ ID NO:63 or SEQ ID NO:73, the CD28 hinge domain of SEQ ID NO:51, the CD28 transmembrane domain of SEQ ID NO:57, the CD28 costimulatory domain of SEQ ID NO:60, the CD3 ⁇ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
  • the CD 19 CAR may additionally comprise a signal peptide (e.g., a CD 8 a signal peptide) as described.
  • the pay load agent is a CD 19 CAR as encoded by the sequence set forth in SEQ ID NO:74 or a sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO:74 (see Table 11).
  • the encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO:75 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:75, with the following components: CD8a signal peptide, FMC63 scFv (VL- Whitlow linker-Vn), CD8a hinge domain, CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3 ⁇ signaling domain.
  • the pay load agent is a commercially available embodiment of a CD 19 CAR.
  • CD 19 CARs include tisagenlecleucel, lisocabtagene maraleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel.
  • the CAR is tisagenlecleucel or portions thereof.
  • Tisagenlecleucel comprises a CD19 CAR with the following components: CD8a signal peptide, FMC63 scFv (VL-3XG4S linker-Vn), CD8a hinge domain, CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3 ⁇ signaling domain.
  • CD8a signal peptide CD8a signal peptide
  • FMC63 scFv VL-3XG4S linker-Vn
  • CD8a hinge domain CD8a transmembrane domain
  • 4-1BB costimulatory domain CD3 ⁇ signaling domain.
  • the CAR is lisocabtagene maraleucel or portions thereof.
  • Lisocabtagene maraleucel comprises a CD19 CAR with the following components: GMCSFR-a or CSF2RA signal peptide, FMC63 scFv (VL- Whitlow linker-Vn), IgG4 hinge domain, CD28 transmembrane domain, 4-1BB costimulatory domain, and CD3 ⁇ signaling domain.
  • the nucleotide and amino acid sequence of the CD 19 CAR in lisocabtagene maraleucel are provided in Table 9, with annotations of the sequences provided in Table 13.
  • the CAR is axicabtagene ciloleucel or portions thereof.
  • Axicabtagene ciloleucel comprises a CD19 CAR with the following components: GMCSFR-a or CSF2RA signal peptide, FMC63 scFv (V
  • the nucleotide and amino acid sequence of the CD 19 CAR in axicabtagene ciloleucel are provided in Table 11, with annotations of the sequences provided in Table 14.
  • the CAR is brexucabtagene autoleucel or portions thereof.
  • Brexucabtagene autoleucel comprises a CD 19 CAR with the following components: GMCSFR- a signal peptide, FMC63 scFv, CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain, and CD3 ⁇ signaling domain.
  • the CAR is encoded by the sequence set forth in SEQ ID NO: 76, 78, or 80, or a sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 76, 78, or 80.
  • the encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 77, 79, or 81, respectively, or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 77, 79, or 81, respectively.
  • the CAR is encoded by the sequence set forth in SEQ ID NO: 76, 78, or 80, or a sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 76, 78, or 80.
  • the encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 77, 79, or 81, respectively, is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 77, 79, or 81, respectively.
  • CD20 CARs are at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 77, 79, or 81, respectively.
  • the CAR is a CD20 CAR (“CD20-CAR”).
  • CD20 is an antigen found on the surface of B cells as early at the pro-B phase and progressively at increasing levels until B cell maturity, as well as on the cells of most B-cell neoplasms. CD20 positive cells are also sometimes found in cases of Hodgkins disease, myeloma, and thymoma.
  • the CD20 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD20, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and/or an intracellular signaling domain in tandem.
  • the signal peptide of the CD20 CAR comprises a CD 8 a signal peptide.
  • the CD8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:47 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 47.
  • the signal peptide comprises an IgK signal peptide.
  • the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:48 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:48.
  • the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide.
  • the GMCSFR-a or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:49 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 49.
  • the extracellular binding domain of the CD20 CAR is specific to CD20, for example, human CD20.
  • the extracellular binding domain of the CD20 CAR can be codon- optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain.
  • the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.
  • the extracellular binding domain of the CD20 CAR is derived from an antibody specific to CD20, including, for example, Leul6, IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab.
  • the extracellular binding domain of the CD20 CAR can comprise or consist of the VH, the VL, and/or one or more CDRs of any of the antibodies.
  • the extracellular binding domain of the CD20 CAR comprises an scFv derived from the Leu 16 monoclonal antibody, which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of Leul6 connected by a linker.
  • the linker is a 3xG4S linker.
  • the linker is a Whitlow linker as described herein.
  • the amino acid sequences of different portions of the entire Leul6-derived scFv also referred to as Leu 16 scFv
  • Table 15 the amino acid sequences of different portions of the entire Leul6-derived scFv
  • the CD20-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:82, 83, or 87, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:82, 83, or 87.
  • the CD20-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 84-86, 88, 89, and 144.
  • the CD20-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 84-86. In some embodiments, the CD20-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 88, 89, and 144.
  • the CD20-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified.
  • the extracellular binding domain of the CD20 CAR comprises or consists of the one or more CDRs as described herein.
  • the hinge domain of the CD20 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain.
  • the CD8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:50 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:50.
  • the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain.
  • the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:51 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:51.
  • the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain.
  • the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:53 or SEQ ID NO:54, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:53 or SEQ ID NO:54.
  • the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain.
  • the IgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:55 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:55.
  • the transmembrane domain of the CD20 CAR comprises a CD 8 a transmembrane domain, for example, a human CD8a transmembrane domain.
  • the CD 8 a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:56 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:56.
  • the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain.
  • the CD28 transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:57 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:57.
  • the intracellular costimulatory domain of the CD20 CAR comprises a 4-1BB costimulatory domain, for example, a human 4-1BB costimulatory domain.
  • the 4-1BB costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:59 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:59.
  • the intracellular costimulatory domain comprises a CD28 costimulatory domain, for example, a human CD28 costimulatory domain.
  • the CD28 costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:60 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:60.
  • the intracellular signaling domain of the CD20 CAR comprises a CD3 zeta (Q signaling domain, for example, a human CD3 ⁇ signaling domain.
  • the CD3 ⁇ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:61 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:61.
  • the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the CD8a hinge domain of SEQ ID NO:50, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3 ⁇ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
  • a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the CD8a hinge domain of SEQ ID NO:50, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB
  • the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the CD28 hinge domain of SEQ ID NO:51, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3 ⁇ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
  • a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the CD28 hinge domain of SEQ ID NO:51, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB costim
  • the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the IgG4 hinge domain of SEQ ID NO:53 or SEQ ID NO:54, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3 ⁇ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
  • a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the IgG4 hinge domain of SEQ ID NO:53 or SEQ ID NO:54, the CD8a transme
  • the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the CD8a hinge domain of SEQ ID NO:50, the CD28 transmembrane domain of SEQ ID NO:57, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3 ⁇ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
  • a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the CD8a hinge domain of SEQ ID NO:50, the CD28 transmembrane domain of SEQ ID NO:57, the 4-1BB costim
  • the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the CD28 hinge domain of SEQ ID NO:51, the CD28 transmembrane domain of SEQ ID NO:57, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3 ⁇ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
  • a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the CD28 hinge domain of SEQ ID NO:51, the CD28 transmembrane domain of SEQ ID NO:57, the 4-1BB costimulatory
  • the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the IgG4 hinge domain of SEQ ID NO:53 or SEQ ID NO:54, the CD28 transmembrane domain of SEQ ID NO:57, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3 ⁇ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
  • CD20 CARs comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the IgG4 hinge domain of SEQ ID NO:53 or SEQ ID NO:54, the CD28 transmembrane
  • the CAR is a CD22 CAR (“CD22-CAR”).
  • CD22 which is a transmembrane protein found mostly on the surface of mature B cells that functions as an inhibitory receptor for B cell receptor (BCR) signaling.
  • BCR B cell receptor
  • CD22 is expressed in 60-70% of B cell lymphomas and leukemias (e.g., B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma) and is not present on the cell surface in early stages of B cell development or on stem cells.
  • B-chronic lymphocytic leukemia e.g., hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma
  • the CD22 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD22, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and/or an intracellular signaling domain in tandem.
  • the signal peptide of the CD22 CAR comprises a CD 8 a signal peptide.
  • the CD8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:47 or an amino acid sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 47.
  • the signal peptide comprises an IgK signal peptide.
  • the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:48 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:48.
  • the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide.
  • the GMCSFR-a or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:49 or an amino acid sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 49.
  • the extracellular binding domain of the CD22 CAR is specific to CD22, for example, human CD22.
  • the extracellular binding domain of the CD22 CAR can be codon- optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain.
  • the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.
  • the extracellular binding domain of the CD22 CAR is derived from an antibody specific to CD22, including, for example, SM03, inotuzumab, epratuzumab, moxetumomab, and pinatuzumab.
  • the extracellular binding domain of the CD22 CAR can comprise or consist of the Vu, the VL, and/or one or more CDRs of any of the antibodies.
  • the extracellular binding domain of the CD22 CAR comprises an scFv derived from the m971 monoclonal antibody (m971), which comprises the heavy chain variable region (Vu) and the light chain variable region (VL) of m971 connected by a linker.
  • the linker is a 3xG4S linker.
  • the Whitlow linker may be used instead.
  • the amino acid sequences of the entire m971 -derived scFv (also referred to as m971 scFv) and its different portions are provided in Table 16 below.
  • the CD22-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:90, 91, or 95, or an amino acid sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:90, 91, or 95.
  • the CD22-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 92-94 and 96-98.

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Abstract

Provided herein are targeted lipid particles, such as targeted lentiviral particles, having a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3, and related polynucleotides, cells, and methods for preparation and use of the targeted lipid particles, such as lenti viral particles. Also provided herein are methods involving mobilizing bone marrow cells in combination with administering targeted or non-targeted lipid particles, such as lentiviral particles, for treatment of a disease or condition in a subject.

Description

VIRAL PARTICLES TARGETING HEMATOPOIETIC STEM CELLS
Cross-Reference to Related Applications
[0001] This application claims priority from U.S. provisional application No. 63/415,975, filed October, 13, 2022, entitled “VIRAL PARTICLES TARGETING HEMATOPOIETIC STEM CELLS”, U.S. provisional application No. 63/460,296, filed April 18, 2023, entitled “VIRAL PARTICLES TARGETING HEMATOPOIETIC STEM CELLS”, to U.S. provisional application No. 63/466,683, filed May 15, 2023, entitled “VIRAL PARTICLES TARGETING HEMATOPOIETIC STEM CELLS”, and U.S. provisional application No. 63/468,526, filed May 23, 2023, entitled “VIRAL PARTICLES TARGETING HEMATOPOIETIC STEM CELLS”, the contents of which are incorporated by reference in their entirety.
Incorperation by Reference of Sequence Listing
[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 18615-20080.40.xml created October 12, 2023, which is 751,853 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.
Field
[0003] The present disclosure relates to targeted lipid particles, such as targeted lentiviral particles, having a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3, and related polynucleotides, cells, and methods for preparation and use of the targeted lipid particles. The present disclosure also relates to methods involving mobilizing bone marrow cells in combination with administering targeted or non-targeted lipid particles, such as lentiviral particles, for treatment of a disease or condition in a subject.
Background
[0004] Lipid particles, including viral and virus-like particles, such as lentiviral particles, are commonly used for delivery of exogenous agents to cells. However, delivery to certain target cells can be challenging. Improved lipid particles, such as lentiviral particles, and methods for delivery are needed. The provided disclosure addresses this need.
Summary
[0005] The embodiments herein provide for lipid particles that allow for efficient targeting and delivery of an exogenous agent contained therein to hematopoietic stem/progenitor cells (HSPC), including to naive hematopoietic stem cells (HSCs). In some embodiments, the targeting can be to such stem or progenitor cells in a human subject, such as human HSPC. In provided embodiments, the lipid particles can include viral vectors such as lentiviral vectors that are pseudotyped with a fusogen with a preferential ability to target HSPC. In some embodiments, the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof, such as the exemplarly fusogens BaEVTR or BaEVRLess. In some embodiments, the lipid particle includes a targeting agent to target the fusogen to HSPCs, such as to CD34+ cells. In some embodiments, the targeting agent is fused to a fusogen, such as a viral envelope attachment glycoprotein, exposed on the lipid bilayer to provide a retargeted fusogen.
[0006] Provided herein in some embodiments is a targeted lipid particle, comprising a lipid bilayer, a fusogen, and a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3, wherein the fusogen and the targeting agent are exposed on the surface of the lipid bilayer.
[0007] In some embodiments, the targeting agent is fused to a transmembrane domain incorporated into the lipid bilayer. In some embodiments, the targeting agent is fused to the fusogen.
[0008] In some of any embodiments, the particle is a viral particle or virus-like particle. In some of any embodiments, the lipid bilayer is derived from a membrane of a producer cell used for producing a viral particle or virus-like particle. In some of any embodiments, the viral particle or virus-like particle is a retroviral particle or retrovirus-like particle. In some of any embodiments, the viral particle or virus-like particle is a lentivirus-like particle. In some of any embodiments, the viral particle or virus-like particle is a lentiviral particle.
[0009] Provided herein in some embodiments is a targeted lentiviral particle, comprising a fusogen and a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3, wherein the fusogen and the targeting agent are exposed on the surface of the lentiviral particle.
[0010] In some of any embodiments, the particle is pseudotyped with the fusogen.
[0011] In some of any embodiments, the targeting agent is fused to the fusogen. In some of any embodiments, the targeting agent is fused to the fusogen directly. In some of any embodiments, the targeting agent is fused to the fusogen via a linker.
[0012] In some of any embodiments, the targeting agent is fused to a transmembrane domain incorporated into the viral envelope.
[0013] In some of any embodiments, the particle is prepared by a method including transducing a producer cell with plasmids encoding the fusogen and targeting agent and a Gag-pol and Rev. In some of any embodiments, the producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells. In some of any embodiments, the producer cell is a 293T cell.
[0014] In some of any embodiments, the particle is replication defective.
[0015] In some of any embodiments, the particle comprises a viral nucleic acid. In some embodiments, the viral nucleic acid is a retroviral nucleic acid. In some of any embodiments, the viral nucleic acid is a lentiviral nucleic acid. In some of any embodiments, the viral nucleic acid comprises one or more, optionally all, of the following nucleic acid sequences: 5’ LTR (e.g., containing U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT)/central termination sequence (CTS) (e.g., DNA flap), Poly A tail sequence, a posttranscriptional regulatory element (e.g., WPRE), a Rev response element (RRE), and 3’ LTR (e.g., containing U5 and lacking a functional U3).
[0016] In some of any embodiments, the particle is devoid of viral genomic nucleic acids.
[0017] In some of any embodiments, wherein the target molecule is ASCT2. In some of any embodiments, the target molecule is CD 117.
[0018] In some of any embodiments, the fusogen is a viral fusion protein. In some of any embodiments, the fusogen is a viral envelope protein.
[0019] In some of any embodiments, the fusogen is a VSV-G protein or a functional variant thereof.
[0020] In some of any embodiments, the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof. In some of any embodiments, the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild-type BaEV inhibitory R peptide. In some of any embodiments, the fusogen is set forth in any of SEQ ID NO:254-260. In some of any embodiments, the fusogen is set forth in SEQ ID NO:261 (BaEVRLess). In some of any embodiments, the fusogen is set forth in SEQ ID NO:262 (BaEVTR). Provided herein is a targeted lipid particle, comprising a lipid bilayer, a fusogen, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof and is exposed on the surface of the lipid bilayer.
[0021] In some of any embodiments, the fusogen is a Cocal virus G protein or a functional variant thereof.
[0022] In some of any embodiments, the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
[0023] In some of any embodiments, the fusogen is a Paramyxovirus fusion protein (e.g., a Morbilli virus or Henipavirus) or a functional variant thereof. In some of any embodiments, the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbilli virus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof. In some of any embodiments, the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof. In some of any embodiments, the fusogen is a Nipah virus fusion protein or a functional variant thereof. In some of any embodiments, the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
[0024] In some embodiments, the targeting agent is fused to the NiV-G protein or biologically active portion thereof. In some of any embodiments, the targeting agent is fused to the C-terminus of the NiV-G protein or biologically active portion thereof.
[0025] In some of any embodiments, the NiV-G or biologically active portion thereof is a wildtype NiV-G protein or a functionally active variant or biologically active portion thereof. In some of any embodiments, the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof does not include an initial methionine. In some of any embodiments, NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
[0026] In some of any embodiments, the fusogen comprises one or modifications to reduce binding to its native receptor.
[0027] In some of any embodiments, the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3. In some of any embodiments, the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
[0028] In some of any embodiments, the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof. In some of any embodiments, the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30. In some embodiments, the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
[0029] In some of any embodiments, the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
[0030] In some of any embodiments, the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule. In some of any embodiments, the targeting agent is a single domain antibody that binds to the target molecule. In some of any embodiments, the targeting agent is a VHH that binds to the target molecule. In some of any embodiments, the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
[0031] In some of any embodiments, the target molecule is expressed on a target cell.
[0032] In some of any embodiments, the particle comprises an exogenous agent for delivery to a target cell. In some embodiments, the target molecule is expressed on the target cell. In some of any of the provided embodiments, the particle comprises a fusion protein between a viral structural protein and an exogenous agent. In some of any of the provided embodiments, the fusion protein is a cleavable fusion protein comprising a cleavable linker positioned between the viral structural protein and the exogenous agent. In some of any of the provided embodiments, the fusion protein comprises one or more nuclear localization sequences. In some of any of the provided embodiments, the fusion protein comprises one or more nuclear export sequences.
[0033] In some of any embodiments, the exogenous agent is present in the lumen. In some of any embodiments, the exogenous agent is a protein or a nucleic acid. In some of any embodiments, the exogenous agent is or encodes a therapeutic agent or a diagnostic agent.
[0034] In some of any embodiments, the exogenous agent is or encodes a factor associated with gene editing. In some of any embodiments, the exogenous agent is or encodes a genome-modifying protein for gene editing a target gene encoding an endogenous protein. In some embodiments, the genome-modifying protein is associated with gene editing by a sequence-specific nuclease, a CRISPR- associated transposase (CAST), prime editing, or Programmable Addition via Site-specific Targeting Elements (PASTE).
[0035] In some of any embodiments, the genome -modifying protein is a sequence-specific nuclease. In some of any embodiments, the sequence-specific nuclease is selected from the group consisting of an RNA-guided nuclease, a meganuclease, a transcription activator-like effector nuclease (TALEN), and a zinc-finger nuclease (ZFN). In some of any embodiments, the sequence-specific nuclease is an RNA-guided nuclease. In some of any embodiments, the RNA-guided nuclease is TnpB. In some of any embodiments, the RNA-guided nuclease comprises a Cas nuclease and a guide RNA (CRISPR-Cas combination). In some embodiments, the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex containing the gRNA and the Cas nuclease. In some of any embodiments, the Cas nuclease is a Type II or Type V Cas protein. In some of any embodiments, the genome-modifying protein is selected from the group consisting of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxll, Csyl, Csy2, Csy3, Mad7. In some of any embodiments, the Cas is a Cas9 or a Cas 12.
[0036] In some of any embodiments, the exogenous agent is or encodes a factor associated with base editing or prime editing (e.g., target-primed reverse transcription (TPRT)). In some of any embodiments, the exogenous agent is or encodes a transposase, integrase, or recombinase. In some of any embodiments, the exogenous agent is or encodes a DNA polymerase, RNA polymerase, or reversetranscriptase.
[0037] In some of any embodiments, the exogenous agent is for use in gene therapy to correct a genetic deficiency in the target cell. In some embodiments, the exogenous agent is a nucleic acid containing a payload gene for correcting the genetic deficiency.
[0038] In some of any embodiments, the exogenous agent is or encodes a membrane protein. In some of any embodiments, the membrane protein is an antigen receptor. In some embodiments, the antigen receptor binds to an antigen expressed on cells associated with a disease or condition. In some embodiments, the disease or condition is a cancer. In some of any embodiments, the antigen receptor binds to an antigen expressed on tumor cells. In some of any embodiments, the antigen receptor is a chimeric antigen receptor (CAR). In some of any embodiments, the antigen receptor is an engineered T cell receptor (TCR).
[0039] In some of any embodiments, the target cell is a hematopoietic cell. In some of any embodiments, the target cell is CD34+. In some of any embodiments, the target cell is a CD34+ progenitor cell. In some of any embodiments, the target cell is a hematopoietic stem cell.
[0040] In some of any embodiments, the delivery to target cells expressing the target molecule is increased by or by greater than 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5- fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, or more compared to delivery to non-target cells not expressing the target molecule.
[0041] Provided herein in some embodiments is a polynucleotide comprising a nucleic acid sequence encoding a fusogen and a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3.
[0042] In some embodiments the polynucleotide encodes a transmembrane domain that is fused to the targeting agent.
[0043] In some embodiments, the targeting agent is fused to the fusogen. In some of any embodiments, the targeting agent is fused to the fusogen directly. In some of any embodiments, the targeting agent is fused to the fusogen via a linker.
[0044] In some of any embodiments, the target molecule is ASCT2. In some of any embodiments, the target molecule is CD 117.
[0045] In some of any embodiments, the fusogen is a viral fusion protein. In some of any embodiments, the fusogen is a viral envelope protein.
[0046] In some of any embodiments, the fusogen is a VSV-G protein or a functional variant thereof.
[0047] In some of any embodiments, the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof. In some of any embodiments, the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild-type BaEV inhibitory R peptide. In some of any embodiments, the fusogen is set forth in any of SEQ ID NO:254-260. In some of any embodiments, the fusogen is set forth in SEQ ID NO:261 (BaEVRLess). In some of any embodiments, the fusogen is set forth in SEQ ID NO:262 (BaEVTR). Provided herein is a nucleic acid sequence encoding a fusogen, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof. [0048] In some of any embodiments, the fusogen is a Cocal virus G protein or a functional variant thereof.
[0049] In some of any embodiments, the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
[0050] In some of any embodiments, the fusogen is a Paramyxovirus fusion protein (e.g., a Morbilli virus or Henipavirus) or a functional variant thereof. In some of any embodiments, the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbilli virus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof. In some of any embodiments, the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof. In some of any embodiments, the fusogen is a Nipah virus fusion protein or a functional variant thereof. In some of any embodiments, the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
[0051] In some embodiments, the targeting agent is fused to the NiV-G protein or biologically active portion thereof. In some of any embodiments, the targeting agent is fused to the C-terminus of the NiV-G protein or biologically active portion thereof.
[0052] In some of any embodiments, the NiV-G or biologically active portion thereof is a wildtype NiV-G protein or a functionally active variant or biologically active portion thereof. In some of any embodiments, the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof does not include an initial methionine. In some of any embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
[0053] In some of any embodiments, the fusogen comprises one or modifications to reduce binding to its native receptor.
[0054] In some of any embodiments, the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3. In some of any embodiments, the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
[0055] In some of any embodiments, the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof. In some of any embodiments, the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C- terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
[0056] In some of any embodiments, the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
[0057] In some of any embodiments, the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule. In some of any embodiments, the targeting agent is a single domain antibody that binds to the target molecule. In some of any embodiments, the targeting agent is a VHH that binds to the target molecule. In some of any embodiments, the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
[0058] In some of any embodiments, the polynucleotide is codon optimized.
[0059] In some of any embodiments, the polynucleotide comprises at least one promoter that is operatively linked to control expression of the targeting agent and fusogen. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter.
[0060] Provided herein in some embodiments is a plasmid, comprising the polynucleotide of some of any embodiments. In some embodiments, the plasmid comprises one or more nucleic acids encoding proteins for lentivirus production.
[0061] Provided herein in some embodiments is a vector, comprising the polynucleotide of any of some of any embodiments.
[0062] Provided herein in some embodiments is a cell comprising the polynucleotide of some of any embodiments, the plasmid of some of any embodiments, or the vector of some of any embodiments. Also provided here is a cell comprising (i) a first polynucleotide encoding a nucleic acid sequence encoding a fusogen, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
[0063] Provided herein in some embodiments is a cell comprising (i) a first polynucleotide containing a nucleic acid sequence encoding a fusogen and (ii) a second polynucleotide containing a nucleic acid sequence encoding a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3.
[0064] In some embodiments, the second polynucleotide encodes a transmembrane domain that is fused to the targeting agent.
[0065] In some of any embodiments, the first and/or second polynucleotide is a plasmid. In some embodiments, the plasmid of the first and/or second polynucleotide comprises one or more nucleic acids encoding proteins for lentivirus production.
[0066] In some of any embodiments, the first and/or second polynucleotide is a vector.
[0067] In some of any embodiments, the first and/or second polynucleotide is codon optimized.
[0068] In some of any embodiments, the first polynucleotide comprises at least one promoter that is operatively linked to control expression of the fusogen; and/or the second polynucleotide comprises at least one promoter that is operatively linked to control expression of the targeting agent. In some embodiments, the promoter of the first and/or second polynucleotide is a constitutive promoter. In some embodiments, the promoter of the first and/or second polynucleotide is an inducible promoter.
[0069] In some of any embodiments, the cell is a producer cell used for producing a viral particle or virus-like particle. In some embodiments, the viral particle or virus-like particle is a retroviral particle or retrovirus-like particle. In some of any embodiments, the viral particle or virus-like particle is a lentiviral particle or lentivirus-like particle. In some of any embodiments, the cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells. In some of any embodiments, the cell is a 293T cell.
[0070] In some of any embodiments, the cell comprises a viral nucleic acid. In some embodiments, the viral nucleic acid is a retroviral nucleic acid. In some of any embodiments, the viral nucleic acid is a lentiviral nucleic acid. In some of any embodiments, the viral nucleic acid lacks one or more genes involved in viral replication. In some of any embodiments, the viral nucleic acid comprises a nucleic acid sequence encoding a viral packaging protein selected from one or more of Gag, Pol, and Rev. In some of any embodiments, the viral nucleic acid comprises one or more, optionally all, of the following nucleic acid sequences: 5’ LTR (e.g., containing U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT)/central termination sequence (CTS) (e.g., DNA flap), Poly A tail sequence, a posttranscriptional regulatory element (e.g., WPRE), a Rev response element (RRE), and 3’ LTR (e.g., containing U5 and lacking a functional U3).
[0071] In some of any embodiments, the target molecule is ASCT2. In some of any embodiments, the target molecule is CD 117.
[0072] In some of any embodiments, the fusogen is a viral fusion protein. In some of any embodiments, the fusogen is a viral envelope protein.
[0073] In some of any embodiments, the fusogen is a VSV-G protein or a functional variant thereof.
[0074] In some of any embodiments, the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof. In some of any embodiments, the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild-type BaEV inhibitory R peptide. In some of any embodiments the fusogen is set forth in any of SEQ ID NO:254-260. In some of any embodiments, the fusogen is set forth in SEQ ID NO:261 (BaEVRLess). In some of any embodiments, the fusogen is set forth in SEQ ID NO:262 (BaEVTR).
[0075] In some of any embodiments, the fusogen is a Cocal virus G protein or a functional variant thereof.
[0076] In some embodiments, the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
[0077] In some of any embodimtents, the fusogen is a Paramyxovirus fusion protein (e.g., a Morbilli virus or Henipavirus) or a functional variant thereof. In some of any embodiments, the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbilli virus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof. In some of any embodiments, the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof. In some of any embodiments, the fusogen is a Nipah virus fusion protein or a functional variant thereof. In some of any embodiments, the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
[0078] In some embodiments, the NiV-G or biologically active portion thereof is a wild-type NiV- G protein or a functionally active variant or biologically active portion thereof. In some of any embodiments, the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof does not include an initial methionine. In some of any embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
[0079] In some of any embodiments, the fusogen comprises one or more modifications to reduce binding to its native receptor.
[0080] In some of any embodiments, the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3. In some of any embodiments, the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof comprises the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:18.
[0081] In some of any embodiments, the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof. In some of any embodiments, the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C- terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
[0082] In some of any embodiments, the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
[0083] In some of any embodiments, the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule. In some of any embodiments, the targeting agent is a single domain antibody that binds to the target molecule. In some of any embodiments, the targeting agent is a VHH that binds to the target molecule. In some of any embodiments, the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
[0084] Provided herein in some embodiments is a method of making a targeted lipid particle, comprising: (a) introducing into a source cell the polynucleotide of any of some of any embodiments, the plasmid of some of any embodiments or the vector of some of any embodiments; (b) culturing the source cell under conditions for producing a lipid particle containing a lipid bilayer and the targeting agent and fusogen exposed on the surface of the lipid bilayer; and (c) separating, enriching, or purifying the lipid particle from the source cell, thereby making the lipid particle.
[0085] Provided herein in some embodiments is a method of making a targeted lipid particle, comprising: (a) introducing into a source cell (i) a first polynucleotide containing a nucleic acid sequence encoding a fusogen and (ii) a second polynucleotide containing a nucleic acid sequence encoding a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3; (b) culturing the source cell under conditions for producing a lipid particle containing a lipid bilayer and the targeting agent and fusogen exposed on the surface of the lipid bilayer; and (c) separating, enriching, or purifying the lipid particle from the source cell, thereby making the lipid particle.
[0086] In some embodiments, the second polynucleotide encodes a transmembrane domain that is fused to the targeting agent.
[0087] In some of any embodiments, the first and/or second polynucleotide is a plasmid. In some embodiments, the plasmid of the first and/or second polynucleotide comprises one or more nucleic acids encoding proteins for lentivirus production.
[0088] In some of any embodiments, the first and/or second polynucleotide is a vector.
[0089] In some of any embodiments, the first and/or second polynucleotide is codon optimized.
[0090] In some of any embodiments, the first polynucleotide comprises at least one promoter that is operatively linked to control expression of the fusogen; and/or the second polynucleotide comprises at least one promoter that is operatively linked to control expression of the targeting agent. In some embodiments, the promoter of the first and/or second polynucleotide is a constitutive promoter. In some embodiments, the promoter of the first and/or second polynucleotide is an inducible promoter.
[0091] In some of any embodiments, the source cell is a mammalian cell. In some of any embodiments, the source cell is a producer cell used for producing a viral particle or virus-like particle. In some embodiments, the particle is a viral particle or virus-like particle. In some of any embodiments, the viral particle or virus-like particle is a retroviral particle or retrovirus-like particle. In some of any embodiments, the viral particle or virus-like particle is a lentivirus-like particle. In some of any embodiments, the viral particle or virus-like particle is a lentiviral particle. In some of any embodiments, the particle is pseudotyped with the fusogen. In some of any embodiments, the producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells. In some of any embodiments, the producer cell is a 293T cell.
[0092] In some of any embodiments, the method comprises transducing the source cell with packaging plasmids encoding a Gag-pol and Rev.
[0093] In some of any embodiments, the target molecule is ASCT2. In some of any embodiments, the target molecule is CD 117. [0094] In some of any embodiments, the fusogen is a viral fusion protein. In some of any embodiments, the fusogen is a viral envelope protein.
[0095] In some of any embodiments, the fusogen is a VSV-G protein or a functional variant thereof.
[0096] In some of any embodiments, the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof. In some of any embodiments, the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild-type BaEV inhibitory R peptide. In some of any embodiments, the fusogen is set forth in any of SEQ ID NO:254-260. In some of any embodiments, the fusogen is set forth in SEQ ID NO:261 (BaEVRLess). In some of any embodiments, the fusogen is set forth in SEQ ID NO:262 (BaEVTR).
[0097] In some of any embodiments, the fusogen is a Cocal virus G protein or a functional variant thereof.
[0098] In some of any embodiments, the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
[0099] In some of any embodiments, the fusogen is a Paramyxovirus fusion protein (e.g., a Morbilli virus or Henipavirus) or a functional variant thereof. In some of any embodiments, the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbilli virus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof. In some of any embodiments, the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof. In some of any embodiments, the fusogen is a Nipah virus fusion protein or a functional variant thereof. In some of any embodiments, the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
[0100] In some embodiments, the NiV-G or biologically active portion thereof is a wild-type NiV- G protein or a functionally active variant or biologically active portion thereof. In some of any embodiments, the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof does not include an initial methionine. In some of any embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
[0101] In some of any embodiments, the fusogen comprises one or modifications to reduce binding to its native receptor.
[0102] In some of any embodiments, the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3. In some of any embodiments, the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
[0103] In some of any embodiments, the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof. In some of any embodiments, the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C- terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21. [0104] In some of any embodiments, the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
[0105] In some of any embodiments, the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule. In some of any embodiments, the targeting agent is a single domain antibody that binds to the target molecule. In some of any embodiments, the targeting agent is a VHH that binds to the target molecule. In some of any embodiments, the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
[0106] Provided herein in some embodiments is a targeted lipid particle produced by the method of some of any embodiments.
[0107] Provided herein in some embodiments is a targeted lentiviral particle produced by the method of some of any embodiments.
[0108] Provided herein in some embodiments is a composition comprising a plurality of the lipid particle of some of any embodiments.
[0109] Provided herein in some embodiments is a composition comprising a plurality of the lentiviral particle of some of any embodiments.
[0110] In some of any embodiments, the composition further comprises a pharmaceutically acceptable excipient.
[0111] Provided herein in some embodiments is a method of transducing a target cell, the method containing contacting a target cell with the lentiviral particle of some of any embodiments or the composition of some of any embodiments.
[0112] In some embodiments, the particle comprises an exogenous agent, and the transduction introduces the exogenous agent into the target cell.
[0113] Provided herein in some embodiments is a method of delivering an exogenous agent to a target cell, the method containing contacting a target cell with the particle of some of any embodiments or the composition of some of any embodiments, the particle or plurality of particles containing an exogenous agent for delivery of the target cell.
[0114] In some of any embodiments, the contacting is in vitro or ex vivo.
[0115] In some of any embodiments, wherein the contacting is in vivo in a subject.
[0116] Provided herein in some embodiments is a method of delivering an exogenous agent to a target cell in a subject, which includes administering to a subject the particle of some of any embodiments or the composition of some of any embodiments, the particle or plurality of particles containing an exogenous agent for delivery to a target cell in the subject. [0117] In some of any embodiments, the exogenous agent is or encodes a therapeutic agent or a diagnostic agent. In some of any embodiments, the exogenous agent is for treating a disease or condition in the subject.
[0118] Provided herein in some embodiments is a method of treating a disease or condition in a subject, which includes administering to a subject the particle of some of any embodiments or the composition of some of any embodiments, the particle or plurality of particles containing an exogenous agent for treating a disease or condition in the subject.
[0119] In some of any embodiments, the method comprises administering to the subject one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood.
[0120] In some of any embodiments, the subject has previously been administered one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood.
[0121] Provided herein in some of any embodiments is a method of treating a disease or condition in a subject, which includes administering to a subject: (a) one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood; and (b) a lipid particle containing a lipid bilayer, a fusogen exposed on the surface of the lipid bilayer, and an exogenous agent for treating a disease or condition in the subject.
[0122] In some of any embodiments, the administration of at least one of the one or more agents that stimulate mobilization is initiated prior to the administration of the particle.
[0123] Provided herein in some of any embodiments is a method of treating a disease or condition in a subject, containing administering to a subject a lipid particle containing a lipid bilayer, a fusogen exposed on the surface of the lipid bilayer, and an exogenous agent for treating a disease or condition in the subject, wherein the subject has previously been administered one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood.
[0124] In some of any embodiments, the exogenous agent is for delivery to a target cell for treating the disease or condition in the subject.
[0125] In some of any embodiments, the particle is a viral particle or virus-like particle. In some of any embodiments, the lipid bilayer is derived from a membrane of a producer cell used for producing a viral particle or virus-like particle. In some of any embodiments, the particle is prepared by a method containing transducing a producer cell with plasmids encoding the fusogen and a Gag-pol and Rev. In some of any embodiments, the viral particle or virus-like particle is a retroviral particle or retrovirus-like particle. In some of any embodiments, the viral particle or virus-like particle is a lentivirus-like particle. In some of any embodiments, the viral particle or virus-like particle is a lentiviral particle.
[0126] In some of any embodiments, the particle is pseudotyped with the fusogen. [0127] In some of any embodiments, the producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In some of any embodiments, the producer cell is a 293T cell.
[0128] In some of any embodiments, the particle is replication defective.
[0129] In some of any embodiments, the particle comprises a viral nucleic acid. In some embodiments, the viral nucleic acid is a retroviral nucleic acid. In some of any embodiments, the viral nucleic acid is a lentiviral nucleic acid. In some of any embodiments, the viral nucleic acid comprises one or more, optionally all, of the following nucleic acid sequences: 5’ LTR (e.g., containing U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT)/central termination sequence (CTS) (e.g., DNA flap), Poly A tail sequence, a posttranscriptional regulatory element (e.g., WPRE), a Rev response element (RRE), and 3’ LTR (e.g., containing U5 and lacking a functional U3).
[0130] In some of any embodiments, the particle is devoid of viral genomic nucleic acids.
[0131] In some of any embodiments, the fusogen is a viral fusion protein. In some of any embodiments, the fusogen is a viral envelope protein.
[0132] In some of any embodiments, the fusogen is a VSV-G protein or a functional variant thereof.
[0133] In some of any embodiments, the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof. In some of any embodiments, the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild-type BaEV inhibitory R peptide. In some of any embodiments, the fusogen is set forth in any of SEQ ID NO:254-260. In some of any embodiments, the fusogen is set forth in SEQ ID NO:261 (BaEVRLess). In some of any embodiments, the fusogen is set forth in SEQ ID NO:262 (BaEVTR).
[0134] In some of any embodiments, the fusogen is a Cocal virus G protein or a functional variant thereof.
[0135] In some of any embodiments, the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
[0136] In some of any embodiments, the fusogen is a Paramyxovirus fusion protein (e.g., a Morbilli virus or Henipavirus) or a functional variant thereof. In some of any embodiments, the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbilli virus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof. In some of any embodiments, the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof. In some of any embodiments, the fusogen is a Nipah virus fusion protein or a functional variant thereof. In some of any embodiments, the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
[0137] In some embodiments, the NiV-G or biologically active portion thereof is a wild-type NiV- G protein or a functionally active variant or biologically active portion thereof. In some of any embodiments, the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof does not include an initial methionine. In some of any embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
[0138] In some of any embodiments, the fusogen comprises one or modifications to reduce binding to its native receptor.
[0139] In some of any embodiments, the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3. In some of any embodiments, the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4. In some of any embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17. In some of any embodiments, the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18. In some of any embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18. [0140] In some of any embodiments, the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof. In some of any embodiments, the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C- terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16. In some of any embodiments, the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21. In some of any embodiments, the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
[0141] In some of any embodiments, the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
[0142] In some of any embodiments, the bone marrow cells are CD34+. In some of any embodiments, the bone marrow cells are CD34+ progenitor cells. In some of any embodiments, the bone marrow cells are hematopoietic stem cells.
[0143] In some of any embodiments, the one or more agents that stimulate mobilization are selected from the group consisting of stem cell factor (SCF), small molecule VLA-4 inhibitor BI05192, BOP (N-(benzenesnlfonyl)-L-prolyl-L-0-(l-pyrrolidinylcarbonyl)tyrosine), heparin, granulocyte colonystimulating factor (G-CSF), MGTA-145, and plerixafor (AMD3100).
[0144] In some of any embodiments, the one or more agents that stimulate mobilization include G- CSF. In some embodiments, the G-CSF is administered to the subject daily on the two days, three days, four days, or five days prior to the administration of the first dose of the particle. In some of any embodiments, the G-CSF is administered to the subject on the day of the administration of the first dose of the particle. In some of any embodiments, the G-CSF is administered to the subject on the day of administration of a second or later dose of the particle.
[0145] In some of any embodiments, the one or more mobilization agents comprise plerixafor. In some of any embodiments, the plerixafor is administered to the subject on the day of the administration of the first dose of the particle. In some of any embodiments, the plerixafor is administered to the subject on the day of the administration of a second or later dose of the particle.
[0146] In some of any embodiments, the one or more agents that stimulate mobilization are G-CSF and plerixafor. In some embodiments, the G-CSF is administered to the subject daily on the four days prior to the administration of the first dose of the particle; and the plerixafor is administered to the subject on the day of the administration of the first dose of the particle. In some embodiments, the G-CSF is administered to the subject (i) daily on the two days prior to the administration of the particle; (ii) on the day of the administration of the first dose of the particle; and (iii) on the day of administration of a second or later dose of the particle; and the plerixafor is administered to the subject on the day of administration of a second or later dose of the particle.
[0147] In some of any embodiments, the method comprises administering to the subject a transduction adjuvant. In some of any embodiments, the transduction adjuvant is administered to the subject on the day of the administration of at least one dose of the particle. In some of any embodiments, the transduction adjuvant is administered to the subject on the days of the administration of at least two doses of the particle.
[0148] In some of any embodiments, the transduction adjuvant is a cationic peptide. In some embodiments, the transduction adjuvant is a histidine-rich cationic peptide. In some of any embodiments, the transduction adjuvant is a cationic amphipathic peptide. In some of any embodiments, the transduction adjuvant is derived from the LAH4 peptide family. In some of any embodiments, the transduction adjuvant is Vectofusin-1.
[0149] In some embodiments, the target molecule is expressed on the target cell. In some of any embodiments, the target cell is a hematopoietic cell. In some of any embodiments, the target cell is CD34+. In some of any embodiments, the target cell is a CD34+ progenitor cell. In some of any embodiments, the hematopoietic cell is a hematopoietic stem cell.
[0150] In some of any embodiments, the exogenous agent is present in the lumen. In some of any embodiments, the exogenous agent is a protein or a nucleic acid.
[0151] In some of any embodiments, the exogenous agent is or encodes a factor associated with gene editing. In some of any embodiments, the exogenous agent is or encodes a genome-modifying protein for gene editing a target gene encoding an endogenous protein. In some embodiments, the exogenous agent is a fusion protein between a viral structural protein and an exogenous agent. In some of any of the provided embodiments, the fusion protein is a cleavable fusion protein comprising a cleavable linker positioned between the viral structural protein and the exogenous agent. In some of any of the provided embodiments, the fusion protein comprises one or more nuclear localization sequences. In some of any of the provided embodiments, the fusion protein comprises one or more nuclear export sequences. [0152] In some embodiments, the genome-modifying protein is associated with gene editing by a sequence-specific nuclease, a CRISPR-associated transposase (CAST), prime editing, or Programmable Addition via Site-specific Targeting Elements (PASTE).
[0153] In some of any embodiments, the genome -modifying protein is a sequence-specific nuclease. In some of any embodiments, the sequence-specific nuclease is selected from the group consisting of an RNA-guided nuclease, a meganuclease, a transcription activator-like effector nuclease (TALEN), and a zinc-finger nuclease (ZFN). In some of any embodiments, the sequence-specific nuclease is an RNA-guided nuclease. In some of any embodiments, the RNA-guided nuclease is TnpB. In some of any embodiments, the RNA-guided nuclease comprises a Cas nuclease and a guide RNA (CRISPR-Cas combination). In some embodiments, the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex containing the gRNA and the Cas nuclease. In some of any embodiments, the Cas nuclease is a Type II or Type V Cas protein. In some of any embodiments, the genome-modifying protein is selected from the group consisting of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxll, Csyl, Csy2, Csy3, Mad7. In some of any embodiments, the Cas is a Cas9 or a Cas 12.
[0154] In some of any embodiments, the exogenous agent is or encodes a factor associated with base editing or prime editing (e.g., target-primed reverse transcription (TPRT)). In some of any embodiments, the exogenous agent is or encodes a transposase, integrase, or recombinase. In some of any embodiments, the exogenous agent is or encodes a DNA polymerase, RNA polymerase, or reversetranscriptase.
[0155] In some of any embodiments, the exogenous agent is for use in gene therapy to correct a genetic deficiency in the target cell. In some embodiments, the exogenous agent is a nucleic acid containing a payload gene for correcting the genetic deficiency.
[0156] In some of any embodiments, the exogenous agent is or encodes a membrane protein. In some embodiments, the membrane protein is an antigen receptor. In some embodiments, the antigen receptor binds to an antigen expressed on cells associated with a disease or condition in the subject. In some embodiments, the disease or condition is a cancer. In some of any embodiments, the antigen receptor binds to an antigen expressed on tumor cells in the subject. In some of any embodiments, the antigen receptor is a chimeric antigen receptor (CAR). In some of any embodiments, the antigen receptor is an engineered T cell receptor (TCR).
[0157] In some of any embodiments, the delivery to target cells expressing the target molecule is increased by or by greater than 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5- fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, or more compared to delivery to non-target cells not expressing the target molecule. In some of any embodiments, the subject is a human subject.
[0158] In some of any of the provided embodiments, the target molecule is CD 133. In some of any of the provided embodiments, the fusogen is fused to a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3. In some of any of the provided embodiments, the target molecule is ASCT2. In some of any of the provided embodiments, is CD117.
[0159] In some of any of the provided embodiments, the target molecule is CD 133. In some of any of the provided embodiments, the targeting agent is a CD 133 binding agent that is an antibody or antigen-binding fragment comprising a variable heavy (VH) chain and a variable light (VL) chain selected from a) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 271, 272, 273, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 280, 281, 282, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 284, 285, and 286, respectively; (c) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 298, 299, and 300, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively; (d) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 307, 308, and 309, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 311, 312, and 313, respectively; or (e) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 298, 290, and 291, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 293, 294, and 295, respectively. In some of any of the provided embodiments, the targeting agent is a CD 133 binding agent that is an antibody or antigen-binding fragment comprising a variable heavy (VH) chain and a variable light (VL) chain selected from a) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 314, 315, and 273, respectively, and a VL comprising a CDR- Ll, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 316, 317, and 282, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 284, 285, and 286, respectively; (c) a VH comprising a a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 318, 319, and 300, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR- L3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively; (d) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 320, 321, and 309, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 311, 312, and 313, respectively; and (e) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 322, 323, and 291, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 293, 294, and 295, respectively.
[0160] In some of any of the provided embodiments, the CD 133 binding agent is selected from a) a CD 133 binding agent comprising a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 270, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 274, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; b) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 279, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 283, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; c) a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 297, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 301, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; d) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 306, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 310, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; e) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 288, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 292, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
[0161] In some of any of the provided embodiments, the CD 133 binding agent is a scFv. In some of any of the provided embodiments, the CD 133 binding agent comprises the amino acid sequence set forth in any one of SEQ ID NOs: 269, 278, 287, 296 or 305, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. [0162] In some of any of the provided embodiments, the targeting agent is a CD117 binding agent that is a VHH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the CD117 binding agent comprises the amino acid sequence set froth in any one of SEQ ID NOS: 324-374.
[0163] Provided herein is an anti-CDl 17 binding agent comprising a VHH single domain antibody comprising a CDR-H1, a CDR-H2, and a CDR-H3 contained within an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374.
[0164] Provided herein is an anti-CDl 17 binding agent comprising a VHH single domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
[0165] In some of any of the provided embodiments, the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOS: 324-374.
[0166] Provided herein is a viral fusion protein comprising a viral envelope protein and the antiCD 117 binding agent.
[0167] Provided herein is a viral fusion protein comprising a viral envelope protein and an antiCD 133 binding agent, wherein the anti-CDl 33 binding agent is an antibody or antigen-binding fragment comprising a variable heavy (VH) chain and a variable light (VL) chain selected from a) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 314, 315, and 273, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 316, 317, and 282, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 284, 285, and 286, respectively; (c) a VH comprising a a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 318, 319, and 300, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively; (d) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 320, 321, and 309, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 311, 312, and 313, respectively; and (e) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 322, 323, and 291, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 293, 294, and 295, respectively. [0168] In some of any of the provided embodiments, the CD 133 binding agent is selected from a) a CD 133 binding agent comprising a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 270, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 274, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; b) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 279, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 283, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; c) a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 297, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 301, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; d) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 306, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 310, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; e) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 288, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 292, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
[0169] In some of any of the provided embodiments, the CD 133 binding agent is a scFv. In some of any of the provided embodiments, the CD 133 binding agent comprises the amino acid sequence set forth in any one of SEQ ID NOs: 269, 278, 287, 296 or 305, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the viral envelope protein is a VSV-G protein or a functional variant thereof. In some of any of the provided embodiments, the viral envelope protein is a Cocal virus G protein or a functional variant thereof. In some of any of the provided embodiments, the viral envelope protein is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof. In some of any of the provided embodiments, the viral envelope protein is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof. In some of any of the provided embodiments, the viral envelope protein is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof. In some of any of the provided embodiments, the viral envelope protein is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
[0170] In some of any of the provided embodiments, the viral envelope protein is a Nipah virus fusion protein or a functional variant thereof. In some of any of the provided embodiments, the viral envelope protein comprises a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof. In some of any of the provided embodiments, the binding agent is fused to the C-terminus of the NiV-G protein or biologically active portion thereof. In some of any of the provided embodiments, the NiV-G or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof does not include an initial methionine. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 42. In some of any of the provided embodiments, the viral envelope protein comprises one or more modifications to reduce binding to its native receptor. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17. In some of any of the provided embodiments, the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18. In some of any of the provided embodiments, the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18. Brief Description of The Drawings
[0171] FIG. 1A depicts a schematic of experiments performed to assess the capacity of VSV-G and BaEVTR vectors to transduce human hematopoietic stem cells (HSCs), blood lineage cells, and hepatocytes in vitro.
[0172] FIG. IB shows results for transduction of hematopoietic cells with VSV-G and BaEVTR lentiviral vectors.
[0173] FIG. 1C shows results of transduction of hematopoietic stem cells with increasing dilutions with a BaEVTR or VSV-G lentiviral vector.
[0174] FIG. ID shows additional results of transduction of hematopoietic stem cells with increasing dilutions with a BaEVTR or VSV-G lentiviral vector.
[0175] FIG. IE shows results of transduction of hematopoietic stem cells with two different Multiplicity of infection values of BaEVTR vector.
[0176] FIG. 2 shows results for the ex vivo transduction of hematopoietic cells and subsequent transgene engraftment in a mouse model.
[0177] FIG. 3 shows the phenotypic distribution of hematopoietic cells following humanization of two mouse models.
[0178] FIG.4A shows an exemplary humanization protocol for mouse experiments described herein in Example 4. FIG. 4B shows distribution of hematopoietic cells following early-stage humanization of a mouse models.
[0179] FIG. 5A shows the effect of dose schedules on transduction of hematopoietic cells with a BaEVTR vector or a VSV-G vector.
[0180] FIG.5B shows the effect of mobilization on transduction of hematopoietic cells with a BaEVTR vector.
[0181] FIG. 6A shows the effect of mobilization on in vivo distribution of hematopoietic cells following early-stage humanization.
[0182] FIG.6B shows additional results of the effect of mobilization on in vivo distribution of hematopoietic cells following early-stage humanization.
[0183] FIG. 6C shows the effect of mobilization on transduction ofhematopoietic cells with a BaEVTR vector in early-stage humanized mice.
[0184] FIG.6D shows additional results of the effect of mobilization on transduction of hematopoietic cells with a BaEVTR vector in early-stage humanized mice.
[0185] FIG. 7A shows the effect of co-infusion of BaEVTR vectors and cord-blood CD34+ cells on bone marrow transduction. FIG. 7B depicts the frequency of various cell types in a huCD34+ cell population. [0186] FIG.8A shows the effect of co-infusion of BaEVTR vectors and cord-blood CD34+ cells on myeloid cell expression.
[0187] FIG.8B shows the effect of co-infusion of BaEVTR vectors blood CD34+ cells on stable transduction of hematopoietic cells after 16 weeks.
[0188] FIG.8C shows additional results of the the effect of co-infusion of BaEVTR vectors and cord-blood CD34+ cells on hematopoietic cell and progenitor cells sub-populations.
[0189] FIG. 9 shows the effect of different administration schedules on transduction of hematopoietic cells with VSV-G and BaEVTR vectors.
[0190] FIG. 10A shows results for transduction of hematopoietic cells with VSV-G and BaEVTR vectors and retargeted Nipah vectors.
[0191] FIG. 10B shows additional results for transduction of hematopoietic cells with VSV-G and BaEVTR vectors and retargeted Nipah vectors.
[0192] FIG. 11 shows the phenotypic distribution of hematopoietic cells transduced with BaEVTR vectors and retargeted Nipah vectors.
[0193] FIG. 12 shows an additional phenotypic distribution of hematopoietic cells transduced with BaEVTR vectors and retargeted Nipah vectors.
[0194] FIG. 13 shows the phenotypic distribution of peripheral blood hematopoietic cells following humanization.
[0195] FIG. 14 shows the results for transduction of hematopoietic cells with BaEVTR virus-like particles.
[0196] FIG. 15A shows a representative flow plot of B2M gene editing in human hematopoietic cells with the BaEVTR virus-like particles.
[0197] FIG. 15B shows the results for transduction of hematopoietic cells with BaEVTR virus-like particles in three humanized mouse models.
[0198] FIG. 16A depicts percentage of cells which were subjected to gene editing in vitro as a function of increasing dose of BaEVTR virus-like particles carrying a gene editing cargo. Percent edited cells are similarly shown for in vivo gene editing of cells of the bone marrow and Lin-/CD34+ progenitor cells in FIG. 16B.
[0199] FIG. 16 C shows , the in vivo knockout of B2M gene (e.g., B2M editing) in different HSPC subpopulations. FIG. 16D shows an exemplary long-term humanization protocol for mice experiments herein in Example 12. FIG. 16E shows the level of editing in long-term humanized mice. FIG. 16F shows true in vivo B2M editing in huCD45+ cells.
[0200] FIG. 17A shows an exemplary study design for an experiment described in Example 13. FIG. 17B shows in vivo cell transduction of a GFP transgene.
[0201] A model study design for non-human primates is shown in FIG. 18. Detailed Description
[0202] Provided herein in some embodiments are lipid particles and methods of delivering the same to a target cell. In some embodiments, the target cell is a hematopoietic stem/progenitor cell (HSPC), such as a CD34+ cell. In some embodiments, the lipid particle is a targeted lipid particle that preferentially targets a target molecule expressed on the target cell. Provided herein in some embodiments are targeted lipid particles having a targeting agent that binds to a target molecule. In some embodiments, the target molecule is expressed on a target cell. In some embodiments, the provided particle preferentially targets a target cell compared to a non-target cell. Among provided embodiments, the lipid particles allow for targeted delivery to hematopoietic stem/progenitor cells (HSPC), including to naive hematopoietic cells (HSCs).
[0203] Exogenous agent delivery that is preferentially targeted to certain target cells, such as for the preferential genetic engineering of the target cells, can result in long-term and/or systemic genetic engineering of the target cells and/or cells derived therefrom, for instance when engineered target cells are able to differentiate into a variety of cell types. As an example, CD34+ progenitor cells, which are a heterogeneous cell population that include a subpopulation of hematopoietic stem cells (HSCs), pluripotent stem cells, and cells in the early stages of lineage commitment, can differentiate to produce all hematopoietic cell types found in circulation. While CD34 is expressed in a diversity of hematopoietic progenitor cells at various stages of differentiation across the hematopoietic tree, CD34+ progenitor cells that are HSCs or multipotent progenitors (MPPs) at the top of the hematopoietic tree may be the most appealing cell targets for achieving long-term and/or systemic genetic engineering across a broad range of hematopoietic cell types. Yet other appealing cell targets for genetic engineering among CD34+ progenitor cells include myeloid progenitors and lymphoid progenitors, such as myelo-lymphoid progenitors (MLPs), early T progenitors (ETPs), megakaryo-erythroid progenitors (MEPs), common myeloid progenitors (CMPs), and granulo-monocyte progenitors (GMPs). There is a need for reagents and methods for targeted delivery of exogenous agents to highly pluripotent subpopulations of hematopoietic cells, such as to CD34+ progenitor cells, including HSCs and MPPs. Achieving in vivo genetic engineering of hematopoietic stem/progenitor cells (HSPC) would dramatically expand patients’ access to gene therapy.
[0204] The provided disclosure addresses this need. The embodiments herein provide for lipid particles that allow for efficient targeting and delivery of an exogenous agent contained therein to human HSPC. In provided embodiments, the lipid particles can include viral vectors such as lentiviral vectors that are pseudotyped with a fusogen with a preferential ability to target HSPC. In some embodiments, the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof, such as the exemplarly fusogens BaEVTR or BaEVRLess. In some embodiments, the lipid particle includes a targeting agent to target the fusogen to HSPCs, such as to CD34+ cells. In some embodiments, the targeting agent is fused to a fusogen, such as a viral envelope attachment glycoprotein, exposed on the lipid bilayer to provide a retargeted fusogen. Results herein demonstrate that such lipid particles can efficiently target HSPC, including naive HSC. Notably, results show that the lipid particles can be delivered in vivo not only to cells in the peripheral blood but that they also can establish basal accesss to human HPSC in the bone marrow (BM) and peripheral blood without relying on high vector doses or selective enrichment. The provided delivery methods also can be combined with mobilization agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood in the subject. Results herein show that improved transduction of cells in peripheral blood can be achieved when combined with mobilization. Moreover, combined mobilization strategies also allow transduction to naive HSC in the bone marrow even where there is not substantial egress of human HSPC into the peripheral blood. For instance, examples herein show that embodiments using a provided lipid particle able to target HSPC (e.g. BaEVTR fusogen) allow about 2% targeting of phenotypic HSC compared to undetectable transduction without mobilization.
[0205] In some embodiments, the fusogen of a provided lipid particle is not fused to a targeting agent. For instance, observations herein indicate that a lipid particle (e.g. lentiviral vector) pseudotyped with a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof, such as the exemplarly fusogens BaEVTR, exhibits highly efficient targeting of HSPC. In some embodiments, such lipid particles can additionally include a targeting agent.
[0206] In some embodiments, the fusogen of a provided lipid particle is a retargeted fusogen in which a viral envelope attachment glycoprotein is linked or fused to a targeted agent. In such embodiments, a retargeted fusogen is engineered to recognize receptors on chosen target cell types resulting in highly cell-specific in vivo gene transfer. For example, provided embodiments relate to lipid particles (e.g. lentiviral vector) pseudotyped with a retargeted fusogenof a parmamyxovirus envelope attachment glycoprotein, such as a Nipah G protein or functional variant thereof, linked or fused with a targeting agent. In some aspects, the use of a retargeted fusogen can reduce off-target delivery. In some embodiments, a retargeted fusogen as shown herein can exhibit striking (e.g. lOOx) specificity increase over other broadly tropic pseudotyped lipid particles in targeting a receptor-positive population, including in target cells exclusively localized in the bone marrow even where such cells make up as little as 0.15% of total human cells.
[0207] In some embodiments, the target molecule bound by the targeting agent of the provided particle is expressed on CD34+ progenitor cells. In some embodiments, the target cell is a CD34+ progenitor cell. In some embodiments, the target molecule is expressed on all CD34+ progenitor cells. In some embodiments, the target molecule is expressed on at least a subset of CD34+ progenitor cells. In some embodiments, the target molecule is expressed on HSCs. In some embodiments, the target molecule is expressed on MPPs. [0208] In some embodiments, the target molecule is selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3. As demonstrated herein, preferential genetic engineering, e.g., transduction, of CD34+ progenitor cells, e.g., subpopulations thereof that included highly pluripotent HSCs and MPPs, as well as myeloid progenitors that included MEPs, CMPs, and GMPs, was achieved with exemplary targeted lipid particles, e.g., targeted lentiviral particles, having a targeting agent that binds to a target molecule selected from the foregoing, e.g., CD117. Also demonstrated herein are such effects for lymphoid progenitors, such as MLPs and ETPs, using exemplary targeted lipid particles, e.g., targeted lentiviral particles, having a targeting agent that binds to a target molecule selected from the foregoing, e.g., ASCT2.
[0209] In some embodiments, the provided particle is any described in Section II. In some embodiments, the provided particle is a lipid particle, such as any described in Section II-A. In some embodiments, the provided particle is a lipid particle that is a viral particle, such as any described in Section II-A-1. In some embodiments, the provided particle is a targeted lentiviral particle. In some embodiments, the provided particle is a lipid particle that is a virus-like particle, such as any described in Section II-A-2. In some embodiments, the provided particle is a lipid particle that is a cell-based particle, such as any described in Section II-A-3.
[0210] In some embodiments, the provided particle has a fusogen, such as any described in Section II-B. In some embodiments, the fusogen contains a mammalian protein, such as any described in Section II-B-1. In some embodiments, the fusogen contains a viral protein, such as any described in Section II-B- 2. In particular embodiments, the fusogen is a baboon endogenous retrovirus, such as BaEVTR.In some embodiments, the targeting agent of the provided particle is any described in Section II-C. In some embodiments, the target molecule or target cell for any of the provided particles is any described in Section II-C.
[0211] In some embodiments, the provided particle contains an exogenous agent. In some embodiments, the exogenous agent is for delivery to the target cell. In some embodiments, the provided particle exhibits preferential delivery of the exogenous agent to a target cell compared to a non-target cell. In some embodiments, the delivery to target cells is increased by or by greater than 1.1 -fold, 1.2- fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4- fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, or more compared to delivery to non-target cells.
[0212] In some embodiments, the exogenous agent of the provided particle is any described in Section II-D. In some embodiments, the exogenous agent is a nucleic acid, such as any described in Section II-D-1. In some embodiments, the nucleic acid contains a payload gene encoding a payload agent, for instance a protein, such as any described in Section II-D-2. In some embodiments, the exogenous agent is a protein, such as any described in Section II-D-2. In some embodiments, the protein is a payload agent. In some embodiments, the exogenous agent is a small molecule, such as any described in Section II-D-3.
[0213] In some embodiments, the pay load agent encoded or contained by the exogenous agent is any described in Section III. In some embodiments, the exogenous agent is or encodes an engineered receptor, such as any described in Section III-A. In some embodiments, the exogenous agent is or encodes a gene-editing agent, such as any described in Section III-B.
[0214] Also provided herein in some embodiments are polynucleotides, such as any described in Section IV-A, encoding the fusogen and targeting agent of any of the provided particles. Also provided herein in some embodiments are cells, such as any described in Section IV-B, containing polynucleotides encoding one or both of the fusogen and targeting agent of any of the provided particles, for instance any of the provided polynucleotides, such as any described in Section IV-A. Also provided herein in some embodiments are methods, such as any described in Section IV-B, of making a targeted lipid particle, such as any described in Section II, using polynucleotides encoding one or both of the fusogen and targeting agent of any of the provided particles, for instance any of the provided polynucleotides, such as any described in Section IV-A. In some embodiments, the provided methods are for making any of the provided particles containing an exogenous agent, such as any described in Section II-D.
[0215] Also provided herein in some embodiments are compositions, such as any described in Section V, containing a plurality of any of the provided particles. In some embodiments, the provided composition contains a plurality of any of the provided lenti viral particles.
[0216] Also provided herein in some embodiments are methods, such as any described in Section VI, of using any of the provided particles. In some embodiments, the provided method involves administering any of the provided particles or compositions to a subject. In some embodiments, the provided method involves administering to the subject an agent that stimulates mobilization of bone marrow cells from the bone marrow to the peripheral blood in the subject. In some embodiments, the bone marrow cells are CD34+ progenitor cells. As demonstrated herein using the provided targeted particles, e.g., ASCT2- and CD117-targeted lentiviral particles, the mobilization of bone marrow cells to the peripheral blood prior to the in vivo genetic engineering, e.g., transduction, of cells improved the transduction of CD34+ progenitor cells, including HSCs and MPPs, following intravenous administration of the particles. Such effects are also demonstrated herein using non-targeted particles, e.g., not containing a targeting agent, such as those with fusogens having natural tropism for bone marrow cells like CD34+ progenitor cells, e.g., BaEV envelope glycoproteins. During steady state, CD34+ progenitors including HSCs reside in specialized niches within the bone marrow (Hoggatt et al. Stem Cell Res Ther. 2011 Mar 14;2(2): 13). The mobilization of bone marrow cells may increase the number of CD34+ progenitor cells in the peripheral blood that are accessible to the intravenously administered particles, thereby improving transduction. [0217] Also provided herein in some embodiments are methods involving administering to a subject (i) an agent that stimulates mobilization of bone marrow cells from the bone marrow to the peripheral blood in the subject and (ii) a particle, such as any described in Section II, either with or without any of the targeting agents described herein. In some embodiments, the method is any described in Section VI.
[0218] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.
[0219] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
I. DEFINITIONS
[0220] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
[0221] As used herein, the articles “a” and “an” refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
[0222] As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein, “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
[0223] By “wild type” or “WT” or “native” herein is meant an amino acid sequence that is found in nature, including allelic variations. A wild type protein or polypeptide has an amino acid sequence that has not been intentionally modified.
[0224] As used herein, “lipid particle” refers to any biological or synthetic particle that contains a bilayer of amphipathic lipids enclosing a lumen or cavity. Typically, a lipid particle does not contain a nucleus. Examples of lipid particles include solid particles such as nanoparticles, viral-derived particles, and cell-derived particles. Exemplary lipid particles also include viral-based particles, such as virus-like particles or viral particles (e.g., lentiviral particles), exosomes, enucleated cells, various vesicles, such as a microvesicle, a membrane vesicle, an extracellular membrane vesicle, a plasma membrane vesicle, a giant plasma membrane vesicle, an apoptotic body, a mitoparticle, a pyrenocyte, or a lysosome. In some embodiments, a lipid particle can be a fusosome. In some embodiments, the lipid particle is not a platelet. In some embodiments, the lipid particle is derived from a source cell. A lipid particle also may include an exogenous agent, which may be present in the lumen of the lipid particle.
[0225] The term “viral-based particle” can be any type of lipid particle that is derived from a virus or from viral protein, for example viral particles and virus-like particles.
[0226] The terms “viral vector”, “viral particle”, and “viral vector particle” are used interchangeably herein. A viral particle can be any type of lipid particle which comprises one or more viral structural proteins in addition to at least one non-structural viral genomic component or functional fragment thereof (e.g., a polymerase, an integrase, a protease or other non-structural component).
[0227] The term “virus-like particle” or VLP can be any type of particle that features at least one viral structural protein and is devoid of viral genetic material.
[0228] The term “retroviral particle” refers to a viral particle that contains retroviral nucleic acid or is derived from a retrovirus. A retroviral particle includes the following components: a vector genome (retrovirus nucleic acid), a nucleocapsid encapsidating the nucleic acid, and a membrane envelope surrounding the nucleocapsid. Typically, a retroviral particle contains sufficient retroviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle capable of infecting a target cell. Infection of the target cell may include reverse transcription and integration into the target cell genome. A retroviral particle may be a recombinant retroviral particle that is replication defective and lacks genes essential for replication, such as a functional gag-pol and/or env gene and/or other genes essential for replication. A retroviral particle also may be a self-inactivating (SIN) vector.
[0229] As used herein, a “lentiviral particle” or LV refers to a viral particle that contains lentiviral nucleic acid or is derived from a lentivirus. A lentiviral particle includes the following components: a vector genome (lentivirus nucleic acid), a nucleocapsid encapsidating the nucleic acid, and a membrane stirrounding the nucleocapsid. Typically, a lentiviral particle contains sufficient lentiviral genetic information to allow packaging of an RN genome, in the presence of packaging components, Into a viral particle capable of infecting a target cell. Infection of the target cell may include reverse transcription and integration into the target cell genome. A lentiviral particle may be a recombinant lentiviral particle that is replication defective and lacks genes essential for replication, such as a functional gag-pol and/or env gene and/or other genes essential for replication. A lentiviral particle also may be a self-inactivating (SIN) vector.
[0230] As used herein, a “retroviral nucleic acid,” refers to a nucleic acid containing at least the minimal sequence requirements for packaging into a retroviral particle, alone or in combination with a helper cell, helper virus, or helper plasmid. In the case of “lentiviral nucleic acid” the nucleic acid refers to at least the minimal sequence requirements for packaging into a lentiviral particle, alone or in combination with a helper cell, helper virus, or helper plasmid. In some embodiments, the viral nucleic acid comprises one or more of (e.g., all of) a 5’ LTR (e.g., to promote integration), U3 (e.g., to activate viral genomic RNA transcription), R (e.g., a Tat-binding region), U5, a 3’ LTR (e.g., to promote integration), a packaging site (e.g., psi ( )), RRE (e.g., to bind to Rev and promote nuclear export). The viral nucleic acid can comprise RNA (e.g., when part of a virion) or DNA (e.g., when being introduced into a source cell or after reverse transcription in a recipient cell). In some embodiments, the viral nucleic acid is packaged using a helper cell, helper virus, or helper plasmid which comprises one or more of (e.g., all of) gag, pol, and env.
[0231] As used herein, “fusosome” refers to a particle containing a bilayer of amphipathic lipids enclosing a lumen or cavity and a fusogen that interacts with the amphipathic lipid bilayer. In some embodiments, the fusosome comprises an exogenous agent. In some embodiments, the exogenous agent is a nucleic acid (e.g., DNA or RNA), a peptide, or a protein. In some embodiments, the fusosome is a membrane enclosed preparation. In some embodiments, the fusosome is derived from a source cell.
[0232] As used herein, “fusosome composition” refers to a composition comprising one or more fusosomes.
[0233] As used herein, “fusogen” refers to an agent or molecule that creates an interaction between two membranes, including membrane enclosed lumens. In embodiments, the fusogen facilitates fusion of the membranes. In other embodiments, the fusogen creates a connection, e.g., a pore, between two membranes or lumens (e.g., a lumen of a retroviral particle and a cytoplasm of a target cell). In some embodiments, the fusogen comprises a complex of two or more proteins, e.g., wherein neither protein has fusogenic activity alone. In some embodiments, the fusogen comprises a targeting domain.
[0234] As used herein, a “re-targeted fusogen” refers to a fusogen that comprises a targeting moiety having a sequence that is not part of the naturally-occurring form of the fusogen. In embodiments, the fusogen comprises a different targeting agent relative to the targeting agent in the naturally-occurring form of the fusogen. In embodiments, the naturally-occurring form of the fusogen lacks a targeting domain, and the re-targeted fusogen comprises a targeting agent that is absent from the naturally- occurring form of the fusogen. In embodiments, the fusogen is modified to comprise a targeting agent. In embodiments, the fusogen comprises one or more sequence alterations outside of the targeting agent relative to the naturally-occurring form of the fusogen, e.g., in a transmembrane domain, fusogenically active domain, or cytoplasmic domain.
[0235] As used herein, a “target cell” refers to a cell of a type to which it is desired that a targeted lipid particle or viral vector delivers an exogenous agent. In embodiments, a target cell is a cell of a specific tissue type or class. In some embodiments, the targeting agent or fusogen, e.g., re-targeted fusogen, leads to preferential delivery of the exogenous agent to a target cell compared to a non-target cell. [0236] As used herein a “non-target cell” refers to a cell of a type to which it is not desired that a targeted lipid particle or viral particle delivers an exogenous agent. In some embodiments, a non-target cell is a cell of a specific tissue type or class. In some embodiments, the targeting agent or fusogen, e.g., re-targeted fusogen leads to lower delivery of the exogenous agent to a non-target cell compared to a target cell.
[0237] As used herein a “biologically active portion,” such as with reference to a protein such as a G protein or an F protein, refers to a portion of the protein that exhibits or retains an activity or property of the full-length of the protein. For example, a biologically active portion of an F protein retains fusogenic activity in conjunction with the G protein when each are embedded in a lipid bilayer. A biologically active portion of the G protein retains fusogenic activity in conjunction with an F protein when each is embedded in a lipid bilayer. The retained activity can include 10%-150% or more of the activity of a full-length or wild-type F protein or G protein. Examples of biologically active portions of F and G proteins include proteins with truncations of the cytoplasmic domain, such as any of the described NiV-F with a truncated cytoplasmic tail.
[0238] As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
[0239] An amino acid substitution may include but are not limited to the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 19. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, for example, retained/improved binding.
Table 19
[0240] Amino acids may be grouped according to common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
[0241] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
[0242] The term, “corresponding to” with reference to positions of a protein, such as recitation that nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions in a disclosed sequence, such as set forth in the Sequence listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. For example, corresponding residues of a similar sequence (e.g. fragment or species variant) can be determined by alignment to a reference sequence by structural alignment methods. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.
[0243] The term “isolated” as used herein refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or produced. For example, a polypeptide is referred to as “isolated” when it is separated from at least some of the components of the cell in which it was produced. Where a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be “isolating” the polypeptide. Similarly, a polynucleotide is referred to as “isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as “isolated”.
[0244] The term “effective amount” as used herein means an amount of a pharmaceutical composition which is sufficient enough to significantly and positively modify the symptoms and/or conditions to be treated (e.g., provide a positive clinical response). The effective amount of an active ingredient for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically-acceptable excipient(s) and/or carrier(s) utilized, and like factors with the knowledge and expertise of the attending physician.
[0245] An “exogenous agent” as used herein with reference to a viral particle refers to an agent that is neither comprised by nor encoded in the corresponding wild-type virus or fusogen made from a corresponding wild-type source cell. In some embodiments, the exogenous agent does not naturally exist, such as a protein or nucleic acid that has a sequence that is altered (e.g., by insertion, deletion, or substitution) relative to a naturally occurring protein. In some embodiments, the exogenous agent does not naturally exist in the source cell. In some embodiments, the exogenous agent exists naturally in the source cell but is exogenous to the virus. In some embodiments, the exogenous agent does not naturally exist in the recipient cell. In some embodiments, the exogenous agent exists naturally in the recipient cell, but is not present at a desired level or at a desired time. In some embodiments, the exogenous agent comprises nucleic acids or protein.
[0246] As used herein, a “promoter” refers to a cis- regulatory DNA sequence that, when operably linked to a gene coding sequence, drives transcription of the gene. The promoter may comprise a transcription factor binding sites. In some embodiments, a promoter works in concert with one or more enhancers which are distal to the gene.
[0247] As used herein, “operably linked” or “operably associated” includes reference to a functional linkage of at least two sequences. For example, operably linked includes linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Operably associated includes linkage between an inducing or repressing element and a promoter, wherein the inducing or repressing element acts as a transcriptional activator of the promoter.
[0248] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, nonaqueous or any combination thereof.
[0249] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
[0250] As used herein, the term “pharmaceutical composition” refers to a mixture of at least one particle with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
[0251] A “disease” or “disorder” as used herein refers to a condition where treatment is needed and/or desired.
[0252] As used herein, the terms “treat,” “treating,” or “treatment” refer to ameliorating a disease or disorder, e.g., slowing or arresting or reducing the development of the disease or disorder or reducing at least one of the clinical symptoms thereof. For purposes of this disclosure, ameliorating a disease or disorder can include obtaining a beneficial or desired clinical result that includes, but is not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (for example, metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total).
[0253] The terms “individual” and “subject” are used interchangeably herein to refer to an animal; for example a mammal. The term patient includes human and veterinary subjects. In some embodiments, methods of treating mammals, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are provided. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some examples, an “individual” or “subject” refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject to receive the treatment can be a patient, designating the fact that the subject has been identified as having a disorder of relevance to the treatment, or being at adequate risk of contracting the disorder. In particular embodiments, the subject is a human, such as a human patient.
[0254] As used herein, the terms “effective amount” and “pharmaceutically effective amount” refer to a nontoxic but sufficient amount of an agent or drug to provide the desired biological result. That result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease or disorder, imaging or monitoring of an in vitro or in vivo system (including a living organism), or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. [0255] As used herein, the terms “nuclear export sequence” (NES) or “nuclear export signal” (NES) refer to a nuclear export signal or other sequence or domain that is present in a protein and capable of targeting the protein for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. A nuclear export domain can be fused (e.g., fused in-frame) with a polypeptide.
[0256] As used herein, the terms “nuclear localization siequence” (NLS) or “nuclear localization sequence” (NLS) refer to a nuclear localization signal or other seuqence or domain that is present in a protien and capable of targeting the protein for import from the cytoplams to the cell nucleus through the nuclear pore complex using nuclear transport. A nucelar lozalization can be fused (e.g., fused in-frame) with a polypeptide.
II. TARGETED PARTICLES
[0257] Provided herein are lipid particles that allow for efficient targeting and delivery of an exogenous agent contained therein to hematopoietic stem/progenitor cells (HSPC), including to naive hematopoietic stem cells (HSCs). In some embodiments, the targeting can be to such stem or progenitor cells in a human subject, such as human HSPC. In provided embodiments, the lipid particles can include viral vectors such as lentiviral vectors that are pseudotyped with a fusogen with a preferential ability to target HSPC. In some embodiments, the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof, such as the exemplarly fusogens BaEVTR or BaEVRLess. In some embodiments, the lipid particle includes a targeting agent to target the fusogen to HSPCs, such as to CD34+ cells. In some embodiments, the targeting agent is fused to a fusogen, such as a viral envelope attachment glycoprotein, exposed on the lipid bilayer to provide a retargeted fusogen.
[0258] Provided herein in some embodiments are targeted particles having a targeting agent that binds to a target molecule. In some embodiments, the target molecule is expressed on a target cell. In some embodiments, the provided particle preferentially targets a target cell compared to a non-target cell.
[0259] In some embodiments, the provided particle is a lipid particle, such as any described in Section II-A. In some embodiments, the provided particle is a lipid particle that is a viral particle, such as any described in Section II-A-1. In some embodiments, the provided particle is a targeted lentiviral particle. In some embodiments, the provided particle is a lipid particle that is a virus-like particle, such as any described in Section II-A-2. In some embodiments, the provided particle is a lipid particle that is a cell-based particle, such as any described in Section II-A-3.
[0260] In some embodiments, the provided particle has a fusogen, such as any described in Section II-B. In some embodiments, the fusogen promotes mixing between lipids in the particle and lipids in the target cell. In some embodiments, the fusogen facilitates the fusion of the particle to a membrane of the target cell. In some embodiments, the particle integrates into the membrane of the target cell. In some embodiments, the membrane is the plasma membrane of the target cell. In some embodiments, the fusogen promotes formation of one or more pores between the interior of the particle and the cytosol of the target cell.
[0261] In some embodiments, the fusogen is endogenous to the particle. In some embodiments, the particle is pseudotyped with the fusogen, such as when the particle is a viral particle or virus-like particle.
[0262] In some embodiments, the fusogen is exposed on the surface of the particle. In some embodiments, the fusogen is exposed on the surface of the lipid bilayer of the particle. In some embodiments, a portion of the fusogen is embedded in the lipid bilayer of the particle.
[0263] In some embodiments, the particle is a viral particle or a virus-like particle. In some embodiments, the fusogen is exposed on the surface of the viral envelope of the particle. In some embodiments, a portion of the fusogen is embedded in the viral envelope of the particle.
[0264] In some embodiments, the fusogen contains a mammalian protein, such as any described in Section II-B-1. In some embodiments, the fusogen contains a viral protein, such as any described in Section II-B-2.
[0265] In some embodiments, the lipid particle includes a targeting agent to target the fusogen to HSPCs. In some embodiments, the targeting agent of the provided particle is any described in Section II- C. Exemplary target molecules and target cells for any of the provided particles are also described in Section II-C. In some embodiments, the fusogen is fused to the targeting agent of the particle. In some aspects, the fusogen is retargeted by the targeting agent to display altered tropism. In other embodiments, the targeting agent is fused to a separate transmembrane domain incorporated into the lipid bilayer, not the fusogen.
[0266] In some embodiments, the provided particle contains an exogenous agent. In some embodiments, the exogenous agent is in the lumen of the provided particle. In some embodiments, the sequence of the exogenous agent is exogenous to the particle. In some embodiments, the sequence of the exogenous agent is non-viral. In some embodiments, the exogenous agent is for delivery to the target cell. In some embodiments, the exogenous agent of the provided particle is any described in Section II-D. In some embodiments, the exogenous agent is a nucleic acid, such as any described in Section II-D-1. In some embodiments, the nucleic acid contains a payload gene encoding a payload agent. In some embodiments, the exogenous agent is a protein, such as any described in Section II-D-2. In some embodiments, the protein is a payload agent. In some embodiments, the exogenous agent is a small molecule, such as any described in Section II-D-3.
[0267] In some embodiments, the pay load agent encoded or contained by the exogenous agent is any described in Section III. In some embodiments, the exogenous agent is or encodes an engineered receptor, such as any described in Section III-A. In some embodiments, the exogenous agent is or encodes a gene-editing agent, such as any described in Section III-B. A. Lipid Particle
[0268] In some embodiments, the particle is a lipid particle. In some embodiments, the particle includes a bilayer of amphipathic lipids that encloses a lumen or cavity. In some embodiments, the particle has a lipid bilayer as the outermost surface. In some embodiments, the particle is spherical. In some embodiments, the particle is of elongated or irregular shape.
[0269] In some embodiments, the lipid bilayer encloses a lumen. In some embodiments, the lumen is aqueous. In some embodiments, the lumen is in contact with the hydrophilic head groups on the interior of the lipid bilayer. In some embodiments, the lumen is a cytosol. In some embodiments, the cytosol contains cellular components present in a source cell. In some embodiments, the cytosol does not contain components present in a source cell. In some embodiments, the lumen is a cavity. In some embodiments, the cavity contains an aqueous environment. In some embodiments, the cavity does not contain an aqueous environment.
[0270] In some embodiments, the particle comprises several different types of lipids. In some embodiments, the lipids are amphipathic lipids. In some embodiments, the amphipathic lipids are phospholipids. In some embodiments, the phospholipids comprise phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine. In some embodiments, the lipids comprise phospholipids such as phosphocholines and phosphoinositols. In some embodiments, the lipids comprise DMPC, DOPC, and DSPC.
[0271] In some embodiments, a particle or composition of particles can be assessed for one or more features related to their size, including diameter, average (e.g., mean or median) diameter, range of variation thereof above and below an average (e.g., mean or median) value of the diameter, coefficient of variation, polydispersity index, or other measure of size of particles. Various methods for particle characterization can be used, including laser diffraction, dynamic light scattering (DLS; also known as photon correlation spectroscopy) or image analysis, such as microscopy or automated image analysis.
[0272] In some embodiments, the particle has a diameter of less than about 3 pm, less than about 2 pm, less than about 1 pm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 m, less than about 400 nm, less than about 300, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 50 nm, or less than about 20 nm. In some embodiments, the particle has a diameter of less than about 400 nm. In some embodiments, the particle has a diameter of less than about 150 nm. In some embodiments, the particle has a diameter of between at or about 2 pm and at or about 1 pm, between at or about 1 pm and at or about 900 nm, between at or about 900 nm and at or about 800 nm, between at or about 800 and at or about 700 nm, between at or about 700 nm and at or about 600 nm, between at or about 600 nm and at or about 500 nm, between at or about 500 nm and at or about 400 nm, between at or about 400 nm and at or about 300 nm, between at or about 300 nm and at or about 200 nm, between at or about 200 and at or about 100 nm, between at or about 100 and at or about 50 nm, or between at or about 20 nm and at or about 50 nm. [0273] In some embodiments, the particle has a diameter between at or about 10 nm and at or about 1000 nM, between at or about 25 nm and at or about 500 nm, between at or about 40 nm and at or about 300 nm, between at or about 50 nm and at or about 250 nm, between at or about 60 nm and at or about 225 nm, between at or about 70 nm and at or about 200 nm, between at or about 80 nm and at or about 175 nm, or between at or about 90 nm and at or about 150 nm.
[0274] In some embodiments, the particle has a hydrodynamic radius, e.g., as determined by DLS, of about 100 nm to about two microns. In some embodiments, the particle has a hydrodynamic radius between at or about 2 pm and at or about 1 pm, between at or about 1 pm and at or about 900 nm, between at or about 900 nm and at or about 800 nm, between at or about 800 and at or about 700 nm, between at or about 700 nm and at or about 600 nm, between at or about 600 nm and at or about 500 nm, between at or about 500 nm and at or about 400 nm, between at or about 400 nm and at or about 300 nm, between at or about 300 nm and at or about 200 nm, between at or about 200 and at or about 100 nm, between at or about 100 and at or about 50 nm, or between at or about 20 nm and at or about 50 nm.
[0275] In some embodiments, the particle has a geometric radius, e.g., as determined by a multiangle light scattering, of about 100 nm to about two microns. In some embodiments, the particle has a geometric radius between at or about 2 pm and at or about 1 pm, between at or about 1 pm and at or about 900 nm, between at or about 900 nm and at or about 800 nm, between at or about 800 and at or about 700 nm, between at or about 700 nm and at or about 600 nm, between at or about 600 nm and at or about 500 nm, between at or about 500 nm and at or about 400 nm, between at or about 400 nm and at or about 300 nm, between at or about 300 nm and at or about 200 nm, between at or about 200 and at or about 100 nm, between at or about 100 and at or about 50 nm, or between at or about 20 nm and at or about 50 nm.
[0276] Various particles are known, any of which can be generated in accord with the provided embodiments. Non-limiting examples of particles include any as described in, or containing any features as described in, International Published PCT Application No. WO 2017/095946; WO 2017/095944; WO 2017/095940; WO 2019/157319; WO 2018/208728; WO 2019/113512; WO 2019/161281; WO 2020/102578; WO 2019/222403; WO 2020/014209; WO 2020/102485; WO 2020/102499; WO 2020/102503; WO 2013/148327; WO 2017/182585; WO 2011/058052; or WO 2017/068077, each of which are incorporated by reference in their entirety.
[0277] In some embodiments, the particle is a viral-based particle, a viral particle, a virus-like particle (VLP), a nanoparticle, a vesicle, an exosome, a dendrimer, a lentiviral particle, an enucleated cell, a microvesicle, a membrane vesicle, an extracellular membrane vesicle, a plasma membrane vesicle, a giant plasma membrane vesicle, an apoptotic body, a mitoparticle, a pyrenocyte, a lysosome, another membrane enclosed vesicle, or a cell based particle
[0278] In some embodiments, the lipid bilayer includes a synthetic lipid complex. In some embodiments, the synthetic lipid complex is a liposome. In some embodiments, the lipid bilayer is a vesicular structure characterized by a phospholipid bilayer membrane and an inner aqueous medium. In some embodiments, the lipid bilayer has multiple lipid layers separated by aqueous medium. In some embodiments, the lipid bilayer forms spontaneously when phospholipids are suspended in an excess of aqueous solution. In some embodiments, the lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers
[0279] In some aspects, the lipid bilayer is derived from a source cell during a process to produce a lipid-containing particle. Exemplary methods for producing lipid-containing particles are described in Section IV. In some embodiments, the lipid bilayer includes membrane components of the host cell from which the lipid bilayer is derived, e.g., phospholipids, membrane proteins, etc. In some embodiments, the lipid bilayer includes a cytosol that includes components found in the cell from which the vehicle is derived, e.g., solutes, proteins, nucleic acids, etc., but not all of the components of a cell, e.g., lacking a nucleus. In some embodiments, the lipid bilayer is considered to be exosome-like.
[0280] In particular embodiments, the particle is virally derived. In some embodiments, the particle is a viral or virus-like particle, such as any described in Section II-A-1. In some embodiments, the particle can be a viral-based particle, such as a viral particle (e.g., lentiviral particle) or a virus-like particle (e.g., a lentiviral-like particle). In some embodiments, the provided particle is a targeted lentiviral particle. In some embodiments, the lipid bilayer is a viral envelope. In some embodiments, the viral envelope is obtained from a host cell. In some embodiments, the viral particle includes one or more viral structural proteins (e.g., capsid proteins). In some embodiments, the lipid bilayer is obtained from a membrane other than the plasma membrane of a host cell. In some embodiments, the viral envelope lipid bilayer is embedded with viral proteins, including viral glycoproteins.
[0281] In particular embodiments, the lipid particle is not virally derived. In some embodiments, the lipid particle can be a nanoparticle, a vesicle, an exosome, a dendrimer, an enucleated cell, a microvesicle, a membrane vesicle, an extracellular membrane vesicle, a plasma membrane vesicle, a giant plasma membrane vesicle, an apoptotic body, a mitoparticle, a pyrenocyte, a lysosome, another membrane enclosed vesicle, or a cell derived particle. In some embodiments, the provided particle is a cell-based particle, such as any described in Section II-A-3.
1. Viral Particle
[0282] In some embodiments, the particle is a viral particle, e.g., a retroviral particle, e.g., a lentiviral particle. In some embodiments, the viral particle is recombinant. In some embodiments, the particle is a recombinant lentiviral particle.
[0283] In some embodiments, the lipid bilayer of the particle is or comprises the viral envelope. The viral envelope may comprise a fusogen that is endogenous to the virus or is a pseudotyped fusogen. In some embodiments, the particle’s lumen or cavity comprises a viral nucleic acid, e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid. The viral nucleic acid may be a viral genome. In some embodiments, the particle may further comprise one or more viral non-structural proteins, e.g., in its cavity or lumen. In some embodiments, the particles are lenti viral particles. In some embodiments, the lenti viral particle is Human Immunodeficiency Virus-1 (HIV-1).
[0284] In some aspects, the particle is limited in the number of polynucleotides that can be packaged. In some embodiments, nucleotides encoding polypeptides to be packaged can be modified such that they retain functional activity with fewer nucleotides in the coding region than that which encodes for the wild-type peptide. Such modifications can include truncations, or other deletions. In some embodiments, more than one polypeptide can be expressed from the same promoter, such that they are fusion polypeptides. In some embodiments, the insert size to be packaged (i.e., viral genome, or portions thereof; or heterologous polynucleotides as described) can be between 500-1000, 1000-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, or 7000-8000 nucleotides in length. In some embodiments, the insert can be over 8000 nucleotides, such as 9000, 10,000, or 11,000 nucleotides in length.
[0285] In some embodiments, the particle, such as retroviral particle, comprises one or more of gag polyprotein, polymerase (e.g., pol), integrase (e.g., a functional or non-functional variant), protease, and a fusogen. In some embodiments, the particle further comprises rev. In some embodiments, one or more of the aforesaid proteins are encoded in the retroviral genome (e.g., the insert as described above), and in some embodiments, one or more of the aforesaid proteins are provided in trans, e.g., by a helper cell, helper virus, or helper plasmid. In some embodiments, the nucleic acid (e.g., retroviral nucleic acid) comprises one or more of the following nucleic acid sequences: 5’ LTR (e.g., comprising U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT) Promoter operatively linked to a payload gene encoding a payload agent, payload gene (optionally comprising an intron before the open reading frame), Poly A tail sequence, WPRE, and 3’ LTR (e.g., comprising U5 and lacking a functional U3). In some embodiments, the nucleic acid further comprises a retroviral cisacting RNA packaging element and a cPPT/CTS element. In some embodiments the nucleic acid further comprises one or more insulator element. In some embodiments, the recognition sites are situated between the poly A tail sequence and the WPRE.
[0286] In some embodiments, the particle comprises supramolecular complexes formed by viral proteins that self-assemble into capsids. In some embodiments, the particle is a viral particle derived from viral capsids. In some embodiments, the particle is a viral particle derived from viral nucleocapsids. In some embodiments, the particle comprises nucleocapsid-derived proteins that retain the property of packaging nucleic acids.
[0287] In some embodiments, the particle packages one or more viral nucleic acids (e.g., retroviral nucleic acids) during the expression process. In some embodiments, the nucleic acids do not encode any genes involved in virus replication. In particular embodiments, the particle is replication defective. [0288] In some embodiments, the retroviral nucleic acid comprises one or more of (e.g., all of): a 5’ promoter (e.g., to control expression of the entire packaged RNA), a 5’ LTR (e.g., that includes R (poly adenylation tail signal) and/or U5 which includes a primer activation signal), a primer binding site, a psi packaging signal, a RRE element for nuclear export, a promoter directly upstream of the payload gene to control payload gene expression, a payload gene, a polypurine tract, and a 3’ LTR (e.g., that includes a mutated U3, a R, and U5). In some embodiments, the retroviral nucleic acid further comprises one or more of a cPPT, a WPRE, and/or an insulator element.
[0289] A retrovirus typically replicates by reverse transcription of its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Illustrative retroviruses suitable for use in particular embodiments include Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spuma virus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentivirus.
[0290] In some embodiments the retrovirus is a Gammretro virus. In some embodiments the retrovirus is an Epsilonretrovirus. In some embodiments the retrovirus is an Alpharetrovirus. In some embodiments the retrovirus is a Betaretro virus. In some embodiments the retrovirus is a Deltaretro virus. In some embodiments the retrovirus is a Lentivirus. In some embodiments the retrovirus is a Spumaretrovirus. In some embodiments the retrovirus is an endogenous retrovirus.
[0291] Illustrative lenti viruses include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In some embodiments, HIV based particle backbones (e.g., HIV cis-acting sequence elements) are used.
[0292] A viral particle can comprise a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that typically facilitate transfer of a nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acids. Viral particles and transfer plasmids can comprise structural and/or functional genetic elements that are primarily derived from a virus. A retroviral particle can comprise a viral particle or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. A lenti viral particle can comprise a viral particle or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus.
[0293] In embodiments, a lentiviral particle may comprise a lentiviral transfer plasmid (e.g., as naked DNA) or an infectious lentiviral particle. With respect to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is to be understood that the sequences of these elements can be present in RNA form in lentiviral particles and can be present in DNA form in DNA plasmids.
[0294] In some particles described herein, at least part of one or more protein coding regions that contribute to or are essential for replication may be absent compared to the corresponding wild- type virus. In some embodiments, this makes the viral particle replication-defective. In some embodiments, the particle is capable of transducing a target non-dividing host cell and/or integrating its genome into a host genome.
[0295] The structure of a wild-type retrovirus genome often comprises a 5' long terminal repeat (LTR) and a 3' LTR, between or within which are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome and gag, pol and env genes encoding the packaging components which promote the assembly of viral particles. More complex retroviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell. In the provirus, the viral genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are involved in pro viral integration and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes. Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome.
[0296] The LTRs themselves are typically similar (e.g., identical) sequences that can be divided into three elements, which are called U3, R and U5. U3 is derived from the sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA and U5 is derived from the sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.
[0297] For the viral genome, the site of transcription initiation is typically at the boundary between U3 and R in one LTR and the site of poly (A) addition (termination) is at the boundary between R and U5 in the other LTR. U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins. Some retroviruses comprise any one or more of the following genes that code for proteins that are involved in the regulation of gene expression: tot, rev, tax and rex. With regard to the structural genes gag, pol and env themselves, gag encodes the internal structural protein of the virus. Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome. The env gene encodes the surface (SU) glycoprotein and the transmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. This interaction promotes infection, e.g., by fusion of the viral membrane with the cell membrane.
[0298] In a replication-defective retroviral genome, gag, pol and env may be absent or not functional. The R regions at both ends of the RNA are typically repeated sequences. U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome respectively.
[0299] Retroviruses may also contain additional genes which code for proteins other than gag, pol and env. Examples of additional genes include (in HIV), one or more of vif, vpr, vpx, vpu, tat, rev and nef. El AV has (amongst others) the additional gene S2. Proteins encoded by additional genes serve various functions, some of which may be duplicative of a function provided by a cellular protein. In EIAV, for example, tat acts as a transcriptional activator of the viral LTR (Derse and Newbold 1993 Virology 194:530-6; Maury et al. 1994 Virology 200:632- 42). It binds to a stable, stem-loop RNA secondary structure referred to as TAR. Rev regulates and co-ordinates the expression of viral genes through rev-response elements (RRE) (Martarano et al. 1994 J. Virol. 68:3102-11). The mechanisms of action of these two proteins are thought to be broadly similar to the analogous mechanisms in the primate viruses. In addition, an EIAV protein, Ttm, has been identified that is encoded by the first exon of tat spliced to the env coding sequence at the start of the transmembrane protein.
[0300] In addition to protease, reverse transcriptase and integrase, non-primate lentiviruses contain a fourth pol gene product which codes for a dUTPase. This may play a role in the ability of these lentiviruses to infect certain non-dividing or slowly dividing cell types.
[0301] In embodiments, a recombinant lenti viral particle (RLV) is a particle with sufficient retroviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle capable of infecting a target cell. Infection of the target cell can comprise reverse transcription and integration into the target cell genome. The RLV typically carries non-viral coding sequences which are to be delivered by the particle to the target cell, such as a nucleic acid encoding a payload agent as described herein. In embodiments, an RLV is incapable of independent replication to produce infectious retroviral particles within the target cell. Usually the RLV lacks a functional gag-pol and/or env gene and/or other genes involved in replication. The particle may be configured as a split-intron particle, e.g., as described in PCT patent application WO 99/15683, which is herein incorporated by reference in its entirety.
[0302] In some embodiments, the lentiviral particle comprises a minimal viral genome, e.g., the viral particle has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell, e.g., as described in WO 98/17815, which is herein incorporated by reference in its entirety.
[0303] A minimal lentiviral genome may comprise, e.g., (5')R-U5-one or more first nucleotide sequences-U3-R(3'). However, the plasmid used to produce the lentiviral genome within a source cell can also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a source cell. These regulatory sequences may comprise the natural sequences associated with the transcribed retroviral sequence, e.g., the 5' U3 region, or they may comprise a heterologous promoter such as another viral promoter, for example the CMV promoter. Some lentiviral genomes comprise additional sequences to promote efficient virus production. For example, in the case of HIV, rev and RRE sequences may be included. Alternatively or combination, codon optimization may be used, e.g., the payload gene encoding the payload agent may be codon optimized, e.g., as described in WO 01/79518, which is herein incorporated by reference in its entirety. Alternative sequences which perform a similar or the same function as the rev/RRE system may also be used. For example, a functional analogue of the rev/RRE system is found in the Mason Pfizer monkey virus. This is known as CTE and comprises an RRE-type sequence in the genome which is believed to interact with a factor in the infected cell. The cellular factor can be thought of as a rev analogue. Thus, CTE may be used as an alternative to the rev/RRE system. In addition, the Rex protein of HTLV-I can functionally replace the Rev protein of HIV-I. Rev and Rex have similar effects to IRE-BP.
[0304] In some embodiments, a retroviral nucleic acid (e.g., a lentiviral nucleic acid, e.g., a primate or non-primate lentiviral nucleic acid) (1) comprises a deleted gag gene wherein the deletion in gag removes one or more nucleotides downstream of about nucleotide 350 or 354 of the gag coding sequence; (2) has one or more accessory genes absent from the retroviral nucleic acid; (3) lacks the tat gene but includes the leader sequence between the end of the 5' LTR and the ATG of gag; and (4) combinations of (1), (2) and (3). In an embodiment, the lentiviral particle comprises all of features (1) and (2) and (3). This strategy is described in more detail in WO 99/32646, which is herein incorporated by reference in its entirety.
[0305] In some embodiments, a primate lentivirus minimal system requires none of the HIV/SIV additional genes vif, vpr, vpx, vpu, tat, rev and nef for either particle production or for transduction of dividing and non-dividing cells. In some embodiments, an EIAV minimal particle system does not require S2 for either particle production or for transduction of dividing and non-dividing cells.
[0306] The deletion of additional genes may permit particles to be produced without the genes associated with disease in lentiviral (e.g. HIV) infections. In particular, tat is associated with disease. Secondly, the deletion of additional genes permits the particle to package more heterologous DNA. Thirdly, genes whose function is unknown, such as S2, may be omitted, thus reducing the risk of causing undesired effects. Examples of minimal lentiviral particles are disclosed in WO 99/32646 and in WO 98/17815.
[0307] In some embodiments, the retroviral nucleic acid is devoid of at least tat and S2 (if it is an EIAV particle system), and possibly also vif, vpr, vpx, vpu and nef. In some embodiments, the retroviral nucleic acid is also devoid of rev, RRE, or both. [0308] In some embodiments the retroviral nucleic acid comprises vpx. The Vpx polypeptide binds to and induces the degradation of the SAMHD1 restriction factor, which degrades free dNTPs in the cytoplasm. Thus, the concentration of free dNTPs in the cytoplasm increases as Vpx degrades SAMHD1 and reverse transcription activity is increased, thus facilitating reverse transcription of the retroviral genome and integration into the target cell genome.
[0309] Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available. An additional description of codon optimization is found, e.g., in WO 99/41397, which is herein incorporated by reference in its entirety.
[0310] Many viruses, including HIV and other lentiviruses, use a large number of rare codons and by changing these to correspond to commonly used mammalian codons, increased expression of the packaging components in mammalian producer cells can be achieved.
[0311] In some embodiments, codon optimization has a number of other advantages. In some embodiments, by virtue of alterations in their sequences, the nucleotide sequences encoding the packaging components may have RNA instability sequences (INS) reduced or eliminated from them. At the same time, the amino acid sequence coding sequence for the packaging components is retained so that the viral components encoded by the sequences remain the same, or at least sufficiently similar that the function of the packaging components is not compromised. In some embodiments, codon optimization also overcomes the Rev/RRE requirement for export, rendering optimized sequences Rev independent. In some embodiments, codon optimization also reduces homologous recombination between different constructs within the particle system (for example between the regions of overlap in the gag-pol and env open reading frames). In some embodiments, codon optimization leads to an increase in viral titer and/or improved safety.
[0312] In some embodiments, only codons relating to INS are codon optimized. In other embodiments, the sequences are codon optimized in their entirety, with the exception of the sequence encompassing the frameshift site of gag-pol.
[0313] The gag-pol gene comprises two overlapping reading frames encoding the gag-pol proteins. The expression of both proteins depends on a frameshift during translation. This frameshift occurs as a result of ribosome "slippage" during translation. This slippage is thought to be caused at least in part by ribosome-stalling RNA secondary structures. Such secondary structures exist downstream of the frameshift site in the gag-pol gene. For HIV, the region of overlap extends from nucleotide 1222 downstream of the beginning of gag (wherein nucleotide 1 is the A of the gag ATG) to the end of gag (nt 1503). Consequently, a 281 bp fragment spanning the frameshift site and the overlapping region of the two reading frames is preferably not codon optimized. In some embodiments, retaining this fragment will enable more efficient expression of the gag-pol proteins. For EIAV, the beginning of the overlap is at nt 1262 (where nucleotide 1 is the A of the gag ATG). The end of the overlap is at nt 1461. In order to ensure that the frameshift site and the gag-pol overlap are preserved, the wild type sequence may be retained from nt 1156 to 1465.
[0314] In some embodiments, derivations from optimal codon usage may be made, for example, in order to accommodate convenient restriction sites, and conservative amino acid changes may be introduced into the gag-pol proteins.
[0315] In some embodiments, codon optimization is based on codons with poor codon usage in mammalian systems. The third and sometimes the second and third base may be changed.
[0316] In some embodiments, due to the degenerate nature of the genetic code, it will be appreciated that numerous gag-pol sequences can be achieved by a skilled worker. Also, there are many retroviral variants described which can be used as a starting point for generating a codon optimized gag-pol sequence. Lentiviral genomes can be quite variable. For example there are many quasi-species of HIV-I which are still functional. This is also the case for EIAV. These variants may be used to enhance particular parts of the transduction process. Examples of HIV-I variants may be found in the HIV databases maintained by Los Alamos National Laboratory. Details of EIAV clones may be found at the NCBI database maintained by the National Institutes of Health.
[0317] It is within the level of a skilled artisan to empirically determine appropriate codon optimization of viral sequences. The strategy for codon optimized sequences, including gag-pol sequences, can be used in relation to any retrovirus, e.g., EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-I and HIV -2. In addition this method could be used to increase expression of genes from HTLV-I, HTLV-2, HFV, HSRV and human endogenous retroviruses (HERV), MLV and other retroviruses.
[0318] In embodiments, the retroviral particle comprises a packaging signal that comprises from 255 to 360 nucleotides of gag in particles that still retain env sequences, or about 40 nucleotides of gag in a particular combination of splice donor mutation, gag and env deletions. In some embodiments, the retroviral particle includes a gag sequence which comprises one or more deletions, e.g., the gag sequence comprises about 360 nucleotides derivable from the N-terminus.
[0319] In some embodiments, the retroviral particle, helper cell, helper virus, or helper plasmid may comprise retroviral structural and accessory proteins, for example gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef proteins or other retroviral proteins. In some embodiments, the retroviral proteins are derived from the same retrovirus. In some embodiments, the retroviral proteins are derived from more than one retrovirus, e.g. 2, 3, 4, or more retroviruses.
[0320] In some embodiments, the gag and pol coding sequences are generally organized as the Gag- Pol Precursor in native lentivirus. The gag sequence codes for a 55-kD Gag precursor protein, also called p55. The p55 is cleaved by the virally encoded protease (a product of the pol gene) during the process of maturation into four smaller proteins designated MA (matrix [pl7]), CA (capsid [p24]), NC (nucleocapsid [p9]) , and p6. The pol precursor protein is cleaved away from Gag by a virally encoded protease, and further digested to separate the protease (plO), RT (p50), RNase H (pl5), and integrase (p31) activities.
[0321] In some embodiments, the lentiviral particle is integration-deficient. In some embodiments, the pol is integrase deficient, such as by encoding due to mutations in the integrase gene. For example, the pol coding sequence can contain an inactivating mutation in the integrase, such as by mutation of one or more of amino acids involved in catalytic activity, i.e. mutation of one or more of aspartic 64, aspartic acid 116 and/or glutamic acid 152. In some embodiments, the integrase mutation is a D64V mutation. In some embodiments, the mutation in the integrase allows for packaging of viral RNA into a lentivirus. In some embodiments, the mutation in the integrase allows for packaging of viral proteins into a lentivirus. In some embodiments, the mutation in the integrase reduces the possibility of insertional mutagenesis. In some embodiments, the mutation in the integrase decreases the possibility of generating replication- competent recombinants (RCRs) (Wanisch et al. 2009. Mol Ther. 1798): 1316-1332). In some embodiments, native Gag-Pol sequences can be utilized in a helper particle (e.g., helper plasmid or helper virus), or modifications can be made. These modifications include chimeric Gag-Pol, where the Gag and Pol sequences are obtained from different viruses (e.g., different species, subspecies, strains, clades, etc.), and/or where the sequences have been modified to improve transcription and/or translation, and/or reduce recombination.
[0322] In some embodiments, the retroviral nucleic acid includes a polynucleotide encoding a 150- 250 (e.g., 168) nucleotide portion of a gag protein that (i) includes a mutated INS1 inhibitory sequence that reduces restriction of nuclear export of RNA relative to wild-type INS1, (ii) contains two nucleotide insertion that results in frame shift and premature termination, and/or (iii) does not include INS2, INS3, and INS4 inhibitory sequences of gag.
[0323] In some embodiments, a particle described herein is a hybrid particle that comprises both retroviral (e.g., lentiviral) sequences and non-lentiviral viral sequences. In some embodiments, a hybrid particle comprises retroviral, e.g., lentiviral, sequences for reverse transcription, replication, integration and/or packaging.
[0324] According to certain specific embodiments, most or all of the viral particle backbone sequences are derived from a lentivirus, e.g., HIV-1. However, it is to be understood that many different sources of retroviral and/or lentiviral sequences can be used, or combined and numerous substitutions and alterations in certain of the lentiviral sequences may be accommodated without impairing the ability of a transfer particle to perform the functions described herein. A variety of lentiviral particles are described in Naldini et ah, (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which may be adapted to produce a retroviral nucleic acid. [0325] At each end of the provirus, long terminal repeats (LTRs) are typically found. An LTR typically comprises a domain located at the ends of retroviral nucleic acid which, in their natural sequence context, are direct repeats and contain U3, R and U5 regions. LTRs generally promote the expression of retroviral genes (e.g., promotion, initiation and poly adenylation of gene transcripts) and viral replication. The LTR can comprise numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences for replication and integration of the viral genome. The viral LTR is typically divided into three regions called U3, R and U5. The U3 region typically contains the enhancer and promoter elements. The U5 region is typically the sequence between the primer binding site and the R region and can contain the polyadenylation sequence. The R (repeat) region can be flanked by the U3 and U5 regions. The LTR is typically composed of U3, R and U5 regions and can appear at both the 5' and 3' ends of the viral genome. In some embodiments, adjacent to the 5' LTR are sequences for reverse transcription of the genome (the tRNA primer binding site) and for efficient packaging of viral RNA into particles (the Psi site).
[0326] In some embodiments, a packaging signal can comprise a sequence located within the retroviral genome which mediate insertion of the viral RNA into the viral capsid or particle, see e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109. Several retroviral particles use a minimal packaging signal (a psi [Y] sequence) for encapsidation of the viral genome.
[0327] In various embodiments, retroviral nucleic acids comprise modified 5' LTR and/or 3' LTRs. Either or both of the LTR may comprise one or more modifications including, but not limited to, one or more deletions, insertions, or substitutions. Modifications of the 3' LTR are often made to improve the safety of lentiviral or retroviral systems by rendering viruses replication-defective, e.g., virus that is not capable of complete, effective replication such that infective virions are not produced (e.g., replicationdefective lentiviral progeny).
[0328] In some embodiments, a particle is a self-inactivating (SIN) particle, e.g., replicationdefective particle, e.g., retroviral or lentiviral particle, in which the right (3') LTR enhancer- promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. In some aspects, provided herein is a replication incompetent (also referred to herein as replication defective) particle, that cannot participate in replication in the absence of the packaging cell (i.e., viral particles are not produced from the transduced cell). In some aspects, this is because the right (3') LTR U3 region can be used as a template for the left (5') LTR U3 region during viral replication and, thus, absence of the U3 enhancer-promoter inhibits viral replication. In embodiments, the 3' LTR is modified such that the U5 region is removed, altered, or replaced, for example, with an exogenous poly(A) sequence The 3' LTR, the 5' LTR, or both 3' and 5' LTRs, may be modified LTRs. Other modifications to the viral particle, i.e., retroviral or lentiviral particle, to render said particle replication incompetent are known in the art. [0329] In some embodiments, the U3 region of the 5' LTR is replaced with a heterologous promoter to drive transcription of the viral genome during production of viral particles. Examples of heterologous promoters which can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. In some embodiments, promoters are able to drive high levels of transcription in a Tat- independent manner. In certain embodiments, the heterologous promoter has additional advantages in controlling the manner in which the viral genome is transcribed. For example, the heterologous promoter can be inducible, such that transcription of all or part of the viral genome will occur only when the induction factors are present. Induction factors include, but are not limited to, one or more chemical compounds or the physiological conditions such as temperature or pH, in which the host cells are cultured.
[0330] In some embodiments, viral particles comprise a TAR (trans-activation response) element, e.g., located in the R region of lentiviral (e.g., HIV) LTRs. This element interacts with the lentiviral trans-activator (tat) genetic element to enhance viral replication. However, this element is not required, e.g., in embodiments wherein the U3 region of the 5' LTR is replaced by a heterologous promoter.
[0331] The R region, e.g., the region within retroviral LTRs beginning at the start of the capping group (i.e., the start of transcription) and ending immediately prior to the start of the poly A tract can be flanked by the U3 and U5 regions. The R region plays a role during reverse transcription in the transfer of nascent DNA from one end of the genome to the other.
[0332] The retroviral nucleic acid can also comprise a FLAP element, e.g., a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, et ah, 2000, Cell, 101:173, which are herein incorporated by reference in their entireties. During HIV-1 reverse transcription, central initiation of the plus-strand DNA at the central polypurine tract (cPPT) and central termination at the central termination sequence (CTS) can lead to the formation of a three- stranded DNA structure: the HIV-1 central DNA flap. In some embodiments, the retroviral or lentiviral particle backbones comprise one or more FLAP elements upstream or downstream of the payload gene encoding the payload agent. For example, in some embodiments a transfer plasmid includes a FLAP element, e.g., a FLAP element derived or isolated from HIV-L
[0333] In embodiments, a retroviral or lentiviral nucleic acid comprises one or more export elements, e.g., a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include the human immunodeficiency virus (HIV) rev response element (RRE) (see e.g., Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), and the hepatitis B virus post-transcriptional regulatory element (HPRE), which are herein incorporated by reference in their entireties. Generally, the RNA export element is placed within the 3' UTR of a gene, and can be inserted as one or multiple copies. [0334] In some embodiments, expression of heterologous sequences (e.g., nucleic acid encoding a payload agent) in viral particles is increased by incorporating one or more of, e.g., all of, posttranscriptional regulatory elements, polyadenylation sites, and transcription termination signals into the particles. A variety of posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid at the protein, e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9:1766), each of which is herein incorporated by reference in its entirety. In some embodiments, a retroviral nucleic acid described herein comprises a posttranscriptional regulatory element such as a WPRE or HPRE
[0335] In some embodiments, a retroviral nucleic acid described herein lacks or does not comprise a posttranscriptional regulatory element such as a WPRE or HPRE.
[0336] Elements directing the termination and polyadenylation of the heterologous nucleic acid transcripts may be included, e.g., to increase expression of the payload agent. Transcription termination signals may be found downstream of the polyadenylation signal. In some embodiments, particles comprise a polyadenylation sequence 3' of a polynucleotide encoding the payload agent. A polyA site may comprise a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency. Illustrative examples of polyA signals that can be used in a retroviral nucleic acid, include AATAAA, ATT AAA, AGTAAA, a bovine growth hormone polyA sequence (BGHpA), a rabbit b- globin polyA sequence (rPgpA), or another suitable heterologous or endogenous polyA sequence.
[0337] In some embodiments, a retroviral or lenti viral particle further comprises one or more insulator elements, e.g., an insulator element described herein.
[0338] In various embodiments, the particles comprise a promoter operably linked to a polynucleotide encoding a payload agent. The particles may have one or more LTRs, wherein either LTR comprises one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The particles may further comprise one of more accessory elements to increase transduction efficiency (e.g., a cPPT/FLAP), viral packaging (e.g., a Psi packaging signal, RRE), and/or other elements that increase payload gene expression (e.g., poly (A) sequences), and may optionally comprise a WPRE or HPRE.
[0339] In some embodiments, a lentiviral nucleic acid comprises one or more of, e.g., all of, e.g., from 5’ to 3’, a promoter (e.g., CMV), an R sequence (e.g., comprising TAR), a U5 sequence (e.g., for integration), a PBS sequence (e.g., for reverse transcription), a DIS sequence (e.g., for genome dimerization), a psi packaging signal, a partial gag sequence, an RRE sequence (e.g., for nuclear export), a cPPT sequence (e.g., for nuclear import), a promoter to drive expression of the payload agent, a payload gene encoding the payload agent, a WPRE sequence (e.g., for efficient transgene expression), a PPT sequence (e.g., for reverse transcription), an R sequence (e.g., for polyadenylation and termination), and a U5 signal (e.g., for integration).
2. Virus-Like Particle
[0340] In some embodiments, the particle is a virus-like particle (VLP) that is derived from virus. In some embodiments, the viral envelope may comprise a fusogen, e.g., a fusogen that is endogenous to the virus or a pseudotyped fusogen. The VLPS include those derived from retroviruses or lentiviruses. While VLPs mimic native virion structure, they lack the viral genomic information necessary for independent replication within a host cell. Therefore, in some aspects, VLPs are non-infectious. In particular embodiments, a VLP does not contain a viral genome. In some embodiments, the VLP’s bilayer of amphipathic lipids is or comprises the viral envelope. In some embodiments, the particle’s bilayer of amphipathic lipids is or comprises lipids derived from a cell. In some embodiments, a VLP contains at least one type of structural protein from a virus. In most cases this protein will form a proteinaceous capsid. In some cases the capsid will also be enveloped in a lipid bilayer originating from the cell from which the assembled VLP has been released (e.g., VLPs comprising a human immunodeficiency virus structural protein such as GAG). In some embodiments, the VLP further comprises a targeting moiety as an envelope protein within the lipid bilayer.
[0341] In some embodiments, the particle comprises supramolecular complexes formed by viral proteins that self-assemble into capsids. In some embodiments, the particle is a virus-like particle derived from viral capsid proteins. In some embodiments, the particle is a virus-like particle derived from viral nucleocapsid proteins. In some embodiments, the particle comprises nucleocapsid-derived proteins that retain the property of packaging nucleic acids. In some embodiments, the particle comprises only viral structural glycoproteins among proteins from the viral genome. In some embodiments, the particle does not contain a viral genome.
[0342] In some embodiments, the particle packages nucleic acids during the expression process, such as a nucleic acid encoding a payload agent. In some embodiments, the nucleic acids do not encode any genes involved in virus replication. In particular embodiments, the particle is a virus-like particle, e.g., retrovirus-like particle, such as a lentivirus-like particle, that is replication defective.
[0343] In some embodiments, the particle is a virus-like particle which comprises a sequence that is devoid of or lacking viral RNA, which may be the result of removing or eliminating the viral RNA from the sequence. In some embodiments, this may be achieved by using an endogenous packaging signal binding site on gag. In some embodiments, the endogenous packaging signal binding site is on pol. In some embodiments, the RNA which is to be delivered will contain a cognate packaging signal. In some embodiments, a heterologous binding domain (which is heterologous to gag) located on the RNA to be delivered, and a cognate binding site located on gag or pol, can be used to ensure packaging of the RNA to be delivered. In some embodiments, the heterologous sequence could be non-viral or it could be viral, in which case it may be derived from a different virus. In some embodiments, the particles could be used to deliver therapeutic RNA, in which case functional integrase and/or reverse transcriptase is not required. In some embodiments, the particles could also be used to deliver a therapeutic gene of interest, in which case pol is typically included.
3. Cell-Based Particle
[0344] In some embodiments, the particle is a cell based particle that comprises a naturally derived membrane. In some embodiments, the naturally derived membrane comprises membrane vesicles prepared from cells or tissues. In some embodiments, the cell based particle comprises a vesicle that is obtainable from a cell. In some embodiments, the cell based particle comprises a microvesicle, an exosome, a membrane enclosed body, an apoptotic body (from apoptotic cells), a particle (which may be derived from e.g. platelets), an ectosome (derivable from, e.g., neutrophiles and monocytes in serum), a prostatosome (obtainable from prostate cancer cells), or a cardiosome (derivable from cardiac cells).
[0345] In some embodiments, the source cell is an endothelial cell, a fibroblast, a blood cell (e.g., a macrophage, a neutrophil, a granulocyte, a leukocyte), a stem cell (e.g., a mesenchymal stem cell, an umbilical cord stem cell, bone marrow stem cell, a hematopoietic stem cell, an induced pluripotent stem cell e.g., an induced pluripotent stem cell derived from a subject’s cells), an embryonic stem cell (e.g., a stem cell from embryonic yolk sac, placenta, umbilical cord, fetal skin, adolescent skin, blood, bone marrow, adipose tissue, erythropoietic tissue, hematopoietic tissue), a myoblast, a parenchymal cell (e.g., hepatocyte), an alveolar cell, a neuron (e.g., a retinal neuronal cell) a precursor cell (e.g., a retinal precursor cell, a myeloblast, myeloid precursor cells, a thymocyte, a meiocyte, a megakaryoblast, a promegakaryoblast, a melanoblast, a lymphoblast, a bone marrow precursor cell, a normoblast, or an angioblast), a progenitor cell (e.g., a cardiac progenitor cell, a satellite cell, a radial gial cell, a bone marrow stromal cell, a pancreatic progenitor cell, an endothelial progenitor cell, a blast cell), or an immortalized cell (e.g., HeEa, HEK293, HFF-1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080, or BJ cell). In some embodiments, the source cell is other than a 293 cell, HEK cell, human endothelial cell, or a human epithelial cell, monocyte, macrophage, dendritic cell, or stem cell.
[0346] In some embodiments, the cell based particle has a density of <1, 1-1.1, 1.05-1.15, 1.1-1.2, 1.15-1.25, 1.2-1.3, 1.25-1.35, or >1.35 g/ml. In embodiments, the cell based particle has a size that is less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, of that of the source cell. In some embodiments, a composition containing a plurality of the cell based particle comprises less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or 10% source cells by protein mass or less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or 10% of cells having a functional nucleus. In some embodiments, the source cell used to make the cell based particle will not be available for testing after the particle is made. [0347] In some embodiments the cell based particle is an extracellular vesicle, e.g., a cell based vesicle comprising a membrane that encloses an internal space and has a smaller diameter than the cell from which it is derived. In embodiments the extracellular vesicle has a diameter from 20 nm to 1000 nm. In some embodiments, the cell based particle is an apoptotic body, a fragment of a cell, a vesicle derived from a cell by direct or indirect manipulation, a vesiculated organelle, and a vesicle produced by a living cell (e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane). In embodiments the extracellular vesicle is derived from a living or dead organism, explanted tissues or organs, or cultured cells.
[0348] In embodiments, the cell based particle is a nanovesicle, e.g., a cell-derived small (e.g., between 20-250 nm in diameter, or 30-150 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from said cell by direct or indirect manipulation. The production of nanovesicles can, in some instances, result in the destruction of the source cell. The nanovesicle may comprise a lipid or fatty acid and polypeptide.
[0349] In embodiments, the cell based particle is an exosome. In embodiments, the exosome is a cell-derived small (e.g., between 20-300 nm in diameter, or 40-200 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from said cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. In embodiments, production of exosomes does not result in the destruction of the source cell. In embodiments, the exosome comprises lipid or fatty acid and polypeptide. Exemplary exosomes and other membrane- enclosed bodies are also described in WO/2017/161010, WO/2016/077639, US20160168572, US20150290343, and US20070298118, each of which is incorporated by reference herein in its entirety.
[0350] In some embodiments, the cell based particle is a microvesicle. In some embodiments the micro vesicle has a diameter of about 100 nm to about 2000 nm.
[0351] In some embodiments, the cell based particle is a cell ghost. In some embodiments, a vesicle is a plasma membrane vesicle, e.g. a giant plasma membrane vesicle.
[0352] In some embodiments, the cell based particle is derived from a source cell with a genetic modification which results in increased expression of an immunomodulatory agent, such as an immunosuppressive agent. In some embodiments, the immunosuppressive agent is on an exterior surface of the cell. In some embodiments, the immunosuppressive agent is incorporated into the exterior surface of the particle. In some embodiments, the particle comprises an immunomodulatory agent attached to the surface of the solid particle by a covalent or non-covalent bond
[0353] In some embodiments, cell based particles are generated by inducing budding of an exosome, microvesicle, membrane vesicle, extracellular membrane vesicle, plasma membrane vesicle, giant plasma membrane vesicle, apoptotic body, mitoparticle, pyrenocyte, lysosome, or other membrane enclosed vesicle. [0354] In some embodiments, cell based particles are generated by inducing cell enucleation.
Enucleation may be performed using assays such as genetic, chemical (e.g., using Actinomycin D, see Bayona-Bafaluyet al., “A chemical enucleation method for the transfer of mitochondrial DNA to p° cells” Nucleic Acids Res. 2003 Aug 15; 31(16): e98), mechanical methods (e.g., squeezing or aspiration, see Lee et al., “A comparative study on the efficiency of two enucleation methods in pig somatic cell nuclear transfer: effects of the squeezing and the aspiration methods.” Anim Biotechnol. 2008;19(2):71- 9), or combinations thereof.
[0355] In some embodiments, the cell based particles are generated by inducing cell fragmentation. In some embodiments, cell fragmentation can be performed using the following methods, including, but not limited to: chemical methods, mechanical methods (e.g., centrifugation (e.g., ultracentrifugation, or density centrifugation), freeze-thaw, or sonication), or combinations thereof.
[0356] In some embodiments, a characteristic of a cell based particle is described by comparison to a reference cell. In embodiments, the reference cell is the source cell. In embodiments, the reference cell is a HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080, or BJ cell. In some embodiments, a characteristic of a composition of cell based particles is described by comparison to a population of reference cells, e.g., a population of source cells, or a population of HeLa, HEK293, HFF- 1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080, or BJ cells.
B. Fusogen
[0357] In some embodiments, the particle has a fusogen. In some embodiments, the particle has more than one fusogen. In some embodiments, the fusogen contains a mammalian protein, such as any described in Section II-B-1. In some embodiments, the fusogen contains a viral protein, such as any described in Section II-B-2.
[0358] In some embodiments, the fusogen promotes mixing between lipids in the particle and lipids in the target cell. In some embodiments, the fusogen facilitates the fusion of the particle to a membrane of the target cell. In some embodiments, the particle integrates into the membrane of the target cell. In some embodiments, the membrane is the plasma membrane of the target cell. In some embodiments, the fusogen promotes formation of one or more pores between the interior of the particle and the cytosol of the target cell.
[0359] In some embodiments, the fusogen is endogenous to the particle. In some embodiments, the particle is pseudotyped with the fusogen, such as when the particle is a viral particle or virus-like particle.
[0360] In some embodiments, the fusogen is exposed on the surface of the particle. In some embodiments, the fusogen is exposed on the surface of the lipid bilayer of the particle. In some embodiments, a portion of the fusogen is embedded in the lipid bilayer of the particle. [0361] In some embodiments, the particle is a viral particle or a virus-like particle. In some embodiments, the fusogen is exposed on the surface of the viral envelope of the particle. In some embodiments, a portion of the fusogen is embedded in the viral envelope of the particle.
[0362] In some embodiments, the fusogen is present at a copy number of at least, or no more than, 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the fusogen comprised by the particle is disposed in the cell membrane. In embodiments, the particle also comprises fusogen internally, e.g., in the cytoplasm or an organelle. In some embodiments, the fusogen comprises (or is identified as comprising) about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, or more, or about 1-30%, 5- 20%, 10- 15%, 12-15%, 13-14%, or 13.6% of the total protein in a particle, e.g., as determined by a mass spectrometry assay. In embodiments, the fusogen comprises (or is identified as comprising) about 13.6% of the total protein in the particle. In some embodiments, the fusogen is (or is identified as being) more or less abundant than one or more additional proteins of interest. In an embodiment, the fusogen has (or is identified as having) a ratio to EGFP of about 140, 145, 150, 151, 152, 153, 154, 155, 156, 157 (e.g., 156.9), 158, 159, 160, 165, or 170. In another embodiment, the fusogen has (or is identified as having) a ratio to CD63 of about 2700, 2800, 2900, 2910 (e.g., 2912), 2920, 2930, 2940, 2950, 2960, 2970, 2980, 2990, or 3000, or about 1000-5000, 2000-4000, 2500-3500, 2900-2930, 2910-2915, or 2912.0, e.g., by a mass spectrometry assay. In an embodiment, the fusogen has (or is identified as having) a ratio to ARRDC1 of about 600, 610, 620, 630, 640, 650, 660 (e.g., 664.9), 670, 680, 690, or 700. In another embodiment, the fusogen has (or is identified as having) a ratio to GAPDH of about 50, 55, 60, 65, 70 (e.g., 69), 75, 80, or 85, or about 1-30%, 5-20%, 10-15%, 12-15%, 13-14%, or 13.6%. In another embodiment, the fusogen has (or is identified as having) a ratio to CNX of about 500, 510, 520, 530, 540, 550, 560 (e.g., 558.4), 570, 580, 590, or 600, or about 300-800, 400-700, 500-600, 520-590, 530-580, 540-570, 550-560, or 558.4, e.g., by a mass spectrometry assay.
[0363] In some embodiments, the fusogen is a protein fusogen. In some embodiments, the fusogen is a native protein or a derivative of a native protein. In some embodiments, the fusogen is a synthetic protein. In some embodiments, the fusogen is a mammalian protein or a homologue of a mammalian protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the mammalian protein), such as any described in Section II-B-1. In some embodiments, the fusogen is a non-mammalian protein. In some embodiments, the fusogen is a viral protein or a homologue of a viral protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the viral protein), such as any described in Section II-B-2. In some embodiments, the fusogen is a fragment of any of the foregoing. In some embodiments, the fusogen is a variant of any of the foregoing. In some embodiments, the fusogen is a protein fusion containing one or more proteins or fragments thereof. In some embodiments, the fusogen is a protein fusion containing one or more of any of the foregoing.
[0364] In some embodiments, the fusogen is mutated to reduce binding for the native binding partner of the fusogen. In some embodiments the fusogen is randomly mutated. In some embodiments the fusogen is rationally mutated. In some embodiments the fusogen is subjected to directed evolution. In some embodiments the fusogen is truncated and only a subset of the peptide is used in the particle.
1. Mammalian Proteins
[0365] In some embodiments, the fusogen is or contains a mammalian protein. Exemplary mammalian fusogens include a SNARE family protein such as vSNAREs or tSNAREs, a syncytin protein such as Syncytin-1 (DOI: 10.1128/JVI.76.13.6442-6452.2002) and Syncytin-2, myomaker (biorxiv.org/content/early/2017/04/02/123158, doi.org/10.1101/123158, doi: 10.1096/fj.201600945R, doi: 10.1038/nature 12343), myomixer (www.nature.com/nature/journal/v499/n7458/full/naturel2343.html, doi: 10.1038/nature 12343), myomerger (science.sciencemag.org/content/early/2017/04/05/science.aam9361, DOI: 10.1126/science.aam9361), FGFRL1 (fibroblast growth factor receptor-like 1), Minion (doi.org/10.1101/122697), an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (e.g., as disclosed in US 6,099,857A), a gap junction protein such as connexin 43, connexin 40, connexin 45, connexin 32, or connexin 37 (e.g., as disclosed in US 2007/0224176), Hap2, any protein capable of inducing syncytium formation between heterologous cells (see Table 2), any protein with fusogenic properties, a homologue thereof, a fragment thereof, a variant thereof, and a protein fusion containing one or more proteins or fragments thereof, such as one or more of any of the foregoing. In some embodiments, the fusogen is encoded by a human endogenous retroviral element (hERV) found in the human genome. Additional exemplary fusogens are disclosed in US 6,099,857A and US 2007/0224176, the entire contents of each of which are hereby incorporated by reference.
2. Viral Proteins
[0366] In some embodiments, the fusogen is or contains a non-mammalian protein. In some embodiments, the fusogen is or contains a viral protein. In some embodiments, the fusogen is a viral fusion protein. In some embodiments, the fusogen is a viral envelope protein.
[0367] In some embodiments, a viral fusogen is a Class I viral membrane fusion protein, a Class II viral membrane protein, a Class III viral membrane fusion protein, a viral membrane glycoprotein, or other viral fusion proteins, or a homologue thereof, a fragment thereof, a variant thereof, or a protein fusion containing one or more proteins or fragments thereof, such as one or more of any of the foregoing. [0368] In some embodiments, Class I viral membrane fusion proteins include Baculovirus F protein, e.g., F proteins of the nucleopolyhedrovirus (NPV) genera, e.g., Spodoptera exigua MNPV (SeMNPV) F protein and Lymantria dispar MNPV (LdMNPV), and paramyxovirus F proteins.
[0369] In some embodiments, Class II viral membrane proteins include tick bone encephalitis E (TBEV E) and Semliki Forest Virus E1/E2.
[0370] In some embodiments, Class III viral membrane fusion proteins include rhabdovirus G (e.g., fusogenic protein G of the Vesicular Stomatatis Virus (VSV-G), Cocal virus G protein), herpesvirus glycoprotein B (e.g., Herpes Simplex virus 1 (HSV-1) gB), Epstein Barr Virus glycoprotein B (EBV gB), thogotovirus G, baculovirus gp64 (e.g., Autographa California multiple NPV (AcMNPV) gp64), and Borna disease virus (BDV) glycoprotein (BDV G).
[0371] Examples of other viral fusogens, e.g., membrane glycoproteins and viral fusion proteins, include viral syncytia proteins such as influenza hemagglutinin (HA) or mutants, or fusion proteins thereof; human immunodeficiency virus type 1 envelope protein (HIV-1 ENV), gpl20 from HIV binding LFA-1 to form lymphocyte syncytium, HIV gp41, HIV gpl60, or HIV Trans- Activator of Transcription (TAT); viral glycoprotein VSV-G, viral glycoprotein from vesicular stomatitis virus of the Rhabdoviridae family; glycoproteins gB and gH-gL of the varicella-zoster virus (VZV); murine leukaemia virus (MLV)-lOAl; Gibbon Ape Leukemia Virus glycoprotein (GaLV); type G glycoproteins in Rabies, Mokola, vesicular stomatitis virus and Togaviruses; murine hepatitis virus JHM surface projection protein; porcine respiratory coronavirus spike- and membrane glycoproteins; avian infectious bronchitis spike glycoprotein and its precursor; bovine enteric coronavirus spike protein; the F and H, HN or G genes of a Morbillivirus (e.g., measles virus (MeV), canine distemper virus, Cetacean morbilli virus, Peste-des-petits-ruminants virus, Phocine distemper virus, Rinderpest virus), Newcastle disease virus, human parainfluenza virus 3, simian virus 41, Sendai virus and human respiratory syncytial virus; gH of human herpesvirus 1 and simian varicella virus, with the chaperone protein gL; human, bovine and cercopithicine herpesvirus gB; envelope glycoproteins of Friend murine leukaemia virus and Mason Pfizer monkey virus; mumps virus hemagglutinin neuraminidase, and glycoproteins Fl and F2; membrane glycoproteins from Venezuelan equine encephalomyelitis; paramyxovirus F protein; SIV gpl60 protein; Ebola virus G protein; or Sendai virus fusion protein, or a homologue thereof, a fragment thereof, a variant thereof, and a protein fusion containing one or more proteins or fragments thereof, such as one or more of any of the foregoing.
[0372] Non-mammalian fusogens include viral fusogens, homologues thereof, fragments thereof, and fusion proteins comprising one or more proteins or fragments thereof, such as one or more of any of the foregoing. Viral fusogens include class I fusogens, class II fusogens, class III fusogens, and class IV fusogens. In some embodiments, class I fusogens such as human immunodeficiency virus (HIV) gp41 have a characteristic postfusion conformation with a signature trimer of a-helical hairpins with a central coiled-coil structure. Class I viral fusion proteins include proteins having a central postfusion six-helix bundle. Class I viral fusion proteins include influenza HA, parainfluenza F, HIV Env, Ebola GP, hemagglutinins from orthomyxoviruses, F proteins from paramyxoviruses (e.g. Measles, (Katoh et al. BMC Biotechnology 2010, 10:37)), ENV proteins from retroviruses, and fusogens of filoviruses and coronaviruses. In some embodiments, class II viral fusogens such as dengue E glycoprotein, have a structural signature of P- sheets forming an elongated ectodomain that refolds to result in a trimer of hairpins. In some embodiments, the class II viral fusogen lacks the central coiled coil. Class II viral fusogen can be found in alphaviruses (e.g., El protein) and flaviviruses (e.g., E glycoproteins). Class II viral fusogens include fusogens from Semliki Forest virus, Sinbis, rubella virus, and dengue virus. In some embodiments, class III viral fusogens such as the vesicular stomatitis virus G glycoprotein, combine structural signatures found in classes I and II. In some embodiments, a class III viral fusogen comprises a helices (e.g., forming a six-helix bundle to fold back the protein as with class I viral fusogens), and sheets with an amphiphilic fusion peptide at its end, reminiscent of class II viral fusogens. Class III viral fusogens can be found in rhabdo viruses and herpesviruses. In some embodiments, class IV viral fusogens are fusion-associated small transmembrane (FAST) proteins (doi:10.1038/sj.emboj.7600767, Nesbitt, Rae L., "Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins" (2012). Electronic Thesis and Dissertation Repository. Paper 388), which are encoded by nonenveloped reoviruses. In some embodiments, the class IV viral fusogens are sufficiently small that they do not form hairpins (doi: 10.1146/annurev-cellbio- 101512-122422, doi:10.1016/j.devcel.2007.12.008).
[0373] In some embodiments, the fusogen is any of the fusogenic moieties described in WO2017/182585; WO2022/164935; WO2021/076788; Hamilton et al. bioRxiv 2022.08.24.505004; Nikolic et al. Nat Commun 9, 1029 (2018); Dobson et al. Nat. Methods. 19, 449-460 (2022); and Yu et al. bioRxiv 2021.12.13.472464, for instance any of the VSV or variant VSV glycoproteins described therein, such as VSV glycoproteins that have reduced binding to native receptors. a. Baboon Endogenous Retrovirus
[0374] In some embodiments, the fusogen is a Baboon Endogenous Retrovirus (BaEV) envelope glycoprotein. Exemplary BaEV envelope glycoproteins and variants thereof are described in PCT/US2022/031459; US9249426; Aguila et al. Journal of Virology 2003 77(2):1281-1291; Bernadin et al. Blood Advances 2019 3(3):461-475; Colamartino et al. Frontiers in Immunology 2019 10:2873; Girard-Gagnepain et al. Blood 2014 124(8): 1221-1231; and Levy et al. Journal of Thrombosis and Haemostasis 2016 14:2478-2492.
[0375] Wild-type BaEV envelope glycoproteins are retroviral envelope proteins containing a C- terminal cytoplasmic tail (e.g., corresponding to amino acids 512-545 of SEQ ID NO:252), a transmembrane domain (e.g., corresponding to amino acids 489-511 of SEQ ID NO:252), and an extracellular domain (e.g., corresponding to amino acids 1-488 of SEQ ID NO:252). Maturation of the precursor protein in the Golgi, which requires the minimal sequence [KR]-X-[KR]-R (wherein X is any amino acid), results in two subunits, the surface unit protein or gp70, and the transmembrane protein p20E. The surface unit protein or gp70 (e.g., corresponding to amino acids 1-358 of SEQ ID NO:252) and the transmembrane protein p20E (e.g., corresponding to amino acids 359-545 of SEQ ID NO:252) remain associated in a labile interaction that may include a disulfide bond. In wild-type BaEV envelope glycoproteins, fusogenicity is controlled by a short, 17 amino acid sequence termed a fusion inhibitory R peptide (e.g., set forth in SEQ ID NO:253), which is localized on the C-terminal of the cytoplasmic tail domain. The fusion inhibitory R peptide harbors the tyrosine endocytosis signal YXXL, and its cleavage by the viral protease is thought to potentiate fusogenic activation through molecular rearrangements in the membrane-spanning domain and the extracellular region of the envelope glycoprotein (Salamango et al (2015) Journal of virology 89(24): 12492-12500). In wild-type BaEV envelope glycoproteins, the gp70 mediates receptor binding to the ASCT-2 and ASCT-1 receptors on host cells. In some embodiments, the glycoprotein 70 (g70) subunit or a biologically active portion thereof binds the ASCT-2 and ASCT-1 receptors. In wild-type BaEV envelope glycoproteins, the p20E acts as a class I viral fusion protein. The interaction of the gp70 subunit with a host cell membrane triggers refolding of the p20E and is believed to activate the fusogenic potential by unmasking the fusion peptide.
[0376] In some embodiments, the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
[0377] In some embodiments, the fusogen is a truncated BaEV envelope glycoprotein. Exemplary BaEV envelope glycoproteins and truncates thereof are described in PCT/US2022/031459. In some embodiments, the truncated BaEV envelope glycoprotein comprises a cytoplasmic tail with a partial fusion inhibitory R peptide relative to a wild-type BaEV envelope glycoprotein, wherein the R peptide contains a contiguous portion of the inhibitory R peptide but lacks the full length R peptide of wild-type BaEV envelope glycoprotein. In some embodiments, the truncated BaEV envelope glycoprotein has a cytoplasmic tail that is composed of a partial inhibitory R peptide with at least one, at least two, or at least three contiguous amino-terminal amino acids of the inhibitory R peptide but less than the full-length R peptide relative to wild-type BaEV envelope glycoprotein. In some embodiments, the truncated BaEV envelope glycoprotein has a cytoplasmic tail that has a partial inhibitory R peptide composed of 1 to 16 contiguous amino-terminal amino acids of the inhibitory R peptide of the wild- type BaEV envelope glycoprotein, such as is composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 12, 14, 15 or 16 amino-terminal amino acids of the inhibitory R peptide of the wild-type BaEV envelope glycoprotein. In some embodiments, the truncated BaEV envelope glycoprotein is set forth in any of SEQ ID NO:254-260. In som embodiments, the truncated BaEV envelope glycoprotein s BaEV R+8 set fort hin SEQ ID NO:259.
[0378] In some embodiments, the fusogen is a modified BaEV envelope glycoprotein. In some embodiments, the cytoplasmic tail domain of the BaEV envelope glycoprotein is devoid of the fusion inhibitory R peptide. The expression “fusion inhibitory R peptide” refers to the C-terminal portion of the cytoplasmic tail domain of the envelope glycoprotein which harbours a tyrosine endocytosis signal — YXXL — and which is cleaved by viral protease during virion maturation, thus enhancing membrane fusion of the envelope glycoprotein. The fusion inhibitory R peptide of the BaEV envelope glycoprotein is typically located between amino acids 547 and 564 of the wild-type BaEV envelope glycoprotein. In some embodiments, the modified BaEV envelope glycoprotein is set forth in SEQ ID NO: 261 (BaEVRLess).
[0379] In some embodiments, the cytoplasmic tail domain of the BaEV envelope glycoprotein is replaced by the cytoplasmic tail domain of a murine leukemia virus (MLV) envelope glycoprotein. The Murine Leukemia Virus envelope glycoprotein is notably described in Ott et al. (1990) J. Virol. 64:757- 766. In some embodiments, the Murine Leukemia Virus envelope glycoprotein is that of strain 4070A. The term “MLV envelope glycoprotein” refers to the wild-type form of the MLV envelope glycoprotein or to a mutant of said wild-type MLV envelope glycoprotein which is at least 80%, preferably at least 85%, still preferably at least 90%, more preferably at least 95%, still more preferably at least 99% identical to said wild-type MLV envelope glycoprotein, provided that said mutant glycoprotein retains the capacity of the wild-type envelope glycoprotein of interacting with viral core proteins, in particular with lentiviral core proteins. Typically, the cytoplasmic tail domain of the MLV envelope glycoprotein is located between amino acids 622 and 654 of the wild-type MLV envelope glycoprotein. In some embodiments, the fusogen is BaEVTR. In some embodiments, the modified BaEV envelope glycoprotein is set forth in SEQ ID NO: 262 (BaEVTR).
[0380] In some embodiments, the fusogen is able to target the CD34+ progenitor subpopulations indicated in FIG. 7 for BaEVTR. In some embodiments, the fusogen is able to target HSCs, MPPs, CMPs, MLPs, or ETPs. In some embodiments, the fusogen is able to target HSCs, MPPs, CMPs, MLPs, and ETPs. In some embodiments, the target cells are HSCs, MPPs, CMPs, MLPs, or ETPs. In some embodiments, the target cells are MPPs, CMPs, MLPs, and ETPs.
[0381] In some embodiments where a VLP includes a BaEVTR glycoprotein fusogen (e.g., a fusogen comprising a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof or a truncated BaEV envelope glycoprotein or a functional variant thereof), the VLP also comprises a gag protein (e.g. an MLV-gag protein). In some embodiments, the MLV-gag protein is part of a fusion protein, where the the fusion protein is a cleavable fusion protein between (i) a viral structural protein (e.g. GAG (e.g. MLV-gag or HIV-gag)) and (ii) a nuclease protein (e.g. Cas protein (e.g. any of the Cas protein described herein). In some embodiments where the VLP includes a BaEVTR glycoprotein fusogen and a gag protein (e.g. an MLV-gag protein), production of such a VLP includes varying the ratio of polynucleotide encoding the BaEVTR glycoprotein fusogen and the polynucleotide encoding the gag protein. In some embodiments, the ratio of polynucleotide encoding the BaEVTR glycoprotein fusogen to the polynucleotide encoding the gag protein includes a 1:1, 1:2, 1:3, 1:4, or 1:5 ratio. b. G/H Proteins
[0382] In some embodiments, the fusogen is or contains a G or H protein. In some embodiments, the G or H protein is a Paramyxovirus (e.g., Morbillivirus or Henipavirus) G or H protein or a biologically active portion thereof. In some embodiments, the Henipavirus G protein is a Hendr a (HeV) virus G protein, a Nipah (NiV) virus G-protein (NiV-G), a Cedar (CedPV) virus G-protein, a Mojiang virus G- protein, a bat Paramyxovirus G-protein, a Kumasi virus G-protein, a Langya virus G-protein, or a biologically active portion thereof. A non-limited list of exemplary G proteins is shown in Table 1.
[0383] The Henipavirus attachment G proteins are type II transmembrane glycoproteins containing an N-terminal cytoplasmic tail (e.g., corresponding to amino acids 1-49 of SEQ ID NO:1), a transmembrane domain (e.g., corresponding to amino acids 50-70 of SEQ ID NO:1, and an extracellular domain containing an extracellular stalk (e.g., corresponding to amino acids 71-187 of SEQ ID NO:1), and a globular head (corresponding to amino acids 188-602 of SEQ ID NO:1). The N-terminal cytoplasmic domain is within the inner lumen of the lipid bilayer and the C-terminal portion is the extracellular domain that is exposed on the outside of the lipid bilayer. Regions of the stalk in the C- terminal region (e.g. corresponding to amino acids 159-167 of NiV-G) have been shown to be involved in interactions with F protein and triggering of F protein fusion (Liu et al. 2015 J of Virology 89:1838). In wild-type NiV-G protein, the globular head mediates receptor binding to henipavirus entry receptors ephrin B2 and ephrin B3, but is dispensable for membrane fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13)e00577-19).
[0384] In particular embodiments herein, tropism of the G protein is modified. Binding of the G protein to a binding partner can trigger fusion mediated by a compatible F protein or biologically active portion thereof. G protein sequences disclosed herein are predominantly disclosed as expressed sequences including an N-terminal methionine required for start of translation. As such N-terminal methionines are commonly cleaved co- or post-translationally, the mature protein sequences for all G protein sequences disclosed herein are also contemplated as lacking the N-terminal methionine.
[0385] G glycoproteins are highly conserved between henipavirus species. For example, the G protein of NiV and HeV viruses share 79% amino acids identity. Studies have shown a high degree of compatibility among G proteins with F proteins of different species as demonstrated by heterotypic fusion activation (Brandel-Tretheway et al. Journal of Virology. 2019). As described below, a retargeted lipid particle can contain heterologous proteins from different species.
[0386] In some embodiments, the G protein has a sequence set forth in any of SEQ ID NOs: 1-11 or is a functionally active variant or biologically active portion thereof that has a sequence that is at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% identical to any one of SEQ ID NOs:l, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11. In some embodiments, the G protein has a sequence set forth in SEQ ID NO:1 or is a functionally active variant or biologically active portion thereof that has a sequence that is at least at or about 80%, at least at or about 90%, at least at or about 95%, or at least at or about 99% identical to SEQ ID NO:1. In some embodiments, the G protein has a sequence set forth in SEQ ID N0:4 or is a functionally active variant or biologically active portion thereof that has a sequence that is at least at or about 80%, at least at or about 90%, at least at or about 95%, or at least at or about 99% identical to SEQ ID NO:4. In some embodiments, the G protein has a sequence set forth in SEQ ID NO:5 or is a functionally active variant or biologically active portion thereof that has a sequence that is at least at or about 80%, at least at or about 90%, at least at or about 95%, or at least at or about 99% identical to SEQ ID NO:5.
[0387] In particular embodiments, the G protein or functionally active variant or biologically active portion is a protein that retains fusogenic activity in conjunction with a Henipa virus F protein, e.g. NiV-F or HeV-F. Fusogenic activity includes the activity of the G protein in conjunction with a Henipavirus F protein to promote or facilitate fusion of two membrane lumens, such as the lumen of the targeted lipid particle having embedded in its lipid bilayer a henipavirus F and G protein, and a cytoplasm of a target cell, e.g. a cell that contains a surface receptor or molecule that is recognized or bound by the targeted envelope protein. In some embodiments, the F protein and G protein are from the same Henipavirus species (e.g. NiV-G and NiV-F). In some embodiments, the F protein and G protein are from different Henipavirus species (e.g. NiV-G and HeV-F).
[0388] In particular embodiments, the G protein has the sequence of amino acids set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10 or SEQ ID NO: 11 or is a functionally active variant thereof or a biologically active portion thereof that retains fusogenic activity. In some embodiments, the functionally active variant comprises an amino acid sequence having at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 1, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10 or SEQ ID NO: 11 and retains fusogenic activity in conjunction with a Henipavirus F protein (e.g., NiV-F or HeV-F). In some embodiments, the biologically active portion has an amino acid sequence having at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:1, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10 or SEQ ID NO: 11 and retains fusogenic activity in conjunction with a Henipavirus F protein (e.g., NiV-F or HeV-F).
[0389] Reference to retaining fusogenic activity includes activity (in conjunction with a Henipavirus F protein) that is between at or about 10% and at or about 150% or more of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO: 1, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10 or SEQ ID NO: 11 such as at least or at least about 10% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 15% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 20% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 25% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 30% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 35% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 40% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 45% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 50% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 55% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 60% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 65% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 70% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 75% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 80% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 85% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 90% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 95% of the level or degree of fusogenic activity of the corresponding wild-type G protein, such as at least or at least about 100% of the level or degree of fusogenic activity of the corresponding wild-type G protein, or such as at least or at least about 120% of the level or degree of fusogenic activity of the corresponding wild-type G protein.
1) Truncated Paramyxovirus G/H proteins
[0390] In some embodiments the G protein is a mutant G protein that is a functionally active variant or biologically active portion containing one or more amino acid mutations, such as one or more amino acid insertions, deletions, substitutions or truncations. In some embodiments, the mutations described herein relate to amino acid insertions, deletions, substitutions or truncations of amino acids compared to a reference G protein sequence. In some embodiments, the reference G protein sequence is the wild- type sequence of a G protein or a biologically active portion thereof. In some embodiments, the functionally active variant or the biologically active portion thereof is a mutant of a wild-type Hendra (HeV) virus G protein, a wild-type Nipah (NiV) virus G-protein (NiV-G), a wild-type Cedar (CedPV) virus G-protein, a wild-type Mojiang virus G-protein, a wild-type bat Paramyxovirus G-protein, a Kumasi virus G-protein, a Langya virus G-protein, or biologically active portion thereof. In some embodiments, the wild-type G protein has the sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:7, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10 or SEQ ID NO: 11.
[0391] In some embodiments, the G protein is a mutant G protein that is a biologically active portion that is an N-terminally and/or C-terminally truncated fragment of a wild- type Hendra (HeV) virus G protein, a wild-type Nipah (NiV) virus G-protein (NiV-G), a wild-type Cedar (CedPV) virus G- protein, a wild-type Mojiang virus G-protein, a wild-type bat Paramyxovirus G-protein, a Kumasi virus G-protein, or a Langya virus G-protein. In particular embodiments, the truncation is an N-terminal truncation of all or a portion of the cytoplasmic domain. In some embodiments, the mutant G protein is a biologically active portion that is truncated and lacks up to 49 contiguous amino acid residues at or near the N-terminus of the wild-type G protein, such as a wild-type G protein set forth in any one of SEQ ID NO:1, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:7, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11. In some embodiments, the mutant F protein is truncated and lacks up to 49 contiguous amino acids, such as up to 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 30, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 contiguous amino acids at the N-terminus of the wild-type G protein.
[0392] In some embodiments, the G protein is a wild-type Nipah virus G (NiV-G) protein or a Hendra virus G protein, or is a functionally active variant or biologically active portion thereof. In some embodiments, the G protein is a NiV-G protein that has the sequence set forth in SEQ ID NO:1, SEQ ID NOG or SEQ ID NOG, or is a functional variant or a biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99% sequence identity to SEQ ID NO:1, SEQ ID NOG or SEQ ID NOG. In some embodiments, the G protein is a NiV-G protein that has the sequence set forth in SEQ ID NO:1, or is a functional variant or a biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99% sequence identity to SEQ ID NO:1. In some embodiments, the G protein is a NiV-G protein that has the sequence set forth in SEQ ID NO:1. In some embodiments, the G protein is a NiV-G protein that has the sequence set forth in SEQ ID N0:4, or is a functional variant or a biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99% sequence identity to SEQ ID NO:4. In some embodiments, the G protein is a NiV-G protein that has the sequence set forth in SEQ ID NO:4. In some embodiments, the G protein is a NiV-G protein that has the sequence set forth in SEQ ID NO:5, or is a functional variant or a biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99% sequence identity to SEQ ID NO:5. In some embodiments, the G protein is a NiV-G protein that has the sequence set forth in SEQ ID NO:5.
[0393] In some embodiments, the G protein is a mutant NiV-G protein that is a biologically active portion of a wild-type NiV-G. In some embodiments, the biologically active portion is an N- terminally truncated fragment. In some embodiments, the mutant NiV-G protein is truncated and lacks up to 5 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 6 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 7 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 8 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 9 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5) up to 10 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 11 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 12 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), up to 13 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 14 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 15 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5) up to 16 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 17 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 18 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 19 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 20 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 21 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 22 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 23 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 24 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 25 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 26 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 27 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 28 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 29 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 30 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 31 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 32 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 33 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 34 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 35 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 36 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 37 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5) up to 38 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 39 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 40 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 41 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 42 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 43 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 44 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), or up to 45 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5).
[0394] In some embodiments, the mutant NiV-G protein is truncated and lacks 5 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the mutant NiV-G protein is truncated and lacks 10 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:44. In some embodiments, the mutant NiV-G protein is truncated and lacks 15 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:45. In some embodiments, the mutant NiV-G protein is truncated and lacks 20 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the mutant NiV-G protein is truncated and lacks 25 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the mutant NiV-G protein is truncated and lacks 30 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the mutant NiV-G protein is truncated and lacks 34 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:42.
[0395] In some embodiments, the NiV-G protein is a biologically active portion that does not contain a cytoplasmic domain. In some embodiments, the NiV-G protein without the cytoplasmic domain is encoded by SEQ ID NO:22. [0396] In some embodiments, the mutant NiV-G protein comprises a sequence set forth in any of SEQ ID NOS: 12-14, 17, 18 and 22, or 42-45 or is a functional variant thereof that has an amino acid sequence having at least at or 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NOS: 12-14, 17, 18 and 22 or 42-45.
[0397] In some embodiments, the mutant NiV-G protein has a 5 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO: 12 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 12 or such as set forth in SEQ ID NO: 17 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 17. In some embodiments, the mutant NiV-G protein has a 10 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO:44 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:44. In some embodiments, the mutant NiV-G protein has a 20 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO: 13 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 13. In some embodiments, the mutant NiV-G protein has a 25 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO: 14 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 14. In some embodiments, the mutant NiV-G protein has a 33 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO: 17 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 17. In some embodiments, the mutant NiV-G protein has a 34 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO: 18 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:18. In some embodiments, the mutant NiV-G protein has a 48 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), such as set forth in SEQ ID NO:22 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:22.
[0398] In some embodiments, the mutant NiV-G protein has a 15 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), such as set forth in SEQ ID NO:45 or a functional variant thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:45.
[0399] In some embodiments, the mutant NiV-G protein has a 20 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), such as set forth in SEQ ID NO: 13 or a functional variant thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 13.
[0400] In some embodiments, the mutant NiV-G protein has a 25 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), such as set forth in SEQ ID NO: 14 or a functional variant thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 14.
[0401] In some embodiments, the mutant NiV-G protein has a 30 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), such as set forth in SEQ ID NO:43 or a functional variant thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:43.
[0402] In some embodiments, the mutant NiV-G protein has a 34 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:, SEQ ID NO:4, or SEQ ID NO:5), such as set forth in SEQ ID NO:42 or a functional variant thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:42.
[0403] In some embodiments, the mutant NiV-G protein has a 48 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), such as set forth in SEQ ID NO:22 or a functional variant thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:22.
[0404] In some embodiments, the G protein is a mutant HeV-G protein that is a biologically active portion of a wild- type HeV-G. In some embodiments, the biologically active portion is an N-terminally truncated fragment.
[0405] In some embodiments, the G protein is a wild-type HeV-G protein that has the sequence set forth in SEQ ID NO:23 or 24, or is a functional variant or biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at or about 85%, at least at or about 86%, at least at or about 87%, at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:23or 24.
[0406] In some embodiments, the G protein is a mutant HeV-G protein that is a biologically active portion of a wild-type HeV-G (SEQ ID NO:23 or SEQ ID NO:24). In some embodiments, the biologically active portion is an N-terminally truncated fragment. In some embodiments, the mutant HeV-G protein is truncated and lacks up to 5 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 6 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 7 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24) or up to 8 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 9 contiguous amino acid residues at or near the N- terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 10 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 11 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 12 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 13 contiguous amino acid residues at or near the N- terminus of the wild-type HeV-G protein ( SEQ ID NO:23 or 24), up to 14 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 15 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 16 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 17 contiguous amino acid residues at or near the N- terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 18 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 19 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 20 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 21 contiguous amino acid residues at or near the N- terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 22 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 23 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein SEQ ID NO:23 or 24), up to 24 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 25 contiguous amino acid residues at or near the N- terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 26 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 27 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 28 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 29 contiguous amino acid residues at or near the N- terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 30 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 31 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 32 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 33 contiguous amino acid residues at or near the N- terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 34 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 35 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 36 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 37 contiguous amino acid residues at or near the N- terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 38 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 39 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 41 contiguous amino acid residues at or near the N- terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 42 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 43 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), up to 44 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24), or up to 45 contiguous amino acid residues at or near the N- terminus of the wild-type HeV-G protein (SEQ ID NO:23 or 24).
[0407] In some embodiments, the HeV-G protein is a biologically active portion that does not contain a cytoplasmic domain. In some embodiments, the mutant HeV-G protein lacks the N-terminal cytoplasmic domain of the wild-type HeV-G protein (SEQ ID NO:23 or 24), such as set forth in SEQ ID NO:25 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:25. In some embodiments, the mutant HeV-G protein lacks the N-terminal cytoplasmic domain of the wild-type HeV-G protein (SEQ ID NO:23 or 24), such as set forth in SEQ ID NO:26 or a functional variant thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:26.
[0408] In some embodiments, the G protein or the functionally active variant or biologically active portion thereof binds to Ephrin B2 or Ephrin B3. In some aspects, the G protein has the sequence of amino acids set forth in any one of SEQ ID NO:24, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or is a functionally active variant thereof or a biologically active portion thereof that is able to bind to Ephrin B2 or Ephrin B3. In some embodiments, the functionally active variant or biologically active portion has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at or about 86%, at least at or about 87%, at least at or about 88%, or at least at or about 89% , at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to any of SEQ ID NO:24, SEQ ID NO:23, SEQ ID N0:4, SEQ ID N0:6, SEQ ID N0:5, SEQ ID N0:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, and retains binding to Ephrin B2 or B3.
[0409] In some embodiments, the functionally active variant or biologically active portion has an amino acid sequence having at least about 80%, at least about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, and retains binding to Ephrin B2 or B3. Reference to retaining binding to Ephrin B2 or B3 includes binding that is at least or at least about 5% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, 10% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, 15% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, 20% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, 25% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion, 30% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, 35% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, 40% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, 45% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, 50% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, 55% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, 60% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, 65% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10,, or a functionally active variant or biologically active portion thereof, 70% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10 or a functionally active variant or biologically active portion thereof, such as at least or at least about 75% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, such as at least or at least about 80% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, NO:4,SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:10„ or a functionally active variant or biologically active portion thereof, such as at least or at least about 85% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, such as at least or at least about 90% of the level or degree of binding of the corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO: 10, or a functionally active variant or biologically active portion thereof, or such as at least or at least about 95% of the level or degree of binding of the corresponding wild-type protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:10„ or a functionally active variant or biologically active portion thereof. In some embodiments, the G protein is NiV-G or a functionally active variant or biologically active portion thereof and binds to Ephrin B2 or Ephrin B3. In some aspects, the NiV-G has the sequence of amino acids set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, or is a functionally active variant thereof or a biologically active portion thereof that is able to bind to Ephrin B2 or Ephrin B3. In some embodiments, the functionally active variant or biologically active portion has an amino acid sequence having at least about 80%, at least about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27 and retains binding to Ephrin B2 or B3. Exemplary biologically active portions include N-terminally truncated variants lacking all or a portion of the cytoplasmic domain, e.g. 1 or more, such as 1 to 49 contiguous N-terminal amino acid residues. Reference to retaining binding to Ephrin B2 or B3 includes binding that is at least or at least about 5% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 10% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 15% of the level or degree of binding of the corresponding wildtype NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 20% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 25% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 30% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 35% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 40% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 45% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27 50% of the level or degree of binding of the corresponding wildtype NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 55% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 60% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 65% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 70% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, such as at least or at least about 75% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, such as at least or at least about 80% of the level or degree of binding of the corresponding wild-type NIV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, such as at least or at least about 85% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, such as at least or at least about 90% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, or such as at least or at least about 95% of the level or degree of binding of the corresponding wild-type NiV-G, such as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27.
[0410] In some embodiments, the G protein or the biologically thereof is a mutant G protein that exhibits reduced binding for the native binding partner of a wild-type G protein. In some embodiments, the mutant G protein or the biologically active portion thereof is a mutant of wild-type Niv-G and exhibits reduced binding to one or both of the native binding partners Ephrin B2 or Ephrin B3. In some embodiments, the mutant G-protein or the biologically active portion, such as a mutant NiV-G protein, exhibits reduced binding to the native binding partner. In some embodiments, the reduced binding to Ephrin B2 or Ephrin B3 is reduced by greater than at or about 5%, at or about 10%, at or about 15%, at or about 20%, at or about 25%, at or about 30%, at or about 40%, at or about 50%, at or about 60%, at or about 70%, at or about 80%, at or about 90%, or at or about 100%.
[0411] In some embodiments, the mutations described herein can improve transduction efficiency. In some embodiments, the mutations described herein allow for specific targeting of other desired cell types that are not Ephrin B2 or Ephrin B3. In some embodiments, the mutations described herein result in at least the partial inability to bind at least one natural receptor, such has reduce the binding to at least one of Ephrin B2 or Ephrin B3. In some embodiments, the mutations described herein interfere with natural receptor recognition.
[0412] In some embodiments, the G protein is HeV-G or a functionally active variant or biologically active portion thereof and binds to Ephrin B2 or Ephrin B3. In some aspects, the HeV- G has the sequence of amino acids set forth in SEQ ID NO:23 or 24, or is a functionally active variant thereof or a biologically active portion thereof that is able to bind to Ephrin B2 or Ephrin B3. In some embodiments, the functionally active variant or biologically active portion has an amino acid sequence having at least about 80%, at least about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:23 or 24 and retains binding to Ephrin B2 or B3. Exemplary biologically active portions include N- terminally truncated variants lacking all or a portion of the cytoplasmic domain, e.g. 1 or more, such as 1 to 49 contiguous N-terminal amino acid residues. Reference to retaining binding to Ephrin B2 or B3 includes binding that is at least or at least about 5% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 10% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 15% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 20% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 25% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 30% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 35% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 40% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 45% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 50% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 55% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 60% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 65% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, 70% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, such as at least or at least about 75% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, such as at least or at least about 80% of the level or degree of binding of the corresponding wild-type NIV-G, such as set forth in SEQ ID NO:23 or 24, such as at least or at least about 85% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, such as at least or at least about 90% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24, or such as at least or at least about 95% of the level or degree of binding of the corresponding wild-type HeV-G, such as set forth in SEQ ID NO:23 or 24.
[0413] In some embodiments, the G protein or the biologically thereof is a mutant G protein that exhibits reduced binding for the native binding partner of a wild-type G protein. In some embodiments, the mutant G protein or the biologically active portion thereof is a mutant of wildtype Niv-G and exhibits reduced binding to one or both of the native binding partners Ephrin B2 or Ephrin B3. In some embodiments, the mutant G-protein or the biologically active portion, such as a mutant NiV-G protein, exhibits reduced binding to the native binding partner. In some embodiments, the reduced binding to Ephrin B2 or Ephrin B3 is reduced by greater than at or about 5%, at or about 10%, at or about 15%, at or about 20%, at or about 25%, at or about 30%, at or about 40%, at or about 50%, at or about 60%, at or about 70%, at or about 80%, at or about 90%, or at or about 100%.
[0414] In some embodiments, the G protein contains one or more amino acid substitutions in a residue that is involved in the interaction with one or both of Ephrin B2 and Ephrin B3. In some embodiments, the amino acid substitutions correspond to mutations E501 A, W504A, Q530A and E533A with reference to numbering set forth in SEQ ID NO:4.
[0415] In some embodiments, the G protein is a mutant G protein. In some embodiments, the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A and E533A with reference to numbering set forth in SEQ ID NO:4. In some embodiments, the G protein is a mutant G protein that contains one or more amino acid substitutions elected from the group consisting of E501A, W504A, Q530A and E533A with reference to SEQ ID NO:4 and is a biologically active portion thereof containing an N-terminal truncation. In some embodiments, the mutant NiV-G protein or the biologically active portion thereof is truncated and lacks up to 5 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 6 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:4), 7 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 8 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 9 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), up to 10 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 11 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 12 contiguous amino acid residues at or near the N-terminus of the wildtype NiV-G protein (SEQ ID NO:4), 13 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 14 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:4), up to 15 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 16 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 17 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 18 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 19 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), up to 20 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 21 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4) 22 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:4), 23 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 24 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), up to 25 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 26 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 27 contiguous amino acid residues at or near the N-terminus of the wild-type NiV- G protein (SEQ ID NO:4), 28 contiguous amino acid residues at or near the N-terminus of the wildtype NiV-G protein (SEQ ID NO:4), 29 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), up to 30 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), up to 31 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 32 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 33 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4) 34 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), 35 contiguous amino acid residues at or near the N-terminus of the wild-type NiV- G protein (SEQ ID NO:4) up to 36 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), up to 37 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein (SEQ ID NO:4), up to 38 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), up to 39 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:4), or up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4).
[0416] In some embodiments, the mutant NiV-G protein has the amino acid sequence set forth in SEQ ID NO: 17 or 18 or an amino acid sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 17 or 18. In particular embodiments, the G protein has the sequence of amino acids set forth in SEQ ID NO: 17 or 18. In some embodiments, the mutant NiV-G protein has the amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 17. In particular embodiments, the G protein has the sequence of amino acids set forth in SEQ ID NO 17. In some embodiments, the mutant NiV-G protein has the amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 18. In particular embodiments, the G protein has the sequence of amino acids set forth in SEQ ID NO 18.
[0417] In some embodiments, the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A and E533A with reference to numbering set forth in SEQ ID NO:4. In some embodiments, the G protein is a mutant G protein that contains one or more amino acid substitutions elected from the group consisting of E501 A, W504A, Q530A and E533A with reference to SEQ ID NO:4 and is a biologically active portion thereof containing an N-terminal truncation.
2) Mutated Paramyxovirus G/H Proteins
[0418] Provided herein are mutant Paramyxovirus G/H glycoproteins (e.g., variant Paramyxovirus G/H glycoproteins) comprising one or more amino acid mutations that result in decreased glycosylation of the protein. The one or more amino acid mutations, also called deglycosylation mutations, can be one or more amino acid substitutions (also referred to as mutations).
[0419] In some embodiments, the mutant Paramyxovirus G/H glycoprotein comprises an amino acid substitution at one or more amino acid positions that reduce glycosylation of the G/H glycoprotein. In some embodiments, the one or more amino acid substitutions disrupts an N-linked glycosylation site. In some embodiments, the one or more amino acid substitutions disrupts an O-linked glycosylation site.
[0420] In some embodiments, the mutant Paramyxovirus G/H glycoprotein is derived from Morbillivirus (e.g., measles virus (MeV), canine distemper virus, Cetacean morbilli virus, Peste-des- petits-ruminants virus, Phocine distemper virus, Rinderpest virus), Henipavirus (e.g., Hendra (HeV) virus, Nipah (NiV) virus, a Cedar (CedPV) virus, Mojiang virus, a Langya virus or bat Paramyxovirus). In some embodiments, the mutant Paramyxovirus G/H glycoprotein is a mutant of a Paramyxovirus G/H glycoprotein derived from Nipah virus or Measles virus. In some embodiments, the mutant Paramyxovirus G/H protein is selected from the group consisting of SEQ ID NOs: 2-11, 375, and 376, or a modified Paramyxovirus G/H glycoprotein derived from any one of 2-11, 375, and 376 containing an altered cytoplasmic tail . In some embodiments, the mutant Paramyxovirus G/H protein has a sequence of amino acids that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% to any one of SEQ ID NOs: 2-11, 375, and 376 and contains the acid substitution at one or more amino acid positions that reduce glycosylation of the G/H glycoprotein as provided herein.
[0421] The location of precited glycosylation sites can be determined using the sequence of a protein. For example, N-glycosylation often occurs at sites with the sequence N-X-S/T in which “X” is any amino acid except P. Various algorithms and tools are available for prediction of both N- and O- linked glycosylation, including SprintGly (http://sparks-lab.org/server/sprint-gly/), NetNGlyc (https://services.healtlttech.dtu.dk/service.php7NetNGlyc-l.0), NetOGlyc (https://services.healtlttech.dtu.dk/service.php?NetOGlyc-4.0), and GlycoMinestruct
(bttp://glycomine. erc.monash.edu/Lab/GlycoMi e.. Struct/), and methods described in Pitti et al., Sci. Reports, 9:15975 (2019) and Pakhrin et al., Molecules 26:7314 (2021). Any predicted glycosylation site may be substituted as described herein.
[0422] In some embodiments, the Paramyxovirus G/H glycoprotein to which the deglycosylation mutation is made is a NiV-G set forth in SEQ ID NO: 4 or a modified Nipah G glycoprotein (NiV-G) that has an altered cytoplasmic tail compared to native NiV-G (e.g., SEQ ID NO: 4). In some embodiments, the variant Paramyxovirus G/H protein has a sequence of amino acids that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% to SEQ ID NO: 4 and contains the acid substitution at one or more amino acid positions that reduce glycosylation of the G/H glycoprotein as provided herein Exemplary modified NiV- G proteins with altered cytoplasmic tails to which the one or more amino acid substitutions for reducing glycosylation can be incorporated are described in Section II.B.
[0423] Amino acid positions for substitutions are described herein with positions “corresponding to” positions of a reference sequence. It is understood that the amino acid substitutions are not limited to being made in only the reference sequence but also can be made in similar sequences by identification of residues that align or correspond with the reference positions. For instance, positions “corresponding to” to positions of a protein in a reference sequence can be identified upon alignment of a similar sequence with the referenced sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides. For instance, amino acid positions for mutations are described herein with reference to the exemplary truncated NiV-G sequence set forth in SEQ ID NO:42; however, similar amino acid positions for mutations as described can be made in other modified NiV-G sequences, such as any as described in Section II.B, by sequence alignment and identification of the corresponding residues.
[0424] In some embodiments, the one or more amino acid mutations are at positions corresponding to positions 39, 126, 128, 273, 345, 384, 448, and 496 of SEQ ID NO:42. In some embodiments, the variant Paramyxovirus G/H glycoprotein comprises an amino acid mutation at any one of positions 39, 126, 128, 273, 345, 384, 448, and 496 of SEQ ID NO:42. In some embodiments, the variant Paramyxovirus G/H glycoprotein comprises two or more amino acid mutations at any of positions corresponding to positions 39, 126, 128, 273, 345, 384, 448, and 496 of SEQ ID NO:42., such as mutations at 2, 3, 4, 5, 7, or 8 of the positions.
[0425] In some embodiments, the one or more amino acid mutations is at a position corresponding to position 39 of SEQ ID NO:42. In some embodiments, the one or more amino acid mutations is at a position corresponding to position 126 of SEQ ID NO:42. In some embodiments, the one or more amino acid mutations is at a position corresponding to position 128 of SEQ ID NO:42. In some embodiments, the one or more amino acid mutations is at a position corresponding to position 273 of SEQ ID NO: 42. In some embodiments, the one or more amino acid mutations is at a position corresponding to position 345 of SEQ ID NO:42. In some embodiments, the one or more amino acid mutations is at a position corresponding to position 384 of SEQ ID NO:42. In some embodiments, the one or more amino acid mutations is at a position corresponding to position 448 of SEQ ID NO:42. In some embodiments, the one or more amino acid mutations is at a position corresponding to position 496 of SEQ ID NO:42.
[0426] In some embodiments, the native amino acid at the position comprising the amino acid mutation is asparagine or serine. In some embodiments, the amino acid mutation is an amino acid substitution. In some embodiments, the mutation is an asparagine to glutamine substitution. In some embodiments, the mutation is a serine to alanine substitution.
[0427] In some embodiments, the mutation is an asparagine to glutamine substitution at a position corresponding to position 39 (N39Q) of SEQ ID NO:42. In some embodiments, the mutation is an asparagine to glutamine substitution at a position corresponding to position 126 (N126Q) of SEQ ID NO: 42. In some embodiments, the mutation is an asparagine to glutamine substitution at a position corresponding to position 273 (N273Q) of SEQ ID NO: 42. In some embodiments, the mutation is an asparagine to glutamine substitution at a position corresponding to position 345 (N345Q) of SEQ ID NO: 42. In some embodiments, the mutation is an asparagine to glutamine substitution at a position corresponding to position 384 (N384Q) of SEQ ID NO: 42. In some embodiments, the mutation is an asparagine to glutamine substitution at a position corresponding to position 448 (N448Q) of SEQ ID NO: 42. In some embodiments, the mutation is an asparagine to glutamine substitution at a position corresponding to position 496 (N496Q) of SEQ ID NO: 42.
[0428] In some embodiments, the mutation is a serine to alanine substitution at a position corresponding to position 128 (S128A) of SEQ ID NO: 42.
[0429] In some embodiments, the G/H glycoprotein is derived from Nipah virus G protein and the one or more amino acid substitutions are at positions corresponding to positions selected from the group consisting of 39, 126, 128, 273, 345, 384, 448, and 496 of SEQ ID NO: 42. In some embodiments, the one or more amino acid substitutions are selected from N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q or any combination thereof. In some embodiments, the G/H glycoprotein is a mutant NiV-G containing one amino acid substitution from any one of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G/H glycoprotein is a mutant NiV-G containing two amino acid substitutions from any two of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G/H glycoprotein is a mutant NiV-G containing three amino acid substitutions from any three of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G/H glycoprotein is a mutant NiV-G containing four amino acid substitutions from any one of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G/H glycoprotein is a mutant NiV-G containing five amino acid substitutions from any one of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G/H glycoprotein is a mutant NiV-G containing six amino acid substitutions from any one of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G/H glycoprotein is a mutant NiV-G containing seven amino acid substitutions from any one of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G/H glycoprotein is a mutant NiV-G containing eight amino acid substitutions from any one of N39Q, N126Q, S128A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the one or more amino acid substitutions are in the SEQ ID NO: 147 or a or a modified Nipah G glycoprotein (NiV-G) that has an altered cytoplasmic tail compared to native NiV-G (e.g., SEQ ID NO:42). In some embodiments, the amino acid substitutions are in a modified NiV-G protein described in Section II.B. In some embodiments, the amino acid substitutions are in the NiV-G set forth in SEQ ID NO:42.
[0430] In some embodiments, the variant Nipah-G protein comprises at least three amino acid substitutions. In some embodiments, the amino acid substitutions are at positions 273, 384, and 496 of SEQ ID NO:42. In some embodiments, the amino acid substitutions are at positions 273, 345, and 496 of SEQ ID NO:42. In some embodiments, the amino acid substitutions are at positions 39, 126, and 128 of SEQ ID NO:42. In some embodiments, the amino acid substitutions are at positions 39, 273, and 345 of SEQ ID NO:42. In some embodiments, the amino acid substitutions are at positions 39, 384, and 448 of SEQ ID NO:42. In some embodiments, the amino acid substitutions are at positions 39, 448, and 496 of SEQ ID NO:42. In some embodiments, the amino acid substitutions are at positions 39, 128, and 273 of SEQ ID NO:42. In some embodiments, the amino acid substitutions are at positions 39, 345, and 384 of SEQ ID NO:42. In some embodiments, the amino acid substitutions are at positions 39, 384, and 448 of SEQ ID NO:42.
[0431] In some embodiments, the variant Nipah-G protein comprises at least two amino acid substitutions. In some embodiments, the amino acid substitutions are at positions 273, and 496 of SEQ ID NO:42. In some embodiments, the amino acid substitutions are at positions 345, and 496 of SEQ ID NO:42. In some embodiments, the amino acid substitutions are at positions 39 and 128 of SEQ ID
NO:42. In some embodiments, the amino acid substitutions are at positions 39, and 345 of SEQ ID
NO:42. In some embodiments, the amino acid substitutions are at positions 39, and 448 of SEQ ID
NO:42. In some embodiments, the amino acid substitutions are at positions 39 and 496 of SEQ ID
NO:42. In some embodiments, the amino acid substitutions are at positions 39 and 273 of SEQ ID
NO:42. In some embodiments, the amino acid substitutions are at positions 39 and 384 of SEQ ID
NO:42. In some embodiments, the amino acid substitutions are at positions 384 and 448 of SEQ ID NO:42.
[0432] In some embodiments, the amino acid substitution is at position 39 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 126 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 128 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 273 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 345 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 384 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 448 of SEQ ID NO:42. In some embodiments, the amino acid substitution is at position 496 of SEQ ID NO:42.
[0433] In some embodiments, the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 39 of SEQ ID NO:42. In some embodiments, the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 126 of SEQ ID NO:42. In some embodiments, the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 273 of SEQ ID NO:42. In some embodiments, the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 345 of SEQ ID NO:42. In some embodiments, the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 384 of SEQ ID NO:42. In some embodiments, the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 448 of SEQ ID NO:42. In some embodiments, the mutant Nipah-G protein comprises an asparagine to glutamine substitution at position 496 of SEQ ID NO:42. In some embodiments, the mutant Nipah-G protein comprises a serine to alanine substitution at position 128 of SEQ ID NO:42.
[0434] In some embodiments, the mutant Nipah-G protein comprises the sequence selected from the group consisting of any one of SEQ ID NOs: 377-503, such as any exemplary mutant Nipah-G proteins set forth in Table 2 A below. In some embodiments, the mutant Nipah-G protein comprises the sequence of SEQ ID NO: 379. In some embodiments, the variant Nipah-G protein comprises the sequence of SEQ ID NO: 400. In some embodiments, the variant Nipah-G protein comprises the sequence of SEQ ID NO: 404. [0435] In some embodiments, the Paramyxovirus G/H glycoprotein to which the deglycosylation mutations is made is a Measles virus H (Mev-H) protein or a modified MeV-H protein that has an altered cytoplasmic tail compared to native MeV-H (e.g., SEQ ID NO:375). In some embodiments, the mutant Paramyxovirus G/H protein has a sequence of amino acids that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% to SEQ ID NO: 375 and contains the acid substitution at one or more amino acid positions that reduce glycosylation of the G/H glycoprotein as provided herein.
[0436] In some embodiments, the G/H glycoprotein is derived from Measles virus H (Mev-H) protein and the one or more amino acid substitutions are at positions corresponding to positions selected from the group consisting of 168, 187, 200, 215, 238 of SEQ ID NO: 375. In some embodiments, the mutant Mev-H protein comprises at least two amino acid substitutions, such as 2, 3, 4, or 5 substitutions at positions 168, 187, 200, 215, 238 of SEQ ID NO: 375.
[0437] In some embodiments, the Paramyxovirus G/H glycoprotein to which the deglycosylation mutations is made is a Canine distemper virus H (CDV-H) protein or a modified CDV-H protein that has an altered cytoplasmic tail compared to native CDV-H (e.g., SEQ ID NO:376). In some embodiments, the mutant Paramyxovirus G/H protein has a sequence of amino acids that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% to SEQ ID NO: 376 and contains the acid substitution at one or more amino acid positions that reduce glycosylation of the G/H glycoprotein as provided herein.
[0438] In some embodiments, the G/H glycoprotein is derived from Canine distemper virus H (CDV-H) protein and the one or more amino acid substitutions are at positions corresponding to positions selected from the group consisting of 19, 149, 422 of SEQ ID NO: 376. In some embodiments, the variant CDV-H protein comprises at least two amino acid substitutions, such as 2 or 3 substitutions at positions 19, 149, 422 of SEQ ID NO: 376. c. F Proteins
[0439] In some embodiments, the fusogen contains a protein with a hydrophobic fusion peptide domain. In some embodiments, the fusogen is or contains a F protein. In some embodiments, the fusogen contains a henipavirus F protein molecule or biologically active portion thereof. In some embodiments, the Henipavirus F protein is a Hendra (Hev) virus F protein, a Nipah (NiV) virus F-protein, a Cedar (CedPV) virus F protein, a Mojiang virus F protein, a bat Paramyxovirus F protein, a Kumasi virus F protein, a Eangya virus F protein, or a biologically active portion thereof.
[0440] Table 2 provides non-limiting examples of F proteins. In some embodiments, the N- terminal hydrophobic fusion peptide domain of the F protein molecule or biologically active portion thereof is exposed on the outside of lipid bilayer. [0441] F proteins of henipaviruses are encoded as Fo precursors containing a signal peptide (e.g. corresponding to amino acid residues 1-26 of SEQ ID NO:28). Following cleavage of the signal peptide, the mature Fo (e.g. SEQ ID NO:29) is transported to the cell surface, then endocytosed and cleaved by cathepsin L into the mature fusogenic subunits Fl and F2. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, the Fo comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the Fl subunit comprises the sequence amino acid sequence set forth in SEQ ID NO:46. In some embodiments, the F2 subunit comprises the sequence amino acid sequence set forth in SEQ ID NO:39. The Fl and F2 subunits are associated by a disulfide bond and recycled back to the cell surface. The Fl subunit contains the fusion peptide domain located at the N terminus of the Fl subunit, where it is able to insert into a cell membrane to drive fusion. In some aspects, fusion is blocked by association of the F protein with G protein, until the G protein engages with a target molecule resulting in its disassociation from F and exposure of the fusion peptide to mediate membrane fusion.
[0442] Among different henipavirus species, the sequence and activity of the F protein is highly conserved. For examples, the F protein of NiV and HeV viruses share 89% amino acid sequence identity. Further, in some cases, the henipavirus F proteins exhibit compatibility with G proteins from other species to trigger fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13):e00577-19). In some aspects or the provided re-targeted lipid particles, the F protein is heterologous to the G protein, i.e. the F and G protein or biologically active portions are from different henipavirus species. For example, the F protein is from Hendra virus and the G protein is from Nipah virus. In other aspects, the F protein can be a chimeric F protein containing regions of F proteins from different species of Henipavirus. In some embodiments, switching a region of amino acid residues of the F protein from one species of Henipavirus to another can result in fusion to the G protein of the species comprising the amino acid insertion. (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13):e00577-19). In some cases, the chimeric F protein contains an extracellular domain from one henipavirus species and a transmembrane and/or cytoplasmic domain from a different henipavirus species. For example, the F protein contains an extracellular domain of Hendra virus and a transmembrane/cytoplasmic domain of Nipah virus. F protein sequences disclosed herein are predominantly disclosed as expressed sequences including an N-terminal signal sequence. As such N-terminal signal sequences are commonly cleaved co- or post-translationally, the mature protein sequences for all F protein sequences disclosed herein are also contemplated as lacking the N-terminal signal sequence.
[0443] In some embodiments, the F protein is encoded by a nucleotide sequence that encodes the sequence set forth by any one of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or is a functionally active variant or a biologically active portion thereof that has a sequence that is at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% identical to any one of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the F protein is encoded by a nucleotide sequence that encodes the sequence set forth by any one of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
[0444] In particular embodiments, the F protein or the functionally active variant or biologically active portion thereof retains fusogenic activity in conjunction with a Henipavirus G protein, such as a G protein set forth above (e.g. NiV-G or HeV-G). Fusogenic activity includes the activity of the F protein in conjunction with a G protein to promote or facilitate fusion of two membrane lumens, such as the lumen of the targeted lipid particle having embedded in its lipid bilayer a henipavirus F and G protein, and a cytoplasm of a target cell, e.g. a cell that contains a surface receptor or molecule that is recognized or bound by the targeted envelope protein. In some embodiments, the F protein and G protein are from the same Henipavirus species (e.g. NiV-G and NiV-F). In some embodiments, the F protein and G protein are from different Henipavirus species (e.g. NiV-G and HeV-F). In particular embodiments, the F protein of the functionally active variant or biologically active portion retains the cleavage site cleaved by cathepsin L (e.g. corresponding to the cleavage site between amino acids 109-110 of SEQ ID NO: 30).
[0445] In particular embodiments, the F protein has the sequence of amino acids set forth in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or is a functionally active variant thereof or a biologically active portion thereof that retains fusogenic activity. In some embodiments, the functionally active variant comprises an amino acid sequence having at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, and retains fusogenic activity in conjunction with a Henipavirus G protein (e.g., NiV-G or HeV-G). In some embodiments, the biologically active portion has an amino acid sequence having at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
[0446] Reference to retaining fusogenic activity includes activity (in conjunction with a Henipavirus G protein) that between at or about 10% and at or about 150% or more of the level or degree of binding of the corresponding wild-type F protein, such as set forth in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, such as at least or at least about 10% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 15% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 20% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 25% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 30% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 35% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 40% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 45% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 50% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 55% of the level or degree of fusogenic activity of the corresponding wild-type f protein, such as at least or at least about 60% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 65% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 70% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 75% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 80% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 85% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 90% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 95% of the level or degree of fusogenic activity of the corresponding wild-type F protein, such as at least or at least about 100% of the level or degree of fusogenic activity of the corresponding wild-type F protein, or such as at least or at least about 120% of the level or degree of fusogenic activity of the corresponding wild-type F protein.
[0447] In some embodiments, the F protein is a mutant F protein that is a functionally active fragment or a biologically active portion containing one or more amino acid mutations, such as one or more amino acid insertions, deletions, substitutions or truncations. In some embodiments, the mutations described herein relate to amino acid insertions, deletions, substitutions or truncations of amino acids compared to a reference F protein sequence. In some embodiments, the reference F protein sequence is the wild- type sequence of an F protein or a biologically active portion thereof. In some embodiments, the mutant F protein or the biologically active portion thereof is a mutant of a wild-type Hendra (Hev) virus F protein, a Nipah (NiV) virus F-protein, a Cedar (CedPV) virus F protein, a Mojiang virus F protein, a bat Paramyxovirus F protein, a Kumasi virus F protein, or a Langya virus F protein. In some embodiments, the wild-type F protein is encoded by a sequence of nucleotides that encodes any one of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
[0448] In some embodiments, the mutant F protein is a biologically active portion of a wild- type F protein that is an N-terminally and/or C-terminally truncated fragment. In some embodiments, the mutant F protein or the biologically active portion of a wild-type F protein thereof comprises one or more amino acid substitutions. In some embodiments, the mutations described herein can improve transduction efficiency. In some embodiments, the mutations described herein can increase fusogenic capacity. Exemplary mutations include any as described, see e.g. Khetawat and Broder 2010 Virology Journal 7:312; Witting et al. 2013 Gene Therapy 20:997-1005; published international; patent application No. WO/2013/148327.
[0449] In some embodiments, the mutant F protein is a biologically active portion that is truncated and lacks up to 20 contiguous amino acid residues at or near the C-terminus of the wild-type F protein, such as a wild-type F protein encoded by a sequence of nucleotides encoding the F protein set forth in any one of SEQ ID NOS: 28-37. In some embodiments, the mutant F protein is truncated and lacks up to 20 contiguous amino acids, such as up to 19, 18 , 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 contiguous amino acids at the C-terminus of the wild-type F protein. In some embodiments, the mutant F protein comprises the sequence set forth in SEQ ID NO: 15. In some embodiments, the mutant F protein comprises the sequence set forth in SEQ ID NO:20. In some embodiments, the mutant F protein is truncated and lacks up to 19 contiguous amino acids, such as up to 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 contiguous amino acids at the C-terminus of the wild-type F protein.
[0450] In some embodiments, the F protein or the functionally active variant or biologically active portion thereof comprises an Fl subunit or a fusogenic portion thereof. In some embodiments, the Fl subunit is a proteolytically cleaved portion of the Fo precursor. In some embodiments, the Fo precursor is inactive. In some embodiments, the cleavage of the Fo precursor forms a disulfide-linked F1+F2 heterodimer. In some embodiments, the cleavage exposes the fusion peptide and produces a mature F protein. In some embodiments, the cleavage occurs at or around a single basic residue. In some embodiments, the cleavage occurs at Arginine 109 of NiV-F protein. In some embodiments, cleavage occurs at Lysine 109 of the Hendr a virus F protein.
[0451] In some embodiments, the F protein is a wild-type Nipah virus F (NiV-F) protein or is a functionally active variant or biologically active portion thereof. In some embodiments, the Fo precursor is encoded by a sequence of nucleotides encoding the sequence set forth in SEQ ID NO:20. The encoding nucleic acid can encode a signal peptide sequence that has the sequence MVVILDKRCY CNLLILILMI SECSVG (SEQ ID NO:38). In some examples, the F protein is cleaved into an Fl subunit comprising the sequence set forth in SEQ ID NO:46 and an F2 subunit comprising the sequence set forth in SEQ ID NO: 39.
[0452] In some embodiments, the F protein is a NiV-F protein that is encoded by a sequence of nucleotides encoding the sequence set forth in SEQ ID NO:30, or is a functionally active variant or biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at or about 86%, at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:30. In some embodiments, the F protein is a NiV-F protein that is encoded by a sequence of nucleotides encoding the sequence set forth in SEQ ID NO:30. In some embodiments, the NiV-F-protein has the sequence of set forth in 30, or is a functionally active variant or a biologically active portion thereof that has an amino acid sequence having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at or about 86%, at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to 30. In some embodiments, the NiV-F-protein has the sequence of set forth in 30. In In particular embodiments, the F protein or the functionally active variant or biologically active portion thereof retains the cleavage site cleaved by cathepsin L.
[0453] In some embodiments, the F protein or the functionally active variant or the biologically active portion thereof includes an Fl subunit that has the sequence set forth in SEQ ID NO:46, or an amino acid sequence having, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:46.
[0454] In some embodiments, the F protein or the functionally active variant or biologically active portion thereof includes an F2 subunit that has the sequence set forth in SEQ ID NO:39, or an amino acid sequence having, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:39.
[0455] In some embodiments, the F protein or the functionally active variant or the biologically active portion thereof includes an Fl subunit that has the sequence set forth in SEQ ID NO:46, or an amino acid sequence having, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at or about 86%, at least at or about 87%, at least at or about 88%, or at least at or about 89% at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 46.
[0456] In some embodiments, the F protein or the functionally active variant or biologically active portion thereof includes an F2 subunit that has the sequence set forth in SEQ ID NO:39, or an amino acid sequence having, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at or about 86%, at least at or about 87%, at least at or about 88%, or at least at or about 89% at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:39.
[0457] In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof that is truncated and lacks up to 20 contiguous amino acid residues at or near the C- terminus of the wild-type NiV-F protein (e.g. set forth SEQ ID NO:40). In some embodiments, the mutant NiV-F protein comprises an amino acid sequence set forth in SEQ ID NO:20. In some embodiments, the mutant NiV-F protein has a sequence that has at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:20. In some embodiments, the mutant F protein contains an Fl protein that has the sequence set forth in SEQ ID NO:46. In some embodiments, the mutant F protein has a sequence that has at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:46.
[0458] In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof that comprises a 20 amino acid truncation at or near the C-terminus of the wild-type NiV- F protein (SEQ ID NO:40); and a point mutation on an N-linked glycosylation site. In some embodiments, the mutant NiV-F protein comprises an amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the mutant NiV-F protein has a sequence that has at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 15.
[0459] In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof that comprises a 25 amino acid truncation at or near the C-terminus of the wild-type NiV- F protein (SEQ ID NO:40). In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof that comprises a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO:40). In some embodiments, the NiV-F protein is encoded by a nucleotide sequence that encodes the sequence set forth in SEQ ID NO:20. In some embodiments, the NiV-F proteins is encoded by a nucleotide sequence that encodes sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:20.
[0460] In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof that comprises a 22 amino acid truncation at or near the C-terminus of the wild-type NiV- F protein (SEQ ID NO:40). In some embodiments, the NiV-F protein comprises the amino acid sequence set forth in SEQ ID NO:21, or an amino acid sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:21. In some embodiments, the NiV-F protein is encoded by a nucleotide sequence that encodes the sequence set forth in SEQ ID NO:21. In some embodiments, the NiV-F proteins is encoded by a nucleotide sequence that encodes sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:21. [0461] In some embodiments where a VLP includes a fusogen that contains a G or H protein and an F protein (e.g., a G or H protein and F protein from a Paramyxovirus (e.g., Morbillivirus or Henipavirus) G or H protein or a biologically active portion thereof), the VLP also comprises a gag protein (e.g. an MLV-gag protein). In some embodiments, the MLV-gag protein is part of a fusion protein, where the the fusion protein is a cleavable fusion protein between (i) a viral structural protein (e.g. GAG (e.g. MLV- gag or HIV-gag)) and (ii) a nuclease protein (e.g. Cas protein (e.g. any of the Cas protein described herein). In some embodiments where the VLP includes fusogen that contains a G or H protein and an F protein and a gag protein (e.g. an MLV-gag protein), production of such a VLP includes varying the ratio of polynucleotide encoding the G or H protein and an F protein and the polynucleotide encoding the gag protein. In some embodiments, the ratio of polynucleotide encoding the G or H protein and an F protein to the polynucleotide encoding the gag protein includes a 1:1, 1:2, 1:3, 1:4, or 1:5 ratio.
C. Targeting Agent
[0462] In some embodiments, the particle includes a targeting agent that binds to a target molecule. In some embodiments, the target molecule is expressed on a target cell. In some embodiments, the targeting agent targets the particle to the target cell. In some embodiments, the particle preferentially targets a target cell compared to a non-target cell. In some embodiments, the particle preferentially fuses with a target cell compared to a non-target cell.
[0463] In some embodiments, the fusogen is fused to the targeting agent of the particle. In some aspects, the fusogen is retargeted by the targeting agent to display altered tropism.
[0464] In some embodiment, the targeting agent is linked to an exposed domain of the fusogen. In some embodiments, the exposed domain is the N-terminus. In some embodiments, the exposed domain is the C-terminus. In some embodiments, the targeting agent is fused to the fusogen directly. In some embodiments, the targeting agent is fused to the fusogen via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the targeting agent and fusogen are fused together, directly or indirectly, such as via the linker, in a fusion protein.
[0465] In some embodiments, the targeting agent is fused to a Paramyxovirus attachment protein comprised by the fusogen. In some embodiments, the targeting agent is fused to the C-terminus of the Paramyxovirus attachment protein. In some embodiments, the targeting agent is fused to the Paramyxovirus attachment protein directly. In some embodiments, the targeting agent is fused to the Paramyxovirus attachment protein via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the targeting agent and Paramyxovirus attachment protein are fused together, directly or indirectly, such as via the linker, in a fusion protein.
[0466] In some embodiments, the Paramyxovirus attachment protein is a Paramyxovirus G or H protein. In some embodiments, the Paramyxovirus attachment protein is a Paramyxovirus G protein. In some embodiments, the Paramyxovirus attachment protein is a Paramyxovirus H protein. In some embodiments, the Paramyxovirus attachment protein is any described in Section II-B-2-b. In some embodiments, the Paramyxovirus attachment protein is a mutated variant having reduced binding to its native receptor, such as any described in Section II-B-2-b.
[0467] In other embodiments, the targeting agent is fused to a separate transmembrane domain incorporated into the lipid bilayer, not the fusogen. The transmembrane domain may be any naturally- occurring or non-naturally occurring transmembrane domain. The transmembrane domain may be a transmembrane domain of a receptor, a transmembrane protein, preferably a viral transmembrane protein, a fragment of a transmembrane protein, a transmembrane peptide or a variant thereof, such as a genetically modified transmembrane domain of a receptor, a genetically modified transmembrane protein, a genetically modified fragment of a transmembrane protein, or a genetically modified transmembrane peptide. Examples of transmembrane domains are the transmembrane domain (TMD) of the platelet-derived growth factor receptor (PDGFR), the transmembrane domain of CD34, or the VSVG glycoprotein transmembrane domain.
[0468] In some embodiments, the C-terminus of the transmembrane domain is fused, directly or indirectly (for example via a linker, such as a peptide linker), to the N-terminus of the targeting agent.
[0469] In particular embodiments, the linker is a flexible peptide linker. In some such embodiments, the linker is 1-20 amino acids, such as 1-20 amino acids predominantly composed of glycine. In some embodiments, the linker is 1-20 amino acids, such as 1-20 amino acids predominantly composed of glycine and serine. In some embodiments, the linker is a flexible peptide linker containing amino acids Glycine and Serine, referred to as GS -linkers. In some embodiments, the peptide linker includes the sequences GS, GGS, GGGGS (SEQ ID NO:263), GGGGGS (SEQ ID NO:264), or combinations thereof. In some embodiments, the polypeptide linker has the sequence (GGS)n (SEQ IDNO:268), wherein n is 1 to 10. In some embodiments, the polypeptide linker has the sequence (GGGGS)n, (SEQ ID NO:265) wherein n is 1 to 10. In some embodiments, the polypeptide linker has the sequence (GGGGGS)n (SEQ ID NO:266), wherein n is 1 to 6. In some embodiments, the peptide linker is (GmS)n (SEQ ID NO:267), wherein each of m and n is an integer between 1 to 4, inclusive.
[0470] As used herein, a “target cell” refers to a cell of a type that is specifically targeted by the particle. In some embodiments, the target molecule is expressed on the target cell. In some embodiments, the target cell is a hematopoietic cell. In some embodiments, the target cell is CD34+. In some embodiments, the target cell is a CD34+ progenitor cell.
[0471] As used herein, a “non-target cell” refers to a cell of a type to which targeting of the particle is not desired. In some embodiments, the target molecule is expressed at higher levels on target cells than on non-target cells. In some embodiments, the target molecule is not expressed on the non-target cell. In some embodiments, a non-target cell is a non-hematopoietic cell. In some embodiments, the non-target cell is CD34-. In some embodiments, the non-target cell is not a CD34+ progenitor cell. [0472] In some embodiments, the target cell is a hematopoietic lineage cell. Reference to a "hematopoietic cell" includes blood cells, both from the myeloid and the lymphoid lineage. In particular, the term "hematopoietic cell" includes both undifferentiated or poorly differentiated cells, such as hematopoietic stem cells and progenitor cells, and differentiated cells such as T lymphocytes, B lymphocytes, or dendritic cells. In some embodiments, the hematopoietic cells are hematopoietic stem cells (HSCs), CD34+ progenitor cells, in particular peripheral blood CD34+ cells, very early progenitor CD34+ cells, B-cell CD19+ progenitors, myeloid progenitor CD13+ cells, T lymphocytes, B lymphocytes, monocytes, dendritic cells, cancer B cells in particular B-cell chronic lymphocytic leukemia (BCLL) cells and marginal zone lymphoma (MZL) B cells, or thymocytes.
[0473] As known from the skilled person, many hematopoietic cells are produced from bone marrow hematopoietic stem cells.
[0474] In some embodiments, a hematopoietic cell is a hematopoietic stem cell (HSC), which are cells able to replenish all blood cell types and to self-renew. Hematopoietic stem cells may be in particular defined as cells that keep the levels of myeloid, T cells, and B cells at robustly detectable levels (typically more than 1 % of peripheral blood cells) for 16 weeks when injected into the circulation of a recipient mouse with a depleted hematopoietic system (Schroeder (2010) Cell Stem Cell 6:203-207).
[0475] In some embodiments, the hematopoietic cell is a "CD34+ progenitor cell,” which is a heterogeneous cell population that include a subpopulation of HSCs, pluripotent stem cells and cells in the early stages of lineage commitment. CD34+ progenitor cells continuously migrate to and from the bone marrow in normal adult animals. They can differentiate to produce all hematopoietic cell lineages found in the circulation. In some embodiments, the hematopoietic cell is a very early progenitor CD34+ cell which is a subgroup of CD34+ progenitor cells enriched from HSCs.
[0476] In some embodiments, the hematopoietic cell is a "peripheral blood CD34+ cell”, which is a CD34+ cell present in the blood.
[0477] In some embodiments, the hematopoietic cell is a B cell CD 19+ progenitor, which is a population of B-lineage cells that express cell surface CD10, CD34, and CD19.
[0478] In some embodiments, the hematopoietic cell is a myeloid progenitor CD 13+ cells, which is a population of myeloid lineage cells that express cell surface CD34 and CD13, and in some cases, also CD33.
[0479] In some embodiments, the target cell is selected from the group consisting of myeloid- lymphoid balanced hematopoietic lineage cells, myeloid-biased hematopoietic lineage cells, lymphoid- biased hematopoietic lineage cells, a platelet-biased hematopoietic lineage cells, a platelet-myeloid- biased hematopoietic lineage cells, a long-term repopulating hematopoietic lineage cells, an intermediateterm repopulating hematopoietic lineage cells, or a short-term repopulating hematopoietic lineage cells. In some embodiments, the target cell is selected from monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes and platelets. In some embodiments, the target cell is selected from T cells, B cells, natural killer (NK) cells and innate lymphoid cells.
[0480] In some embodiments the target cell is an effector cell, e.g., a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. In some embodiments, a target cell may include one or more of a monocyte, macrophage, neutrophil, dendritic cell, eosinophil, mast cell, platelet, large granular lymphocyte, Langerhans' cell, natural killer (NK) cell, T lymphocyte (e.g., T cell), a Gamma delta T cell, B lymphocyte (e.g., B cell) and may be from any organism including humans, mice, rats, rabbits, and monkeys.
[0481] In some embodiment, the hematopoietic cell is a T cell. In some embodiments, the T cell is a naive T cell. In some embodiments, the T cell is a memory T cell.
[0482] In some embodiments, the hematopoietic cell is a B cell. In some embodiments, the target cell is a resting B cell, such as a naive or a memory B cell. In some embodiments, the target cell is a cancer B cell, such as a B-cell chronic lymphocytic leukemia (BCLL) cell or a marginal zone lymphoma (MZL) B cell.
[0483] In some embodiments, the target cell is a thymocyte. In some embodiments, the target cell is a natural killer (NK) cell. In some embodiments, the thymocyte expresses CD4 or CD8. In some embodiments, the thymocyte does not express CD4 or CD8. In some embodiments, the natural killer (NK) cell is a cell that expresses CD56.
[0484] In some embodiments, the target molecule is expressed on CD34+ progenitor cells. In some embodiments, the target cell is a CD34+ progenitor cell. In some embodiments, the target molecule is expressed on all CD34+ progenitor cells. In some embodiments, the target molecule is expressed on at least a subset of CD34+ progenitor cells.
[0485] In some embodiments, the target molecule is expressed on HSCs. In some embodiments, the target molecule is expressed on MPPs. In some embodiments, the target molecule is expressed on MLPs. In some embodiments, the target molecule is expressed on ETPs. In some embodiments, the target molecule is expressed on MEPs. In some embodiments, the target molecule is expressed on CMPs. In some embodiments, the target molecule is expressed on GMPs. In some embodiments, the target molecule is expressed on any combination of the foregoing CD34+ progenitor subpopulations. In some embodiments, the target molecule is expressed on HSCs and MPPs. In some embodiments, the target molecule is expressed on myeloid progenitors. In some embodiments, the target molecule is expressed on lymphoid progenitors. In some embodiments, the cell surface molecule is expressed on myeloid progenitors. In some embodiments, the cell surface molecule is expressed on HSCs, MPPs, MEPs, CMPs, and GMPs.
[0486] In some embodiments, the target cells are HSCs. In some embodiments, the target cells are MPPs. In some embodiments, the target cells are MLPs. In some embodiments, the target cells are ETPs. In some embodiments, the target cells are MEPs. In some embodiments, the target cells are CMPs. In some embodiments, the target cells are GMPs. In some embodiments, the target cells are any combination of the foregoing CD34+ progenitor subpopulations. In some embodiments, the target cells are HSCs and MPPs. In some embodiments, the target cells are myeloid progenitors. In some embodiments, the target cells are lymphoid progenitors. In some embodiments, the target cells are myeloid progenitors. In some embodiments, the target cells are any one or more, e.g., each of, HSCs, MPPs, MEPs, CMPs, and GMPs.
[0487] In some embodiments, the target molecule is ASCT1.
[0488] In some embodiments, the target molecule is ASCT2. In some embodiments, the target cell is ASCT2+. In some embodiments, the target cells are the CD34+ progenitor subpopulations indicated in FIG. 7 for ASCT2. In some embodiments, the target cells are lymphoid progenitors. In some embodiments, the target cells are MLPs or ETPs. In some embodiments, the target cells are MLPs and ETPs.
[0489] In some embodiments, the target molecule is CD 105. In some embodiments, the target cell is CD105+.
[0490] In some embodiments, the target molecule is CD110. In some embodiments, the target cell is CD110+.
[0491] In some embodiments, the target molecule is CD 117. In some embodiments, the target cell is CD117+. In some embodiments, the target cells are the CD34+ progenitor subpopulations indicated in FIG. 7 for CD117. In some embodiments, the target cells are myeloid progenitors. In some embodiments, the target cells are MPPs, MEPs, CMPs, or GMPs. In some embodiments, the target cells are MPPs, MEPs, CMPs, and GMPs.
[0492] In some embodiments, the target molecule is CD 133. In some embodiments, the target cell is CD133+.
[0493] In some embodiments, the target molecule is CD 146. In some embodiments, the target cell is CD146+.
[0494] In some embodiments, the target molecule is CD 164. In some embodiments, the target cell is CD164+.
[0495] In some embodiments, the target molecule is CD34. In some embodiments, the target cell is CD34+.
[0496] In some embodiments, the target molecule is CD46. In some embodiments, the target cell is CD46+.
[0497] In some embodiments, the target molecule is CD49f. In some embodiments, the target cell is CD49f+. [0498] In some embodiments, the target molecule is CD90. In some embodiments, the target cell is CD90+.
[0499] In some embodiments, the target molecule is EPCR. In some embodiments, the target cell is EPCR+.
[0500] In some embodiments, the target molecule is ITGA3. In some embodiments, the target cell is ITGA3+.
[0501] The targeting agent can include any targeting protein able to confer specific binding to the target molecule expressed on the target cell. In some embodiments, a targeting protein can also include an antibody or an antigen-binding fragment thereof (e.g., Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VE or VH), nanobodies, or camelid VHH domains), an antigen-binding fibronectin type III (Fn3) scaffold such as a fibronectin polypeptide minibody, a ligand, a cytokine, a chemokine, or a T cell receptor (TCRs). In some embodiments, the targeting agent is an antibody or antigen binding fragment thereof.
[0502] In some embodiments, the targeting agent contains a humanized antibody molecule, intact IgA, IgG, IgE or IgM antibody; bi- or multi- specific antibody (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies®; minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KAEBITOR®s.
[0503] In some embodiments, the targeting agent is a single chain molecule. In some embodiments, the targeting agent is a single domain antibody. In some embodiments, the targeting agent is a single chain variable fragment. In particular embodiments, the targeting agent contains an antibody variable sequence (s) that is human or humanized.
[0504] In some embodiments, the single domain antibody is naturally occurring. In some embodiments, the single domain antibody is synthetic.
[0505] In some embodiments, the single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. In some embodiments, the single domain antibody is a heavy chain only antibody variable domain. In some embodiments, the single domain antibody does not include light chains.
[0506] In some embodiments, the heavy chain antibody devoid of light chains is referred to as a VHH. In some embodiments, the single domain antibodies have a molecular weight of 12-15 kDa. In some embodiments, the single domain antibody antibodies include camelid antibodies or shark antibodies. In some embodiments, the single domain antibody is derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca, vicuna and guanaco. In some embodiments, the single domain antibody is referred to as immunoglobulin new antigen receptors (IgNARs) and is derived from cartilaginous fishes. In some embodiments, the single domain antibody is generated by splitting dimeric variable domains of human or mouse IgG into monomers and camelizing critical residues.
[0507] In some embodiments, the single domain antibody can be generated from phage display libraries. In some embodiments, the phage display libraries are generated from a VHH repertoire of camelids immunized with various antigens, as described in Arbabi et al., FEBS Letters, 414, 521-526 (1997); Lauwereys et al., EMBO J., 17, 3512-3520 (1998); Decanniere et al., Structure, 7, 361-370 (1999). In some embodiments, the phage display library is generated comprising antibody fragments of a non-immunized camelid. In some embodiments, single domain antibodies a library of human single domain antibodies is synthetically generated by introducing diversity into one or more scaffolds.
[0508] In some embodiments, the targeting agent can be engineered to bind the Fc region of an antibody that targets an antigen on a target cell, redirecting fusion activity towards cells that display the antibody’s target (DOI: 10.1128/JVI.75.17.8016-8020.2001, doi:10.1038/nml l92). In some embodiments, altered and non-altered fusogens may be displayed on the same retroviral particle or VLP (doi: 10.1016/j.biomaterials.2014.01.051). In some embodiments, a single-chain variable fragment (scFv) can be used as the targeting agent to redirect fusion activity towards target cells that display the scFv binding target (doi:10.1038/nbtl060, DOI 10.1182/blood-2012-l 1-468579, doi:10.1038/nmeth,1514, doi:10.1006/mthe.2002.0550, HUMAN GENE THERAPY 11:817- 826, doi:10.1038/nbt942, doi:10.1371/journal.pone.0026381, DOI 10.1186/sl2896-015-0142-z). In some embodiments, designed ankyrin repeat proteins (DARPin) can be used as the targeting agent to redirect fusion activity towards target cells that display the DARPin binding target (doi:10.1038/mt.2013.16, doi:10.1038/mt.2010.298, doi: 10.4049/jimmunol.1500956), as well as combinations of different DARPins (doi:10.1038/mto.2016.3). In some embodiments, a single domain antibody (e.g., a VHH) can be used as the targeting agent to redirect fusion activity towards target cells that display the sdAb binding target. In some embodiments, receptor ligands and antigens can be used as the targeting agent to redirect fusion activity towards target cells that display the target receptor (DOI: 10.1089/hgtb.2012.054, DOI: 10.1128/JVI.76.7.3558-3563.2002).
[0509] In some embodiments, the target molecule is CD 133. In some embodiments, the target cell is CD133+. In some embodiments, the targeting agent is an anti-CD133 antibody. Exemplary anti-CD133 antibodies include CART133, AC133, 293C3-SDIE, CMab-43, RW03, 293C3H9 (293C3), and W6B3H10 (W6B3); and anti-CD133 antibodies disclosed in US Patent Nos. US8722858, US9249225, US9624303, US10106623, US10711068, US11098109, US11214628, US11352435, and US11220551; US Patent Application Nos. US20130224202; PCT Application Nos. W0200901840, WO2011089211, WO2011149493 , WO2014128185, WO2015121383, WO2016154623, WO2018045880, W02018072025, and WO2022022718; and Canadian Patent Application No. CA2962157.
[0510] In some embodiments, the targeting agent is a CD 133 binding agent that is an antibody or antigen-binding fragment. In some embodiments, the CD 133 targeting agent is a single chain antibody, such as a single chain variable fragment (scFv). In some embodiments, the CD133 binding agent contains (a) a variable heavy (VH) chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 271, 272, 273, respectively, and a variable light (VL) chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 280, 281, 282, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 284, 285, and 286, respectively; (c) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 298, 299, and 300, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively; (d) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 307, 308, and 309, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 311, 312, and 313, respectively; or (e) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 298, 290, and 291, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 293, 294, and 295, respectively.
[0511] In some embodiments, the CD 133 binding agent contains a VH and a VL, e.g. containined in an scFv, selected from (a) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 314, 315, and 273, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 316, 317, and 282, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 284, 285, and 286, respectively; (c) a VH comprising a a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 318, 319, and 300, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR- L3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively; (d) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 320, 321, and 309, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 311, 312, and 313, respectively; and (e) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 322, 323, and 291, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 293, 294, and 295, respectively.
[0512] In some embodiments, the CD 133 binding agent is a CD 133 binding agent comprising a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 270, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 274, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD 133 binding agent is a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 279, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 283, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD 133 binding agent is a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 297, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 301, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD 133 binding agent is a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 306, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 310, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD133 binding agent is a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 288, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 292, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
[0513] In some embodiments, the CD 133 binding agent is a CD 133 binding agent comprising a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 270 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 274. In some embodiments, the CD 133 binding agent is a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 279 and a VL region comprising the amino acid sequence of SEQ ID NO: 283. In some embodiments, the CD133 binding agent is a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 297 and a VL region comprising the amino acid sequence of SEQ ID NO: 301. In some embodiments, the CD133 binding agent is a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 306 and a VL region comprising the amino acid sequence of SEQ ID NO: 310. In some embodiments, the CD133 binding agent is a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 288 and a VL region comprising the amino acid sequence of SEQ ID NO: 292.
[0514] In some embodiments, the CD 133 binding agent is an scFv comprising the amino acid sequence set forth in SEQ ID NO: 269, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD133 binding agent is an scFv comprising the amino acid sequence set forth in SEQ ID NO: 278, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD 133 binding agent is an scFv comprising the amino acid sequence set forth in SEQ ID NO: 287, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD133 binding agent is an scFv comprising the amino acid sequence set forth in SEQ ID NO: 296, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD 133 binding agent is an scFv comprising the amino acid sequence set forth in SEQ ID NO: 305, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
[0515] In some embodiments, the target molecule is CD 105. In some embodiments, the target cell is CD105+. In some embodiments, the targeting agent is an anti-CD105 antibody. Exemplary anti-CD105 antibodies include carotuximab, TRC105, huRH105, and TCR205; and anti-CD105 antibodies disclosed in US Patent Nos. US8221753, US8609094, US9150652, US95181212, US9926375, US9944714, US 10155820, and US 10336831; US Patent Application Nos. US20100098692, US20100196398, US20170007714, and US20220233591; PCT Application Nos. W02010039873, W02011041441, WO2016077451, WO2018067819, W02010032059, WO2012149412, WO2015118031, WO2021118955, and WO2021118957; and Korean Patent No. KR101398707B1.
[0516] In some embodiments, the target molecule is CD 117. In some embodiments, the target cell is CD117+. In some embodiments, the targeting agent is an anti-CD 117 antibody. In some embodiments, the targeting agent is a CD117 binding agent that is an antibody or antigen-binding fragment. In some embodiments, the CD117 binding agent is a single domain antibody, such as a VHH. In some embodiments, the CD117 binding agent comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
[0517] In some embodiments, the CD117 binding agent is a VHH comprising a CDR-H1, a CDR- H2, and a CDR-H3 contained within an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374, wherein the target molecule is CD117.
[0518] In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 324, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 325, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 326, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 327, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 328, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 329, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 330, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 331, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 332, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 333, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 334, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 335, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 336, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 337, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 338, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 339, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 340, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 341, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 342, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 343, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 344, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 345, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 346, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 347, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 348, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 349, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 350, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 351, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 352, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 353, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 354, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 355, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 356, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 357, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 358, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 359, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 360, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 361, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 362, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 363, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 364, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 365, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 366, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 367, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 368, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 369, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 370, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 371, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 372, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 373, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 374, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
[0519] In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 324. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 325. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 326. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 327. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 328. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 329. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 330. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 331. In some embodiments, the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 332. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 333. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 334. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 335. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 336. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 337. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 338. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 339. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 340. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 341. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 342. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 343. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 344. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 345. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 346. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 347. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 348. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 349. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 350. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 351. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 352. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 353. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 354. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 355. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 356. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 357. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 358. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 359. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 360. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 361. In some embodiments, the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 362. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 363. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 364. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 365. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 366. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 367. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 368. In some embodiments, the CD 117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 369. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 370. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 371. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 372. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 373. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 374. [0520] In some embodiments, the target molecule is EPCR. In some embodiments, the target cell is EPCR+. In some embodiments, the targeting agent is an anti-EPCR antibody. Exemplary anti-EPCR antibodies include JRK1494, JRK1535; and anti-EPCR antibodies disclosed in US Patent Application Nos. US20210355231 and US20220127374; and PCT Application Nos. W02020051277 and WO2020161478.
[0521] In some embodiments, the target molecule is CD34. In some embodiments, the target cell is CD34+. In some embodiments, the targeting agent is an anti-CD34 antibody. Exemplary anti-CD34 antibodies include h4C8, 9C5; and anti-CD34 antibodies disclosed in US Patent Nos. US8399249, US8927696, and US10106623; US Patent Application Nos. US20090221003, US20130143238, US20100311955, US20130172533, US20170320966, US20170298148, US20180169177, US20190135945; and PCT Application Nos. W02009079922 and WO2015121383.
[0522] In some embodiments, the target molecule is ASCT1.
[0523] In some embodiments, the target molecule is ASCT2. In some embodiments, the target cell is ASCT2+. In some embodiments, the targeting agent is an anti-ASCT2 antibody. Exemplary anti-ASCT2 antibodies include idactamab, MEDI7247, KM4008, KM4012, KM4018; and anti-ASCT2 antibodies disclosed in US Patent Nos. US8268592, US8501180, US8945870, US8673592, and US10829554; US Patent Application Nos. US20180273617, US20190367605, US 20210024629; and PCT Application Nos. WO2017083451, WO2018089393.
[0524] In some embodiments, the target molecule is CD90. In some embodiments, the target cell is CD90+. In some embodiments, the targeting agent is an anti-CD90 antibody. Exemplary anti-CD90 antibodies include EPR3133, CL1028, CL1040, AF-9, JF10-09, 5E10, 7E1B11; and anti-CD90 antibodies disclosed in US Patent Application No. US20210054068; and PCT Application No.
WO2017214050.
[0525] In some embodiments, the target molecule is CD 164. In some embodiments, the target cell is CD164+. In some embodiments, the targeting agent is an anti-CD164 antibody. Exemplary anti-CD164 antibodies include 67D2, H-4, 32G1, EML2058, 5C5, N6B6, 4B4, and 15-11-14; and anti-CD164 antibodies disclosed in PCT Application No. W02006002438; and German Patent Nos. DE19727813C1 and DE19727815C1.
[0526] In some embodiments, the target molecule is CD49f. In some embodiments, the target cell is CD49f+. In some embodiments, the targeting agent is an anti-CD49f antibody. Exemplary anti-CD49f antibodies include CL6957, GoH3, SR45-00, and MP4F10; and anti-CD49f antibodies disclosed in US Patent Nos. US5538725, US10030071; US Patent Application Nos. US20110301227, US20160194400, US20160280789; and PCT Application Nos. WG2015034052 and WO2018127655.
[0527] In some embodiments, the target molecule is CD 146. In some embodiments, the target cell is CD146+. In some embodiments, the targeting agent is an anti-CD146 antibody. Exemplary anti-CD146 antibodies include imaprelimab, PRX003, ABX-MA1, huAA98, M2H-1, M2J-1, and JM1-24-3; and anti- CD146 antibodies disclosed in US Patent Nos. US6924360, US7067131, US709844, US9447190, US9782500, US10407506, US10414825, US10407507, US10584177, US10905771, US11427648; US Patent Application Nos. US20030147809, US20040115205, US20060246077, US20140314744, US20150239980, US20140227292, US20160206764, US20190192573, US20170002089, US20150259419, US20170037144, US20170129954, US20170101470, US20180271994, US20180105602, US20200010563, US20200165336, US20200216560, US20200262929, US20200100838, US20220041748, US20220041749; and PCT Application Nos. W02003057006, W02003057837, W02003057838, W02012170071, W02014000338, WO2015044218, WO2015136469, WO2015136470, WO2017046776, WO2017046774, WO2017149513, WO2017153953, W02017208210, W02018033630, WO2018220467, WO2018223140, WO2019068842, WO2019133639, WO2019137309, W02020132190, WO2020132232, and W02022082073.
[0528] Further exemplary targeting agents and corresponding target molecules are described in the table below. The targeting agent can be any described in the referenced associated documents that bind to the associated target molecule.
D. Exogenous Agent
[0529] In some embodiments, the particle contains an exogenous agent. In some embodiments, the exogenous agent is in the lumen of the particle. In some embodiments, the exogenous agent is a nucleic acid, such as any described in Section II-D-1. In some embodiments, the nucleic acid contains a payload gene encoding a payload agent. In some embodiments, the exogenous agent encodes a protein, such as any described in Section II-D-2. In some embodiments, the exogenous agent is a protein, such as any described in Section II-D-2. In some embodiments, the protein is a payload agent. In some embodiments, the exogenous agent is a small molecule, such as any described in Section II-D-3.
[0530] In some embodiments, the pay load agent encoded or contained by the exogenous agent is any described in Section III. In some embodiments, the exogenous agent is or encodes an engineered receptor, such as any described in Section III-A. In some embodiments, the exogenous agent is or encodes a gene-editing agent, such as any described in Section III-B. [0531] In some embodiments, the exogenous agent is a protein or a nucleic acid (e.g., a DNA, a chromosome (e.g. a human artificial chromosome), or an RNA, e.g., an mRNA or miRNA). In some embodiments, the exogenous agent is a protein. In some embodiments, the exogenous agent is a nucleic acid (e.g., a DNA, a chromosome (e.g. a human artificial chromosome), or an RNA, e.g., an mRNA or miRNA). In some embodiments, the exogenous agent comprises or encodes a membrane protein. In some embodiments, the exogenous agent comprises or encodes a therapeutic agent. In some embodiments, the therapeutic agent is chosen from one or more of a protein, e.g., an enzyme, a transmembrane protein, a receptor, or an antibody; a nucleic acid, e.g., DNA, a chromosome (e.g. a human artificial chromosome), or an RNA, e.g., mRNA, siRNA, or miRNA; or a small molecule.
[0532] In some embodiments, the particle delivers to a target cell at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the exogenous agent (e.g., an exogenous agent comprising or encoding a therapeutic agent) comprised by the particle. In some embodiments, the particles that contact, e.g., fuse, with the target cell or cells delivers to the target cell or cells an average of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the exogenous agent (e.g., an exogenous agent comprising or encoding a therapeutic agent) comprised by the particles that contact, e.g., fuse, with the target cell or cells. In some embodiments, a composition containing a plurality of the particle delivers to a target tissue at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the exogenous agent (e.g., an exogenous agent comprising or encoding a therapeutic agent) comprised by the composition.
[0533] In some embodiments, the exogenous agent is not expressed naturally in the cell from which the particle is derived. In some embodiments, the exogenous agent is expressed naturally in the cell from which the particle is derived. In some embodiments, the exogenous agent is loaded into the particle via expression in the cell from which the particle is derived (e.g. expression from DNA or mRNA introduced via transfection, transduction, or electroporation). In some embodiments, the exogenous agent is expressed from DNA integrated into the genome or maintained episomally. In some embodiments, expression of the exogenous agent is constitutive. In some embodiments, expression of the exogenous agent is induced. In some embodiments, expression of the exogenous agent is induced immediately prior to generating the particle. In some embodiments, expression of the exogenous agent is induced at the same time as expression of the fusogen of the particle.
[0534] In some embodiments, the exogenous agent is loaded into the particle via electroporation into the particle itself or into the cell from which the particle is derived. In some embodiments, the exogenous agent is loaded into the particle via transfection (e.g., of a DNA or mRNA encoding the exogenous agent) into the particle itself or into the cell from which the particle is derived.
[0535] In some embodiments, the exogenous agent includes one or more nucleic acid sequences, one or more polypeptides, a combination of nucleic acid sequences and/or polypeptides, one or more organelles, or any combination of the foregoing. In some embodiments, the exogenous agent includes one or more cellular components. In some embodiments, the exogenous agent includes one or more cytosolic and/or nuclear components.
[0536] In some embodiments, the exogenous agent includes a mixture of proteins, nucleic acids, or metabolites, e.g., multiple polypeptides, multiple nucleic acids, multiple small molecules; combinations of nucleic acids, polypeptides, and small molecules; ribonucleoprotein complexes (e.g. Cas9-gRNA complex); multiple transcription factors, multiple epigenetic factors, reprogramming factors (e.g. Oct4, Sox2, cMyc, and Klf4); multiple regulatory RNAs; and any combination of the foregoing.
1. Nucleic Acids
[0537] In some embodiments, the exogenous agent includes a nucleic acid. For example, the exogenous agent may comprise RNA to enhance expression of an endogenous protein, or a siRNA or miRNA that inhibits protein expression of an endogenous protein. For example, the endogenous protein may modulate structure or function in the target cells. In some embodiments, the exogenous agent may include a nucleic acid encoding an engineered protein that modulates structure or function in the target cells. In some embodiments, the exogenous agent is a nucleic acid that targets a transcriptional activator that modulate structure or function in the target cells
[0538] In some embodiments, the nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, the nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, the nucleic acid is partly or wholly single stranded; in some embodiments, the nucleic acid is partly or wholly double stranded. In some embodiments the nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. The nucleic acid may include variants, e.g., having an overall sequence identity with a reference nucleic acid of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant nucleic acid does not share at least one characteristic sequence element with a reference nucleic acid. In some embodiments, a variant nucleic acid shares one or more of the biological activities of the reference nucleic acid. In some embodiments, a nucleic acid variant has a nucleic acid sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. In some embodiments, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue as compared to a reference. In some embodiments, a variant nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues that participate in a particular biological activity relative to the reference. In some embodiments, a variant nucleic acid comprises not more than about 15, about 12, about 9, about 3, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant nucleic acid comprises fewer than about 27, about 24, about 21, about 18, about 15, about 12, about 9, about 6, about 3, or fewer than about 9, about 6, about 3, or about 2 additions or deletions as compared to the reference.
[0539] In some embodiments, the exogenous agent includes a nucleic acid, e.g., DNA, nDNA (nuclear DNA), mtDNA (mitochondrial DNA), protein coding DNA, gene, operon, chromosome, genome, transposon, retrotransposon, viral genome, intron, exon, modified DNA, mRNA (messenger RNA), tRNA (transfer RNA), modified RNA, microRNA, siRNA (small interfering RNA), tmRNA (transfer messenger RNA), rRNA (ribosomal RNA), mtRNA (mitochondrial RNA), snRNA (small nuclear RNA), small nucleolar RNA (snoRNA), SmY RNA (mRNA trans-splicing RNA), gRNA (guide RNA), TERC (telomerase RNA component), aRNA (antisense RNA), cis-NAT (Cis-natural antisense transcript), CRISPR RNA (crRNA), IncRNA (long noncoding RNA), piRNA (piwi-interacting RNA), shRNA (short hairpin RNA), tasiRNA (trans-acting siRNA), eRNA (enhancer RNA), satellite RNA, pcRNA (protein coding RNA), dsRNA (double stranded RNA), RNAi (interfering RNA), circRNA (circular RNA), reprogramming RNAs, aptamers, and any combination thereof. In some embodiments, the nucleic acid is a wild- type nucleic acid. In some embodiments, the protein is a mutant nucleic acid. In some embodiments the nucleic acid is a fusion or chimera of multiple nucleic acid sequences
[0540] In embodiments, the nucleic acid encodes one or more (e.g. two or more) inhibitory RNA molecules directed against one or more RNA targets. An inhibitory RNA molecule can be, e.g., a miRNA or an shRNA. In some embodiments, the inhibitory molecule can be a precursor of a miRNA, such as for example, a Pri-miRNA or a Pre-miRNA, or a precursor of an shRNA. In some embodiments, the inhibitory molecule can be an artificially derived miRNA or shRNA. In other embodiments, the inhibitory RNA molecule can be a dsRNA (either transcribed or artificially introduced) that is processed into an siRNA or the siRNA itself. In some embodiments, the inhibitory RNA molecule can be a miRNA or shRNA that has a sequence that is not found in nature, or has at least one functional segment that is not found in nature, or has a combination of functional segments that are not found in nature. In illustrative embodiments, at least one or all of the inhibitory RNA molecules are miR-155. In some embodiments, a retroviral vector described herein encodes two or more inhibitory RNA molecules directed against one or more RNA targets. Two or more inhibitory RNA molecules, in some embodiments, can be directed against different targets. In other embodiments, the two or more inhibitory RNA molecules are directed against the same target. In some embodiments, the exogenous agent comprises a shRNA. A shRNA (short hairpin RNA) can comprise a double-stranded structure that is formed by a single self complementary RNA strand. shRNA constructs can comprise a nucleotide sequence identical to a portion, of either coding or non-coding sequence, of a target gene. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence can also be used. Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene can be used. In certain embodiments, the length of the duplex-forming portion of an shRNA is at least 20, 2 1 or 22 nucleotides in length, e.g., corresponding in size to RNA products produced by Dicer-dependent cleavage. In certain embodiments, the shRNA construct is at least 25, 50, 100, 200, 300 or 400 bases in length. In certain embodiments, the shRNA construct is 400-800 bases in length. shRNA constructs are highly tolerant of variation in loop sequence and loop size. In embodiments, a retroviral vector that encodes an siRNA, an miRNA, an shRNA, or a ribozyme comprises one or more regulatory sequences, such as, for example, a strong constitutive pol III, e.g., human U6 snRNA promoter, the mouse U6 snRNA promoter, the human and mouse H 1 RNA promoter and the human tRNA-val promoter, or a strong constitutive pol II promoter.
[0541] In some embodiments, the nucleic acid is operatively linked to a “positive target cell-specific regulatory element” (or positive TCSRE). In some embodiments, the positive TCSRE is a functional nucleic acid sequence. In some embodiments, the positive TCSRE contains a promoter or enhancer. In some embodiments, the TCSRE is a nucleic acid sequence that increases the level of an exogenous agent in a target cell.
[0542] In some embodiments, the nucleic acid is operatively linked to a “negative target cellspecific regulatory element” (or negative TCSRE). In some embodiments, the negative TCSRE is a functional nucleic acid sequence. In some embodiments, the negative TCSRE is a miRNA recognition site that causes degradation of inhibition of the particle in a non-target cell. In some embodiments, the nucleic acid is operatively linked to a “non-target cell-specific regulatory element” (or NTCSRE). In some embodiments, the NTCSRE comprises a nucleic acid sequence that decreases the level of an exogenous agent in a non-target cell compared to in a target cell. In some embodiments, the NTCSRE comprises a non-target cell-specific miRNA recognition sequence, non-target cell-specific protease recognition site, non-target cell-specific ubiquitin ligase site, non-target cell-specific transcriptional repression site, or non-target cell-specific epigenetic repression site. In some embodiments, the NTCSRE comprises a tissue-specific miRNA recognition sequence, tissue-specific protease recognition site, tissuespecific ubiquitin ligase site, tissue-specific transcriptional repression site, or tissue-specific epigenetic repression site. In some embodiments, the NTCSRE comprises a non-target cell-specific miRNA recognition sequence, non-target cell-specific protease recognition site, non-target cell-specific ubiquitin ligase site, non-target cell-specific transcriptional repression site, or non-target cell-specific epigenetic repression site. In some embodiments, the NTCSRE comprises a non-target cell-specific miRNA recognition sequence and the miRNA recognition sequence is able to be bound by one or more of miR3 1, miR363, or miR29c. In some embodiments, the NTCSRE is situated or encoded within a transcribed region encoding the exogenous agent, optionally wherein an RNA produced by the transcribed region comprises the miRNA recognition sequence within a UTR or coding region.
2. Proteins
[0543] In some embodiments, the exogenous agent is or encodes a protein. In some embodiments, the protein includes moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. In some embodiments, the protein includes more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
[0544] In some embodiments, the protein contains L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs. In some embodiments, the protein contains natural amino acids, non-natural amino acids, synthetic amino acids, or combinations of any of the foregoing. In some embodiments, the protein is selected from antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
[0545] In some embodiments, the protein is a wild-type protein. In some embodiments, the protein is a mutant protein. In some embodiments, the protein includes a polypeptide or its variants, e.g., having an overall sequence identity with a reference polypeptide of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, a variant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, a polypeptide variant has an amino acid sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. In some embodiments, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue as compared to a reference. In some embodiments, a variant polypeptide comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional that participate in a particular biological activity relative to the reference. In some embodiments, a variant polypeptide comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference.
[0546] In some embodiments, the protein includes a polypeptide, e.g., enzymes, structural polypeptides, signaling polypeptides, regulatory polypeptides, transport polypeptides, sensory polypeptides, motor polypeptides, defense polypeptides, storage polypeptides, transcription factors, antibodies, cytokines, hormones, catabolic polypeptides, anabolic polypeptides, proteolytic polypeptides, metabolic polypeptides, kinases, transferases, hydrolases, lyases, isomerases, ligases, enzyme modulator polypeptides, protein binding polypeptides, lipid binding polypeptides, membrane fusion polypeptides, cell differentiation polypeptides, epigenetic polypeptides, cell death polypeptides, nuclear transport polypeptides, nucleic acid binding polypeptides, reprogramming polypeptides, DNA editing polypeptides, DNA repair polypeptides, DNA recombination polypeptides, transposase polypeptides, DNA integration polypeptides, targeted endonucleases (e.g. Zinc -finger nucleases, transcription- activator-like nucleases (TALENs), cas9 and homologs thereof), recombinases, transposases, DNA polymerases, RNA polymerases, reverse transciptases, and any combination of the foregoing.
[0547] In some embodiments, the protein targets a target protein in the cell for degradation. In some embodiments, the protein targets a target protein in the cell for degradation by localizing the protein to the proteasome. In some embodiments, the target protein is a wild-type protein. In some embodiments, the target protein is a mutant protein.
3. Small Molecules
[0548] In some embodiments, the exogenous agent includes a small molecule, e.g., ions (e.g. Ca2+, C1-, Fe2+), carbohydrates, lipids, reactive oxygen species, reactive nitrogen species, isoprenoids, signaling molecules, heme, polypeptide cofactors, electron accepting compounds, electron donating compounds, metabolites, ligands, and any combination of the foregoing. In some embodiments, the small molecule is a pharmaceutical that interacts with a target in the target cell. In some embodiments, the small molecule targets a protein in the target cell for degradation. In some embodiments the small molecule targets a protein in the target cell for degradation by localizing the protein to the proteasome. In some embodiments, the small molecule is a proteolysis targeting chimera molecule (PROTAC).
III. PAYLOAD AGENTS
[0549] In some embodiments, the exogenous agent is a nucleic acid, such as any described in Section II-D-1. In some embodiments, the nucleic acid contains a payload gene encoding a payload agent, for instance a protein, such as any described in Section II-D-2. In some embodiments, the exogenous agent is a protein, such as any described in Section II-D-2. In some embodiments, the protein is a payload agent.
[0550] In some embodiments, the payload agent comprises a cytosolic protein, e.g., a protein that is produced in the recipient cell and localizes to the recipient cell cytoplasm. In some embodiments, the payload agent comprises a secreted protein, e.g., a protein that is produced and secreted by the recipient cell. In some embodiments, the payload agent comprises a nuclear protein, e.g., a protein that is produced in the recipient cell and is imported to the nucleus of the recipient cell. In some embodiments, the payload agent comprises an organellar protein (e.g., a mitochondrial protein), e.g., a protein that is produced in the recipient cell and is imported into an organelle (e.g., a mitochondrial) of the recipient cell. In some embodiments, the protein is a wild-type protein or a mutant protein. In some embodiments the protein is a fusion or chimeric protein.
[0551] In some embodiments, the payload agent comprises a membrane protein. In some embodiments, the membrane protein comprises a chimeric antigen receptor (CAR), a T cell receptor, an integrin, an ion channel, a pore forming protein, a Toll-Like Receptor, an interleukin receptor, a cell adhesion protein, or a transport protein.
A. Engineered Receptors
[0552] In some embodiments, the payload agent is an engineered receptor, such as antigen receptors including chimeric antigen receptors (CARs), and other antigen-binding receptors such as transgenic T cell receptors (TCRs).
1. Chimeric Antigen Receptors (CARs)
[0553] In some embodiments, the pay load agent is a chimeric antigen receptor (CAR) comprising an antigen binding domain. In some embodiments, the CAR is or comprises a first generation CAR comprising an antigen binding domain, a transmembrane domain, and signaling domain (e.g., one, two or three signaling domains). In some embodiments, the CAR comprises a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments, a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some embodiments, the antigen binding domain is or comprises an scFv or Fab.
[0554] In some embodiments, the antigen binding domain targets an antigen characteristic of a cell type. In some embodiments, the antigen binding domain targets an antigen characteristic of a neoplastic cell. In some embodiments, the antigen characteristic of a neoplastic cell is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor, Epidermal Growth Factor Receptors (EGFR) (including ErbBl/EGFR, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), Fibroblast Growth Factor Receptors (FGFR) (including FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF18, and FGF21) Vascular Endothelial Growth Factor Receptors (VEGFR) (including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PIGF), RET Receptor and the Eph Receptor Family (including EphAl, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA9, EphAlO, EphBl, EphB2. EphB3, EphB4, and EphB6), CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CIC- Kb, Bestrophins, TMEM16A, GABA receptor, glycin receptor, ABC transporters, NAV1.1, NAVI.2, NAVI.3, NAVI.4, NAV1.5, NAVI.6, NAV1.7, NAVI.8, NAV1.9, sphingosin-1 -phosphate receptor (S1P1R), NMDA channel, transmembrane protein, multispan transmembrane protein, T-cell receptor motifs; T-cell alpha chains; T-cell P chains; T-cell y chains; T-cell 5 chains; CCR7; CD3; CD4; CD5; CD7; CD8; CDllb; CDllc; CD16; CD19; CD20; CD21 ; CD22; CD25; CD28; CD34; CD35; CD40; CD45RA; CD45RO; CD52; CD56; CD62L; CD68; CD80; CD95; CD117; CD127; CD133; CD137 (4-1 BB); CD163; F4/80; IL-4Ra; Sca-1 ; CTLA-4; GITR; GARP; LAP; granzyme B; LFA-1 ; transferrin receptor; NKp46, perforin, CD4+; Thl; Th2; Thl7; Th40; Th22; Th9; Tfh, Canonical Treg. FoxP3+; Tri; Th3; Tregl7; TREG; CDCP1, NT5E, EpCAM, CEA, gpA33, Mucins, TAG-72, Carbonic anhydrase IX, PSMA, Folate binding protein, Gangliosides (e.g., CD2, CD3, GM2), Lewis-y2, VEGF, VEGFR 1/2/3, aV 3, a5 i, ErbBl/EGFR, ErbBl/HER2, ErB3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANKL, FAP, Tenascin, PDL-1, BAFF, HDAC, ABL, FLT3, KIT, MET, RET, IL-ip, ALK, RANKL, mTOR, CTLA-4, IL-6, IL-6R, JAK3, BRAF, PTCH, Smoothened, PIGF, ANPEP, TIMP1, PLAUR, PTPRJ, LTBR, or ANTXR1, Folate receptor alpha (FRa), ERBB2 (Her2/neu), EphA2, IL-13Ra2, epidermal growth factor receptor (EGFR), Mesothelin, TSHR, CD19, CD123, CD22, CD30, CD171, CS- 1, CLL-1, CD33, EGFRvIII , GD2, GD3, BCMA, MUC16 (CA125), L1CAM, LeY, MSLN, IL13Ral, Ll-CAM, Tn Ag, prostate specific membrane antigen (PSMA), R0R1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, interleukin- 11 receptor a (IL-llRa), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, MUC1, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-1 receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CX0RF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY- BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE- la, MAGE-A1, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Major histocompatibility complex class Lrelated gene protein (MR1), urokinase-type plasminogen activator receptor (uPAR), Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYPIB I, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, a neoantigen, CD133, CD15, CD184, CD24, CD56, CD26, CD29, CD44, HLA-A, HLA-B, HLA-C, (HLA-A,B,C) CD49f, CD151 CD340, CD200, tkrA, trkB, or trkC, or an antigenic fragment or antigenic portion thereof.
[0555] In some embodiments, the antigen binding domain targets an antigen characteristic of a T cell. In some embodiments, the antigen characteristic of a T cell is selected from a cell surface receptor, a membrane transport protein (e.g., an active or passive transport protein such as, for example, an ion channel protein, a pore-forming protein, etc.), a transmembrane receptor, a membrane enzyme, and/or a cell adhesion protein characteristic of a T cell. In some embodiments, an antigen characteristic of a T cell may be a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor, AKT1; AKT2; AKT3; ATF2; BCL10; CALM1; CD3D (CD35); CD3E (CD3a); CD3G (CD3y); CD4; CD8; CD28; CD45; CD80 (B7-1); CD86 (B7-2); CD247 (CD3Q; CTLA4 (CD152); ELK1; ERK1 (MAPK3); ERK2; FOS; FYN; GRAP2 (GADS); GRB2; HLA-DRA; HLA- DRB1; HLA-DRB3; HLA-DRB4; HLA-DRB5; HRAS; IKBKA (CHUK); IKBKB; IKBKE; IKBKG (NEMO); IL2; ITPR1; ITK; JUN; KRAS2; LAT; LCK; MAP2K1 (MEK1); MAP2K2 (MEK2); MAP2K3 (MKK3); MAP2K4 (MKK4); MAP2K6 (MKK6); MAP2K7 (MKK7); MAP3K1 (MEKK1); MAP3K3; MAP3K4; MAP3K5; MAP3K8; MAP3K14 (NIK); MAPK8 (JNK1); MAPK9 (JNK2); MAPK10 (JNK3); MAPK11 (p38|3); MAPK12 (p38y); MAPK13 (p385); MAPK14 (p38a); NCK; NFAT1; NFAT2; NFKB1; NFKB2; NFKBIA; NRAS; PAK1; PAK2; PAK3; PAK4; PIK3C2B; PIK3C3 (VPS34); PIK3CA; PIK3CB; PIK3CD; PIK3R1; PKCA; PKCB; PKCM; PKCQ; PLCY1; PRF1 (Perforin); PTEN; RAC1; RAFI; RELA; SDF1; SHP2; SLP76; SOS; SRC; TBK1; TCRA; TEC; TRAF6; VAV1; VAV2; or ZAP70.
[0556] In some embodiments, the antigen binding domain targets an antigen characteristic of a disorder. In some embodiments, the antigen binding domain targets an antigen characteristic of an autoimmune or inflammatory disorder. In some embodiments, the autoimmune or inflammatory disorder is selected from chronic graft-vs-host disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, goodpasture, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, cold agglutinin disease, Pemphigus vulgaris, Grave's disease, autoimmune hemolytic anemia, Hemophilia A, Primary Sjogren's Syndrome, thrombotic thrombocytopenia purrpura, neuromyelits optica, Evan's syndrome, IgM mediated neuropathy, cyroglobulinemia, dermatomyositis, idiopathic thrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquired angioedema, chronic urticarial, antiphospholipid demyelinating polyneuropathy, and autoimmune thrombocytopenia or neutropenia or pure red cell aplasias, while exemplary non-limiting examples of alloimmune diseases include allosensitization (see, for example, Blazar et al., 2015, Am. J. Transplant, 15(4):931 -41) or xenosensitization from hematopoietic or solid organ transplantation, blood transfusions, pregnancy with fetal allosensitization, neonatal alloimmune thrombocytopenia, hemolytic disease of the newborn, sensitization to foreign antigens such as can occur with replacement of inherited or acquired deficiency disorders treated with enzyme or protein replacement therapy, blood products, and gene therapy. In some embodiments, the antigen characteristic of an an autoimmune or inflammatory disorder is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, or histidine kinase associated receptor. In some embodiments, a CAR antigen binding domain binds to a ligand expressed on B cells, plasma cells, plasmablasts, CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, CD28, TNF, interferon receptors, GM-CSF, ZAP-70, LFA-1, CD3 gamma, CD5 or CD2. See US 2003/0077249; WO 2017/058753; WO 2017/058850, the contents of which are herein incorporated by reference.
[0557] In some embodiments, the antigen binding domain targets an antigen characteristic of senescent cells, e.g., urokinase-type plasminogen activator receptor (uPAR). In some embodiments, the CAR may be used for treatment or prophylaxis of disorders characterized by the aberrant accumulation of senescent cells, e.g., liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis.
[0558] In some embodiments, the antigen binding domain targets an antigen characteristic of an infectious disease. In some embodiments, wherein the infectious disease is selected from HIV, hepatitis B virus, hepatitis C virus, Human herpes virus, Human herpes virus 8 (HHV-8, Kaposi sarcoma- associated herpes virus (KSHV)), Human T-lymphotrophic virus-1 (HTLV-1), Merkel cell polyomavirus (MCV), Simian virus 40 (SV40), Eptstein-Barr virus, CMV, human papillomavirus. In some embodiments, the antigen characteristic of an infectious disease is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor, HIV Env, gpl20, or CD4-induced epitope on HIV-1 Env.
[0559] In some embodiments, the CAR transmembrane domain comprises at least a transmembrane region of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or functional variant thereof. In some embodiments, the transmembrane domain comprises at least a transmembrane region(s) of CD8a, CD8P, 4-1BB/CD137, CD28, CD34, CD4, FcaRIy, CD16, OX40/CD134, CD3^, CD3a, CD3y, CD35, TCRa, TCRp, TCR^, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, and FGFR2B, or functional variant thereof.
[0560] In some embodiments, the CAR comprises at least one signaling domain selected from one or more of B7-1/CD80; B7-2/CD86; B7-H1/PD-L1; B7-H2; B7-H3; B7-H4; B7-H6; B7-H7;
BTLA/CD272; CD28; CTLA-4; Gi24/VISTA/B7-H5; ICOS/CD278; PD-1; PD-L2/B7-DC; PDCD6); 4- 1BB/TNFSF9/CD137; 4-1BB Ligand/TNFSF9; BAFF/BLyS/TNFSF13B; BAFF R/TNFRSF13C; CD27/TNFRSF7; CD27 Ligand/TNFSF7; CD30/TNFRSF8; CD30 Ligand/TNFSF8; CD40/TNFRSF5; CD40/TNFSF5; CD40 Ligand/TNFSF5; DR3/TNFRSF25; GITR/TNFRSF18; GITR Ligand/TNFSF18; HVEM/TNFRSF14; LIGHT/TNFSF14; Lymphotoxin-alpha/TNF-beta; OX40/TNFRSF4; 0X40 Ligand/TNFSF4; RELT/TNFRSF19L; TACI/TNFRSF13B; TL1A/TNFSF15; TNF-alpha; TNF RII/TNFRSF1B); 2B4/CD244/SLAMF4; BLAME/SLAMF8; CD2; CD2F-10/SLAMF9;
CD48/SLAMF2; CD58/LFA-3; CD84/SLAMF5; CD229/SLAMF3; CRACC/SLAMF7; NTB- A/SLAMF6; SLAM/CD150); CD2; CD7; CD53; CD82/Kai-1; CD90/Thyl; CD96; CD160; CD200; CD300a/LMIRl; HLA Class I; HLA-DR; Ikaros; Integrin alpha 4/CD49d; Integrin alpha 4 beta 1; Integrin alpha 4 beta 7/LPAM-l; LAG-3; TCL1A; TCL1B; CRTAM; DAP12; Dectin-1/CLEC7A; DPPIV/CD26; EphB6; TIM-l/KIM-l/HAVCR; TIM-4; TSLP; TSLP R; lymphocyte function associated antigen-1 (LFA-1); NKG2C, a CD3 zeta domain, an immunoreceptor tyrosine-based activation motif (ITAM), CD27, CD28, 4-1BB, CD134/OX40, CD30, CD40, PD-1, ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, or functional fragment thereof.
[0561] In some embodiments, the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; and (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof. In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof, and/or (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof. In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; (iii) a 4- IBB domain, or a CD 134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.
[0562] In some embodiments, the CAR further comprises one or more spacers, e.g., wherein the spacer is a first spacer between the antigen binding domain and the transmembrane domain. In some embodiments, the first spacer includes at least a portion of an immunoglobulin constant region or variant or modified version thereof. In some embodiments, the spacer is a second spacer between the transmembrane domain and a signaling domain. In some embodiments, the second spacer is an oligopeptide, e.g., wherein the oligopeptide comprises glycine-serine doublets.
[0563] In some embodiments, the payload agent is or comprises a CAR, e.g., a first generation CAR or a nucleic acid encoding a first generation CAR. In some embodiments, a first generation CAR comprises an antigen binding domain, a transmembrane domain, and signaling domain. In some embodiments a signaling domain mediates downstream signaling during T cell activation.
[0564] In some embodiments, the payload agent is or comprises a second generation CAR or a nucleic acid encoding a second generation CAR. In some embodiments a second generation CAR comprises an antigen binding domain, a transmembrane domain, and two signaling domains. In some embodiments a signaling domain mediates downstream signaling during T cell activation. In some embodiments a signaling domain is a costimulatory domain. In some embodiments, a costimulatory domain enhances cytokine production, CAR T cell proliferation, and or CAR T cell persistence during T cell activation.
[0565] In some embodiments, the payload agent is or comprises a third generation CAR or a nucleic acid encoding a third generation CAR. In some embodiments, a third generation CAR comprises an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments a signaling domain mediates downstream signaling during T cell activation. In some embodiments a signaling domain is a costimulatory domain. In some embodiments, a costimulatory domain enhances cytokine production, CAR T cell proliferation, and or CAR T cell persistence during T cell activation. In some embodiments, a third generation CAR comprises at least two costimulatory domains. In some embodiments, the at least two costimulatory domains are not the same.
[0566] In some embodiments, the payload agent is or comprises a fourth generation CAR or a nucleic acid encoding a fourth generation CAR. In some embodiments a fourth generation CAR comprises an antigen binding domain, a transmembrane domain, and at least two, three, or four signaling domains. In some embodiments a signaling domain mediates downstream signaling during T cell activation. In some embodiments a signaling domain is a costimulatory domain. In some embodiments, a costimulatory domain enhances cytokine production, CAR T cell proliferation, and or CAR T cell persistence during T cell activation.
[0567] In some embodiments, a first, second, third, or fourth generation CAR further comprises a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some embodiments, a cytokine gene is endogenous or exogenous to a target cell comprising a CAR which comprises a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some embodiments a cytokine gene encodes a pro-inflammatory cytokine. In some embodiments a cytokine gene encodes IL-1, IL-2, IL-9, IL-12, IL-18, TNF, or IFN-gamma, or functional fragment thereof. In some embodiments a domain which upon successful signaling of the CAR induces expression of a cytokine gene is or comprises a transcription factor or functional domain or fragment thereof. In some embodiments a domain which upon successful signaling of the CAR induces expression of a cytokine gene is or comprises a transcription factor or functional domain or fragment thereof. In some embodiments a transcription factor or functional domain or fragment thereof is or comprises a nuclear factor of activated T cells (NF AT), an NF-kB, or functional domain or fragment thereof. See, e.g., Zhang. C. et al., Engineering CAR-T cells. Biomarker Research. 5:22 (2017); WO 2016126608; Sha, H. et al. Chimaeric antigen receptor T-cell therapy for tumour immunotherapy. Bioscience Reports Jan 27, 2017, 37 (1).
[0568] In some embodiments, a CAR antigen binding domain is or comprises an antibody or antigen-binding portion thereof. In some embodiments, a CAR antigen binding domain is or comprises an scFv or Fab. In some embodiments a CAR antigen binding domain comprises an scFv or Fab fragment of a T-cell alpha chain antibody; T-cell P chain antibody; T-cell y chain antibody; T-cell 5 chain antibody; CCR7 antibody; CD3 antibody; CD4 antibody; CD5 antibody; CD7 antibody; CD8 antibody; CDl lb antibody; CDl lc antibody; CD16 antibody; CD19 antibody; CD20 antibody; CD21 antibody; CD22 antibody; CD25 antibody; CD28 antibody; CD34 antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45RO antibody; CD52 antibody; CD56 antibody; CD62E antibody; CD68 antibody; CD80 antibody; CD95 antibody; CD117 antibody; CD127 antibody; CD133 antibody; CD137 (4-1 BB) antibody; CD 163 antibody; F4/80 antibody; IE-4Ra antibody; Sca-1 antibody; CTEA-4 antibody; GITR antibody GARP antibody; FAP antibody; granzyme B antibody; LFA-1 antibody; MR1 antibody; uPAR antibody; or transferrin receptor antibody.
[0569] In some embodiments, an antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, a cell surface antigen is characteristic of one type of cell. In some embodiments, a cell surface antigen is characteristic of more than one type of cell.
[0570] In some embodiments a CAR antigen binding domain binds a cell surface antigen characteristic of a T cell. In some embodiments, an antigen characteristic of a T cell may be a cell surface receptor, a membrane transport protein (e.g., an active or passive transport protein such as, for example, an ion channel protein, a pore-forming protein, etc.), a transmembrane receptor, a membrane enzyme, and/or a cell adhesion protein characteristic of a T cell. In some embodiments, an antigen characteristic of a T cell may be a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, or histidine kinase associated receptor.
[0571] In some embodiments a CAR comprises a signaling domain which is a costimulatory domain. In some embodiments a CAR comprises a second costimulatory domain. In some embodiments a CAR comprises at least two costimulatory domains. In some embodiments a CAR comprises at least three costimulatory domains. In some embodiments a CAR comprises a costimulatory domain selected from one or more of CD27, CD28, 4-1BB, CD134/OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.
[0572] In some embodiments, the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (IT AM), or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (IT AM), or functional variant thereof; and (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof. In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (IT AM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (IT AM), or functional variant thereof; (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof, and/or (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof. In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (IT AM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; (iii) a 4- IBB domain, or a CD 134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.
[0573] In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.
[0574] In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
[0575] In some embodiments the intracellular signaling domain includes intracellular components of a 4-1BB signaling domain and a CD3-zeta signaling domain. In some embodiments, the intracellular signaling domain includes intracellular components of a CD28 signaling domain and a CD3zeta signaling domain.
[0576] In some embodiments, the CAR comprises an extracellular antigen binding domain (e.g., antibody or antibody fragment, such as an scFv) that binds to an antigen (e.g. tumor antigen), a spacer (e.g. containing a hinge domain, such as any as described herein), a transmembrane domain (e.g. any as described herein), and an intracellular signaling domain (e.g. any intracellular signaling domain, such as a primary signaling domain or costimulatory signaling domain as described herein). In some embodiments, the intracellular signaling domain is or includes a primary cytoplasmic signaling domain. In some embodiments, the intracellular signaling domain additionally includes an intracellular signaling domain of a costimulatory molecule (e.g., a costimulatory domain).
[0577] In some embodiments, the CAR contains one or more domains that combine an antigen- or ligand-binding domain (e.g. antibody or antibody fragment) that provides specificity for a desired antigen (e.g., tumor antigen) with intracellular signaling domains. In some embodiments, the intracellular signaling domain is a stimulating or an activating intracellular domain portion, such as a T cell stimulating or activating domain, providing a primary activation signal or a primary signal. In some embodiments, the intracellular signaling domain contains or additionally contains a costimulatory signaling domain to facilitate effector functions. In some embodiments, chimeric receptors when genetically engineered into immune cells can modulate T cell activity, and, in some cases, can modulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells with improved longevity, survival and/or persistence in vivo, such as for use in adoptive cell therapy methods.
[0578] Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, W02013/123061, U.S. patent app. Pub. Nos. US2002131960, US2013287748, US20130149337, U.S. Patent Nos. 6,451,995,
7.446.190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762,
7.446.191, 8,324,353, and 8,479,118, and European patent app. No. EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in WO/2014055668. Examples of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US 7,446,190, US 8,389,282, Kochenderfer et al., (2013) Nature Reviews Clinical Oncology, 10, 267-276; Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US 7,446,190, and US 8, 389, 282. The recombinant receptors, such as CARs, generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, generally a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment. In some embodiments, the antigen binding domain of the CAR molecule comprises an antibody, an antibody fragment, an scFv, a Fv, a Fab, a (Fab')2, a single domain antibody (SdAb), a VH or VL domain, or a camelid VHH domain.
[0579] In some embodiments, a CAR antigen binding domain is or comprises an antibody or antigen-binding portion thereof. In some embodiments, a CAR antigen binding domain is or comprises an scFv or Fab. In some embodiments, a CAR antigen binding domain comprises an scFv or Fab fragment of a CD19 antibody; CD22 antibody; T-cell alpha chain antibody; T-cell chain antibody; T- cell y chain antibody; T-cell 5 chain antibody; CCR7 antibody; CD3 antibody; CD4 antibody; CD5 antibody; CD7 antibody; CD8 antibody; CDl lb antibody; CDl lc antibody; CD16 antibody; CD20 antibody; CD21 antibody; CD25 antibody; CD28 antibody; CD34 antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45RO antibody; CD52 antibody; CD56 antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD95 antibody; CD117 antibody; CD127 antibody; CD133 antibody; CD 137 (4-1 BB) antibody; CD 163 antibody; F4/80 antibody; IL-4Ra antibody; Sca-1 antibody; CTLA-4 antibody; GITR antibody GARP antibody; LAP antibody; granzyme B antibody; LFA-1 antibody; MR1 antibody; uPAR antibody; or transferrin receptor antibody. [0580] In some embodiments, a CAR comprises a signaling domain which is a costimulatory domain. In some embodiments, a CAR comprises a second costimulatory domain. In some embodiments, a CAR comprises at least two costimulatory domains. In some embodiments, a CAR comprises at least three costimulatory domains. In some embodiments, a CAR comprises a costimulatory domain selected from one or more of CD27, CD28, 4-1BB, CD134/OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83. In some embodiments, if a CAR comprises two or more costimulatory domains, two costimulatory domains are different. In some embodiments, if a CAR comprises two or more costimulatory domains, two costimulatory domains are the same.
[0581] In addition to the CARs described herein, various chimeric antigen receptors and nucleotide sequences encoding the same are known in the art and would be suitable for fusosomal delivery and reprogramming of target cells in vivo and in vitro as described herein. See, e.g., W02013040557; W02012079000; W02016030414; Smith T, et al., Nature Nanotechnology. 2017. DOI: 10.1038/NNAN0.2017.57, the disclosures of which are herein incorporated by reference.
[0582] In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on the engineered cells.
[0583] In some embodiments, the antigen targeted by the receptor includes antigens associated with a B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD47, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.
[0584] In some embodiments, the CAR binds to CD 19. In some embodiments, the CAR binds to CD22. In some embodiments, the CAR binds to CD 19 and CD22. In some embodiments, the CAR is selected from the group consisting of a first generation CAR, a second generation CAR, a third generation CAR, and a fourth generation CAR. In some embodiments, the CAR includes a single binding domain that binds to a single target antigen. In some embodiments, the CAR includes a single binding domain that binds to more than one target antigen, e.g., 2, 3, or more target antigens. In some embodiments, the CAR includes two binding domains such that each binding domain binds to a different target antigens. In some embodiments, the CAR includes two binding domains such that each binding domain binds to the same target antigen. Detailed descriptions of exemplary CARs including CD 19- specific, CD22-specific and CD19/CD22-bispecific CARs can be found in W02012/079000, WO2016/149578 and W02020/014482, the disclosures including the sequence listings and figures are incorporated herein by reference in their entirety. [0585] In some embodiments, the chimeric antigen receptor includes an extracellular portion containing an antibody or antibody fragment. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv.
[0586] In some embodiments, the antigen targeted by the antigen-binding domain is CD 19. In some aspects, the antigen-binding domain of the recombinant receptor, e.g., CAR, and the antigen-binding domain binds, such as specifically binds or specifically recognizes, a CD19, such as a human CD19. In some embodiments, the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD 19. In some embodiments, the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US 2016/0152723.
[0587] In some embodiments, the antigen is CD 19. In some embodiments, the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD 19. In some embodiments, the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US 2016/0152723.
[0588] In some embodiments, the scFv is derived from FMC63. FMC63 generally refers to a mouse monoclonal IgGl antibody raised against Naim-1 and -16 cells expressing CD19 of human origin (Fing, N. R., et al. (1987). Leucocyte typing III. 302).
[0589] In some embodiments, the antibody portion of the recombinant receptor, e.g., CAR, further includes spacer between the transmembrane domain and extracellular antigen binding domain. In some embodiments, the spacer includes at least a portion of an immunoglobulin constant region, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgGl. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, WO2014031687, U.S. Patent No. 8,822,647 or published app. No. US 2014/0271635. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgGl.
[0590] In some embodiments, the antigen receptor comprises an intracellular domain linked directly or indirectly to the extracellular domain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an IT AM. For example, in some aspects, the antigen recognition domain (e.g. extracellular domain) generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g. scFv) and intracellular signaling domain. Thus, in some embodiments, the antigen-binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains.
[0591] In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
[0592] In some embodiments, the CAR transmembrane domain comprises at least a transmembrane region of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or functional variant thereof. In some embodiments, the transmembrane domain comprises at least a transmembrane region(s) of CD8a, CD8P, 4-1BB/CD137, CD28, CD34, CD4, FcaRIy, CD16, OX40/CD134, CD3^, CD3a, CD3y, CD35, TCRa, TCRp, TCR^, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, and FGFR2B, or functional variant thereof. The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, CD 154. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s). In some aspects, the transmembrane domain contains a transmembrane portion of CD28.
[0593] In some embodiments, the extracellular domain and transmembrane domain can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the receptor contains extracellular portion of the molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion.
[0594] Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
[0595] T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.
[0596] The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine -based activation motifs or IT AMs. Examples of IT AM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
[0597] In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the intracellular component is or includes a CD3-zeta intracellular signaling domain. In some embodiments, the intracellular component is or includes a signaling domain from Fc receptor gamma chain. In some embodiments, the receptor, e.g., CAR, includes the intracellular signaling domain and further includes a portion, such as a transmembrane domain and/or hinge portion, of one or more additional molecules such as CD8, CD4, CD25, or CD 16. For example, in some aspects, the CAR or other chimeric receptor is a chimeric molecule of CD3-zeta (CD3-z) or Fc receptor and a portion of one of CD8, CD4, CD25 or CD16.
[0598] In some embodiments, upon ligation of the CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement. [0599] In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.
[0600] In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, 0X40, DAP10, and ICOS. In some aspects, the same CAR includes both the activating and costimulatory components. In some embodiments, the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB. In some aspects, the T cell costimulatory molecule is 41BB.
[0601] In some embodiments, the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the cells include one or more stimulatory or activating CAR and/or a costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than the one associated with and/or specific for the disease or condition whereby an activating signal delivered through the disease-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.
[0602] In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.
[0603] In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
[0604] In some embodiments the intracellular signaling domain includes intracellular components of a 4-1BB signaling domain and a CD3-zeta signaling domain. In some embodiments, the intracellular signaling domain includes intracellular components of a CD28 signaling domain and a CD3zeta signaling domain. [0605] In some embodiments, a CD19 specific CAR includes an anti-CD19 single-chain antibody fragment (scFv), a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) costimulatory signaling domain, and a CD3^ signaling domain. In some embodiments, a CD22 specific CAR includes an anti-CD22 scFv, a transmembrane domain such as one derived from human CD8a, a 4- 1BB (CD137) co-stimulatory signaling domain, and a CD3^ signaling domain. In some embodiments, a CD19/CD22-bispecific CAR includes an anti-CD19 scFv, an anti-CD22 scFv, a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co-stimulatory signaling domain, and a CD3^ signaling domain.
[0606] In some embodiments, the CAR comprises a commercial CAR construct carried by a T cell. Non-limiting examples of commercial CAR-T cell based therapies include brexucabtagene autoleucel (TECARTUS®), axicabtagene ciloleucel (YESCARTA®), idecabtagene vicleucel (ABECMA®), lisocabtagene maraleucel (BREYANZI®), tisagenlecleucel (KYMRIAH®), Descartes-08 and Descartes- 11 from Cartesian Therapeutics, CTL110 from Novartis, P-BMCA-101 from Poseida Therapeutics, AUTO4 from Autolus Limited, UCARTCS from Cellectis, PBCAR19B and PBCAR269A from Precision Biosciences, FT819 from Fate Therapeutics, and CYAD-211 from Clyad Oncology.
[0607] In some embodiments, the antigen binding domain targets an antigen characteristic of an autoimmune or inflammatory disorder. In some embodiments, the ABD binds an antigen associated with an autoimmune or inflammatory disorder. In some instances, the antigen is expressed by a cell associated with an autoimmune or inflammatory disorder. In some embodiments, the autoimmune or inflammatory disorder is selected from chronic graft-vs-host disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, goodpasture syndrome, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, cold agglutinin disease, Pemphigus vulgaris, Grave's disease, autoimmune hemolytic anemia, Hemophilia A, Primary Sjogren's Syndrome, thrombotic thrombocytopenia purrpura, neuromyelits optica, Evan's syndrome, IgM mediated neuropathy, cryoglobulinemia, dermatomyositis, idiopathic thrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquired angioedema, chronic urticarial, antiphospholipid demyelinating polyneuropathy, and autoimmune thrombocytopenia or neutropenia or pure red cell aplasias, while exemplary nonlimiting examples of alloimmune diseases include allosensitization (see, for example, Blazar et al., 2015, Am. J. Transplant, 15(4) :931 -41) or xenosensitization from hematopoietic or solid organ transplantation, blood transfusions, pregnancy with fetal allosensitization, neonatal alloimmune thrombocytopenia, hemolytic disease of the newborn, sensitization to foreign antigens such as can occur with replacement of inherited or acquired deficiency disorders treated with enzyme or protein replacement therapy, blood products, and gene therapy. In some embodiments, the antigen characteristic of an autoimmune or inflammatory disorder is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, or histidine kinase associated receptor.
[0608] In some embodiments, an antigen binding domain of a CAR binds to a ligand expressed on B cells, plasma cells, or plasmablasts. In some embodiments, an antigen binding domain of a CAR binds to CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, CD28, TNF, interferon receptors, GM-CSF, ZAP-70, LFA-1, CD3 gamma, CD5 or CD2. See, e.g., US 2003/0077249; WO 2017/058753; WO 2017/058850, the contents of which are herein incorporated by reference.
[0609] In some embodiments, the antigen binding domain targets an antigen characteristic of senescent cells, e.g., urokinase-type plasminogen activator receptor (uPAR). In some embodiments, the ABD binds an antigen associated with a senescent cell. In some instances, the antigen is expressed by a senescent cell. In some embodiments, the CAR may be used for treatment or prophylaxis of disorders characterized by the aberrant accumulation of senescent cells, e.g., liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis.
[0610] In some embodiments, the antigen binding domain targets an antigen characteristic of an infectious disease. In some embodiments, the ABD binds an antigen associated with an infectious disease. In some instances, the antigen is expressed by a cell affected by an infectious disease. In some embodiments, wherein the infectious disease is selected from HIV, hepatitis B virus, hepatitis C virus, Human herpes virus, Human herpes virus 8 (HHV-8, Kaposi sarcoma-associated herpes virus (KSHV)), Human T-lymphotrophic virus-1 (HTLV-1), Merkel cell polyomavirus (MCV), Simian virus 40 (SV40), Epstein-Barr virus, CMV, human papillomavirus. In some embodiments, the antigen characteristic of an infectious disease is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme- linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor, HIV Env, gpl20, or CD4-induced epitope on HIV-1 Env.
[0611] In some embodiments, an antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, a cell surface antigen is characteristic of (e.g., expressed by) a particular or specific cell type. In some embodiments, a cell surface antigen is characteristic of more than one type of cell.
[0612] In some embodiments, a CAR antigen binding domain binds a cell surface antigen characteristic of a T cell, such as a cell surface antigen on a T cell. In some embodiments, an antigen characteristic of a T cell may be a cell surface receptor, a membrane transport protein (e.g., an active or passive transport protein such as, for example, an ion channel protein, a pore-forming protein, etc.), a transmembrane receptor, a membrane enzyme, and/or a cell adhesion protein characteristic of a T cell. In some embodiments, an antigen characteristic of a T cell may be a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, or histidine kinase associated receptor. [0613] In some embodiments, the CAR comprises an extracellular antigen binding domain (e.g., antibody or antibody fragment, such as an scFv) that binds to an antigen (e.g. tumor antigen), a spacer (e.g. containing a hinge domain, such as any as described herein), a transmembrane domain (e.g. any as described herein), and an intracellular signaling domain (e.g. any intracellular signaling domain, such as a primary signaling domain or costimulatory signaling domain as described herein). In some embodiments, the intracellular signaling domain is or includes a primary cytoplasmic signaling domain. In some embodiments, the intracellular signaling domain additionally includes an intracellular signaling domain of a costimulatory molecule (e.g., a costimulatory domain). Examples of exemplary components of a CAR are described in Table 5. In provided aspects, the sequences of each component in a CAR can include any combination listed in Table 5.
[0614] In some embodiments, the antigen receptor further includes a marker and/or cells expressing the CAR or other antigen receptor further includes a surrogate marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor. In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor, such as truncated version of such a cell surface receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence.
[0615] In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof. In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self’ by the immune system of the host into which the cells will be adoptively transferred.
[0616] In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.
[0617] In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD 137; in some aspects, a third generation CAR is one that includes multiple costimulatory domains of different costimulatory receptors.
[0618] For example, in some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4- IBB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge -only spacer.
[0619] In some aspects, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgGlIn other embodiments, the spacer is or contains an Ig hinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only. In some embodiments, the spacer is or comprises a glycineserine rich sequence or other flexible linker such as known flexible linkers.
[0620] For example, in some embodiments, the CAR includes an antibody such as an antibody fragment, including scFvs, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28 -derived intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes an antibody or fragment, such as scFv, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-lBB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.
[0621] The recombinant receptors, such as CARs, expressed by the cells administered to the subject generally recognize or specifically bind to a molecule that is expressed in, associated with, and/or specific for the disease or condition or cells thereof being treated. Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an ITAM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition. For example, in some embodiments, the cells express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition. 2. T Cell Receptors (TCRs)
[0622] In some embodiments, the payload agent is a T cell receptor (TCR) or antigen-binding portion thereof that recognizes a peptide epitope or T cell epitope of a target polypeptide, such as an antigen of a tumor, viral or autoimmune protein.
[0623] In some embodiments, a “T cell receptor” or “TCR” is a molecule that contains a variable a and b chains (also known as TCRalpha and TCRbeta, respectively) or a variable g and d chains (also known as TCRalpha and TCRbeta, respectively), or antigen-binding portions thereof, and which is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the ab form. Typically, TCRs that exist in alpha-beta and gamma-delta forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.
[0624] Unless otherwise stated, the term “TCR” should be understood to encompass full TCRs as well as antigen-binding portions or antigen-binding fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, including TCRs in the ab form or gd form. In some embodiments, the TCR is an antigen-binding portion that is less than a full-length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC -peptide complex. In some cases, an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC-peptide complex, to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable b chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of a TCR contain complementarity determining regions involved in recognition of the peptide, MHC and/or MHC-peptide complex.
3. Chimeric Auto-Antibody Receptor (CAAR)
[0625] In some embodiments, the engineered receptor is a chimeric autoantibody receptor (CAAR). In some embodiments, the CAAR binds, e.g., specifically binds, or recognizes, an autoantibody. In some embodiments, a cell expressing the CAAR, such as a T cell engineered to express a CAAR, can be used to bind to and kill autoantibody-expressing cells, but not normal antibody expressing cells. In some embodiments, CAAR-expressing cells can be used to treat an autoimmune disease associated with expression of self-antigens, such as autoimmune diseases. In some embodiments, CAAR-expressing cells can target B cells that ultimately produce the autoantibodies and display the autoantibodies on their cell surfaces, mark these B cells as disease-specific targets for therapeutic intervention. In some embodiments, CAAR-expressing cells can be used to efficiently targeting and killing the pathogenic B cells in autoimmune diseases by targeting the disease-causing B cells using an antigen-specific chimeric autoantibody receptor. In some embodiments, the recombinant receptor is a CAAR, such as any described in U.S. Patent Application Pub. No. US 2017/0051035.
[0626] In some embodiments, the CAAR comprises an autoantibody binding domain, a transmembrane domain, and one or more intracellular signaling region or domain (also interchangeably called a cytoplasmic signaling domain or region). In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain that is capable of stimulating and/or inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component (e.g. an intracellular signaling domain or region of a CD3-zeta) chain or a functional variant or signaling portion thereof), and/or a signaling domain comprising an immunoreceptor tyrosine- based activation motif (IT AM).
[0627] In some embodiments, the autoantibody binding domain comprises an autoantigen or a fragment thereof. The choice of autoantigen can depend upon the type of autoantibody being targeted. For example, the autoantigen may be chosen because it recognizes an autoantibody on a target cell, such as a B cell, associated with a particular disease state, e.g. an autoimmune disease, such as an autoantibody-mediated autoimmune disease. In some embodiments, the autoimmune disease includes pemphigus vulgaris (PV). Exemplary autoantigens include desmoglein 1 (Dsgl) and Dsg3.
4. Additional Descriptions of CARs
[0628] In certain embodiments, the pay load agent is a CAR. CARs (also known as chimeric immunoreceptors, chimeric T cell receptors, or artificial T cell receptors) are receptor proteins that have been engineered to give host cells (e.g., T cells) the new ability to target a specific protein. The receptors are chimeric because they combine both antigen-binding and T cell activating functions into a single receptor. The provided particles may be used to express one or more CARs in a host cell (e.g., a T cell) for use in cell-based therapies against various target antigens. In these embodiments, the CAR may comprise an extracellular binding domain (also referred to as a “binder”) that specifically binds a target antigen, a transmembrane domain, and an intracellular signaling domain. In certain embodiments, the CAR may further comprise one or more additional elements, including one or more signal peptides, one or more extracellular hinge domains, and/or one or more intracellular costimulatory domains. Domains may be directly adjacent to one another, or there may be one or more amino acids linking the domains. The nucleotide sequence encoding a CAR may be derived from a mammalian sequence, for example, a mouse sequence, a primate sequence, a human sequence, or combinations thereof. In the cases where the nucleotide sequence encoding a CAR is non-human, the sequence of the CAR may be humanized. The nucleotide sequence encoding a CAR may also be codon-optimized for expression in a mammalian cell, for example, a human cell. In any of these embodiments, the nucleotide sequence encoding a CAR may be at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the nucleotide sequences disclosed herein. The sequence variations may be due to codon-optimalization, humanization, restriction enzyme-based cloning scars, and/or additional amino acid residues linking the functional domains, etc.
[0629] In certain embodiments, the CAR may comprise a signal peptide at the N-terminus. Nonlimiting examples of signal peptides include CD8a signal peptide, IgK signal peptide, and granulocytemacrophage colony-stimulating factor receptor subunit alpha (GMCSFR-a, also known as colony stimulating factor 2 receptor subunit alpha (CSF2RA)) signal peptide, and variants thereof, the amino acid sequences of which are provided in Table 6 below.
Table 6. Exemplary sequences of signal peptides
[0630] In certain embodiments, the extracellular binding domain of the CAR may comprise one or more antibodies specific to one target antigen or multiple target antigens. The antibody may be an antibody fragment, for example, an scFv, or a single-domain antibody fragment, for example, a VHH. In certain embodiments, the scFv may comprise a heavy chain variable region (Vu) and a light chain variable region (VL) of an antibody connected by a linker. The VH and the VL may be connected in either order, i.e., Vn-linkcr-Vi. or Vi.-linkcr-Vn. Non-limiting examples of linkers include Whitlow linker, (G4S)n (n can be a positive integer, e.g., 1, 2, 3, 4, 5, 6, etc.) linker, and variants thereof. In certain embodiments, the antigen may be an antigen that is exclusively or preferentially expressed on tumor cells, or an antigen that is characteristic of an autoimmune or inflammatory disease. Exemplary target antigens include, but are not limited to, CD5, CD19, CD20, CD22, CD23, CD30, CD70, Kappa, Lambda, and B cell maturation agent (BCMA), G-protein coupled receptor family C group 5 member D (GPRC5D) (associated with leukemias); CS1/SLAMF7, CD38, CD138, GPRC5D, TACI, and BCMA (associated with myelomas); GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRa, IL-13Ra, Mesothelin, MUC1, MUC16, and ROR1 (associated with solid tumors). In any of these embodiments, the extracellular binding domain of the CAR can be codon- optimized for expression in a host cell or have variant sequences to increase functions of the extracellular binding domain.
[0631] In certain embodiments, the CAR may comprise a hinge domain, also referred to as a spacer. The terms “hinge” and “spacer” may be used interchangeably in the present disclosure. Non-limiting examples of hinge domains include CD8a hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge-CH2-CH3 domain, and variants thereof, the amino acid sequences of which are provided in Table
7 below.
Table 7. Exemplary sequences of hinge domains
[0632] In certain embodiments, the transmembrane domain of the CAR may comprise a transmembrane region of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3s. CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a functional variant thereof, including the human versions of each of these sequences. In other embodiments, the transmembrane domain may comprise a transmembrane region of CD8a, CD8P, 4- 1BB/CD137, CD28, CD34, CD4, FcaRIy, CD16, OX40/CD134, CD3^, CD3a, CD3y, CD35, TCRa, TCRP, TCR^, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, and FGFR2B, or a functional variant thereof, including the human versions of each of these sequences. Table 8 provides the amino acid sequences of a few exemplary transmembrane domains.
Table 8. Exemplary sequences of transmembrane domains [0633] In certain embodiments, the intracellular signaling domain and/or intracellular costimulatory domain of the CAR may comprise one or more signaling domains selected from B7-1/CD80, B7- 2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, PDCD6, 4-1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14, Lymphotoxin-alpha/TNFp, OX40/TNFRSF4, 0X40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF13B, TL1A/TNFSF15, TNFa, TNF RII/TNFRSF1B, 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, SLAM/CD150, CD2, CD7, CD53, CD82/Kai-1, CD90/Thyl, CD96, CD160, CD200, CD300a/LMIRl, HLA Class I, HLA-DR, Ikaros, Integrin alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-l, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin- 1/CLEC7A, DPPIV/CD26, EphB6, TIM-l/KIM-l/HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen-1 (LFA-1), NKG2C, CD3^, an immunoreceptor tyrosine-based activation motif (ITAM), CD27, CD28, 4-1BB, CD134/OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and a functional variant thereof including the human versions of each of these sequences. In some embodiments, the intracellular signaling domain and/or intracellular costimulatory domain comprises one or more signaling domains selected from a CD3^ domain, an ITAM, a CD28 domain, 4-1BB domain, or a functional variant thereof. Table 7 provides the amino acid sequences of a few exemplary intracellular costimulatory and/or signaling domains. In certain embodiments, as in the case of tisagenlecleucel as described below, the CD3^ signaling domain of SEQ ID NO:99 may have a mutation, e.g., a glutamine (Q) to lysine (K) mutation, at amino acid position 14 (see SEQ ID NO:62).
Table 9. Exemplary sequences of intracellular costimulatory and/or signaling domains a. CD 19 CARs
[0634] In some embodiments, the CAR is a CD19 CAR (“CD19-CAR”). In some embodiments, the CD19 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD19, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and/or an intracellular signaling domain in tandem.
[0635] In some embodiments, the signal peptide of the CD 19 CAR comprises a CD 8 a signal peptide. In some embodiments, the CD8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:47 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 47. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:48 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:48. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide. In some embodiments, the GMCSFR-a or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:49 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 49.
[0636] In some embodiments, the extracellular binding domain of the CD 19 CAR is specific to CD 19, for example, human CD 19. The extracellular binding domain of the CD 19 CAR can be codon- optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.
[0637] In some embodiments, the extracellular binding domain of the CD19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (Vu) and the light chain variable region (VL) of FMC63 connected by a linker. FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun. 34(16-17): 1157-1165 (1997) and PCT Application Publication No. WO2018/213337, the entire contents of each of which are incorporated by reference herein. In some embodiments, the amino acid sequences of the entire FMC63-derived scFv (also referred to as FMC63 scFv) and its different portions are provided in Table 10 below. In some embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:63, 64, or 69, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 163, 64, or 69. In some embodiments, the CD19- specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 65-67 and 70-72. In some embodiments, the CD19-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 65-67. In some embodiments, the CD19-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 70-72. In any of these embodiments, the CD19-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD 19 CAR comprises or consists of the one or more CDRs as described herein.
[0638] In some embodiments, the linker linking the VH and the VL portions of the scFv is a Whitlow linker having an amino acid sequence set forth in SEQ ID NO: 68. In some embodiments, the Whitlow linker may be replaced by a different linker, for example, a 3xG4S linker having an amino acid sequence set forth in SEQ ID NO: 143, which gives rise to a different FMC63-derived scFv having an amino acid sequence set forth in SEQ ID NO:73. In certain of these embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 73 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:73.
Table 10. Exemplary sequences of anti-CD19 scFv and components
[0639] In some embodiments, the extracellular binding domain of the CD 19 CAR is derived from an antibody specific to CD19, including, for example, SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol. 138(9):2793-2799 (1987)), 4G7 (Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418-427 (1987)), B4 HB12b (Kansas & Tedder, J. Immunol. 147:4094-4102 (1991); Yazawa et al., Proc. Natl. Acad. Sci. USA 102:15178-15183 (2005); Herbst et al., J. Pharmacol. Exp. Ther. 335:213-222 (2010)), BU12 (Callard et al., J. Immunology, 148(10): 2983-2987 (1992)), and CLB-CD19 (De Rie Cell. Immunol. 118:368-381(1989)). In any of these embodiments, the extracellular binding domain of the CD19 CAR can comprise or consist of the Vu, the VL, and/or one or more CDRs of any of the antibodies.
[0640] In some embodiments, the hinge domain of the CD19 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:50 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:50. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:51 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:51. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:53 or SEQ ID NO:54, or an amino acid sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:53 or SEQ ID NO:54. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:55 or an amino acid sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:55.
[0641] In some embodiments, the transmembrane domain of the CD 19 CAR comprises a CD 8 a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the CD 8 a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:56 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:56. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:57 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:57.
[0642] In some embodiments, the intracellular costimulatory domain of the CD 19 CAR comprises a 4-1BB costimulatory domain. 4-1BB, also known as CD137, transmits a potent costimulatory signal to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes. In some embodiments, the 4-1BB costimulatory domain is human. In some embodiments, the 4-1BB costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:59 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:59. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain. CD28 is another co-stimulatory molecule on T cells. In some embodiments, the CD28 costimulatory domain is human. In some embodiments, the CD28 costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:60 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:60. In some embodiments, the intracellular costimulatory domain of the CD19 CAR comprises a 4- IBB costimulatory domain and a CD28 costimulatory domain as described.
[0643] In some embodiments, the intracellular signaling domain of the CD19 CAR comprises a CD3 zeta (Q signaling domain. CD3 zeta associates with T cell receptors (TCRs) to produce a signal and contains immunoreceptor tyrosine-based activation motifs (ITAMs). The CD3 zeta signaling domain refers to amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation. In some embodiments, the CD3 zeta signaling domain is human. In some embodiments, the CD3 zeta signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 61 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:61.
[0644] In some embodiments, the payload agent is a CD19 CAR, including, for example, a CD19 CAR comprising the CD19-specific scFv having sequences set forth in SEQ ID NO:63 or SEQ ID NO:73, the CD8a hinge domain of SEQ ID NO:50, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof. In any of these embodiments, the CD 19 CAR may additionally comprise a signal peptide (e.g., a CD 8 a signal peptide) as described.
[0645] In some embodiments, the payload agent is a CD19 CAR, including, for example, a CD19 CAR comprising the CD19-specific scFv having sequences set forth in SEQ ID NO:63 or SEQ ID NO:73, the IgG4 hinge domain of SEQ ID NO:53 or SEQ ID NO:54, the CD28 transmembrane domain of SEQ ID NO:57, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof. In any of these embodiments, the CD19 CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide) as described.
[0646] In some embodiments, the payload agent is a CD19 CAR, including, for example, a CD19 CAR comprising the CD19-specific scFv having sequences set forth in SEQ ID NO:63 or SEQ ID NO:73, the CD28 hinge domain of SEQ ID NO:51, the CD28 transmembrane domain of SEQ ID NO:57, the CD28 costimulatory domain of SEQ ID NO:60, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof. In any of these embodiments, the CD 19 CAR may additionally comprise a signal peptide (e.g., a CD 8 a signal peptide) as described.
[0647] In some embodiments, the pay load agent is a CD 19 CAR as encoded by the sequence set forth in SEQ ID NO:74 or a sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO:74 (see Table 11). The encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO:75 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:75, with the following components: CD8a signal peptide, FMC63 scFv (VL- Whitlow linker-Vn), CD8a hinge domain, CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3^ signaling domain.
[0648] In some embodiments, the pay load agent is a commercially available embodiment of a CD 19 CAR. Non-limiting examples of commercially available embodiments of CD 19 CARs include tisagenlecleucel, lisocabtagene maraleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel.
[0649] In some embodiments, the CAR is tisagenlecleucel or portions thereof. Tisagenlecleucel comprises a CD19 CAR with the following components: CD8a signal peptide, FMC63 scFv (VL-3XG4S linker-Vn), CD8a hinge domain, CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3^ signaling domain. The nucleotide and amino acid sequence of the CD 19 CAR in tisagenlecleucel are provided in Table 11, with annotations of the sequences provided in Table 12.
[0650] In some embodiments, the CAR is lisocabtagene maraleucel or portions thereof. Lisocabtagene maraleucel comprises a CD19 CAR with the following components: GMCSFR-a or CSF2RA signal peptide, FMC63 scFv (VL- Whitlow linker-Vn), IgG4 hinge domain, CD28 transmembrane domain, 4-1BB costimulatory domain, and CD3^ signaling domain. The nucleotide and amino acid sequence of the CD 19 CAR in lisocabtagene maraleucel are provided in Table 9, with annotations of the sequences provided in Table 13.
[0651] In some embodiments, the CAR is axicabtagene ciloleucel or portions thereof. Axicabtagene ciloleucel comprises a CD19 CAR with the following components: GMCSFR-a or CSF2RA signal peptide, FMC63 scFv (V|_- Whitlow linker-Vn), CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain, and CD3^ signaling domain. The nucleotide and amino acid sequence of the CD 19 CAR in axicabtagene ciloleucel are provided in Table 11, with annotations of the sequences provided in Table 14.
[0652] In some embodiments, the CAR is brexucabtagene autoleucel or portions thereof. Brexucabtagene autoleucel comprises a CD 19 CAR with the following components: GMCSFR- a signal peptide, FMC63 scFv, CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain, and CD3^ signaling domain. [0653] In some embodiments, the CAR is encoded by the sequence set forth in SEQ ID NO: 76, 78, or 80, or a sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 76, 78, or 80. The encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 77, 79, or 81, respectively, or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 77, 79, or 81, respectively.
Table 11. Exemplary sequences of CD19 CARs
Table 12. Annotation of tisagenlecleucel CD19 CAR sequences
Table 13. Annotation of lisocabtagene maraleucel CD19 CAR sequences
Table 14. Annotation of axicabtagene ciloleucel CD19 CAR sequences
[0654] In some embodiments, the CAR is encoded by the sequence set forth in SEQ ID NO: 76, 78, or 80, or a sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 76, 78, or 80. The encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 77, 79, or 81, respectively, is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 77, 79, or 81, respectively. b. CD20 CARs
[0655] In some embodiments, the CAR is a CD20 CAR (“CD20-CAR”). CD20 is an antigen found on the surface of B cells as early at the pro-B phase and progressively at increasing levels until B cell maturity, as well as on the cells of most B-cell neoplasms. CD20 positive cells are also sometimes found in cases of Hodgkins disease, myeloma, and thymoma. In some embodiments, the CD20 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD20, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and/or an intracellular signaling domain in tandem.
[0656] In some embodiments, the signal peptide of the CD20 CAR comprises a CD 8 a signal peptide. In some embodiments, the CD8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:47 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 47. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:48 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:48. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide. In some embodiments, the GMCSFR-a or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:49 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 49.
[0657] In some embodiments, the extracellular binding domain of the CD20 CAR is specific to CD20, for example, human CD20. The extracellular binding domain of the CD20 CAR can be codon- optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.
[0658] In some embodiments, the extracellular binding domain of the CD20 CAR is derived from an antibody specific to CD20, including, for example, Leul6, IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab. In any of these embodiments, the extracellular binding domain of the CD20 CAR can comprise or consist of the VH, the VL, and/or one or more CDRs of any of the antibodies.
[0659] In some embodiments, the extracellular binding domain of the CD20 CAR comprises an scFv derived from the Leu 16 monoclonal antibody, which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of Leul6 connected by a linker. See Wu et al., Protein Engineering. 14(12): 1025-1033 (2001). In some embodiments, the linker is a 3xG4S linker. In other embodiments, the linker is a Whitlow linker as described herein. In some embodiments, the amino acid sequences of different portions of the entire Leul6-derived scFv (also referred to as Leu 16 scFv) and its different portions are provided in Table 15 below. In some embodiments, the CD20-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:82, 83, or 87, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:82, 83, or 87. In some embodiments, the CD20-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 84-86, 88, 89, and 144. In some embodiments, the CD20-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 84-86. In some embodiments, the CD20-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 88, 89, and 144. In any of these embodiments, the CD20-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD20 CAR comprises or consists of the one or more CDRs as described herein.
Table 15. Exemplary sequences of anti-CD20 scFv and components
[0660] In some embodiments, the hinge domain of the CD20 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:50 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:50. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:51 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:51. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:53 or SEQ ID NO:54, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:53 or SEQ ID NO:54. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:55 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:55.
[0661] In some embodiments, the transmembrane domain of the CD20 CAR comprises a CD 8 a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the CD 8 a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:56 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:56. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:57 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:57. [0662] In some embodiments, the intracellular costimulatory domain of the CD20 CAR comprises a 4-1BB costimulatory domain, for example, a human 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:59 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:59. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, for example, a human CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:60 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:60.
[0663] In some embodiments, the intracellular signaling domain of the CD20 CAR comprises a CD3 zeta (Q signaling domain, for example, a human CD3^ signaling domain. In some embodiments, the CD3^ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:61 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:61.
[0664] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the CD8a hinge domain of SEQ ID NO:50, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
[0665] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the CD28 hinge domain of SEQ ID NO:51, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
[0666] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the IgG4 hinge domain of SEQ ID NO:53 or SEQ ID NO:54, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
[0667] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the CD8a hinge domain of SEQ ID NO:50, the CD28 transmembrane domain of SEQ ID NO:57, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
[0668] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the CD28 hinge domain of SEQ ID NO:51, the CD28 transmembrane domain of SEQ ID NO:57, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
[0669] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO: 82, the IgG4 hinge domain of SEQ ID NO:53 or SEQ ID NO:54, the CD28 transmembrane domain of SEQ ID NO:57, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof. c. CD22 CARs
[0670] In some embodiments, the CAR is a CD22 CAR (“CD22-CAR”). CD22, which is a transmembrane protein found mostly on the surface of mature B cells that functions as an inhibitory receptor for B cell receptor (BCR) signaling. CD22 is expressed in 60-70% of B cell lymphomas and leukemias (e.g., B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma) and is not present on the cell surface in early stages of B cell development or on stem cells. In some embodiments, the CD22 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD22, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and/or an intracellular signaling domain in tandem.
[0671] In some embodiments, the signal peptide of the CD22 CAR comprises a CD 8 a signal peptide. In some embodiments, the CD8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:47 or an amino acid sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 47. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:48 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:48. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide. In some embodiments, the GMCSFR-a or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:49 or an amino acid sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 49.
[0672] In some embodiments, the extracellular binding domain of the CD22 CAR is specific to CD22, for example, human CD22. The extracellular binding domain of the CD22 CAR can be codon- optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.
[0673] In some embodiments, the extracellular binding domain of the CD22 CAR is derived from an antibody specific to CD22, including, for example, SM03, inotuzumab, epratuzumab, moxetumomab, and pinatuzumab. In any of these embodiments, the extracellular binding domain of the CD22 CAR can comprise or consist of the Vu, the VL, and/or one or more CDRs of any of the antibodies.
[0674] In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from the m971 monoclonal antibody (m971), which comprises the heavy chain variable region (Vu) and the light chain variable region (VL) of m971 connected by a linker. In some embodiments, the linker is a 3xG4S linker. In other embodiments, the Whitlow linker may be used instead. In some embodiments, the amino acid sequences of the entire m971 -derived scFv (also referred to as m971 scFv) and its different portions are provided in Table 16 below. In some embodiments, the CD22-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:90, 91, or 95, or an amino acid sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:90, 91, or 95. In some embodiments, the CD22-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 92-94 and 96-98. In some embodiments, the CD22-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 92-94. In some embodiments, the CD22-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 96-98. In any of these embodiments, the CD22-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD22 CAR comprises or consists of the one or more CDRs as described herein.
[0675] In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from m971-L7, which is an affinity matured variant of m971 with significantly improved CD22 binding affinity compared to the parental antibody m971 (improved from about 2 nM to less than 50 pM). In some embodiments, the scFv derived from m971-L7 comprises the VH and the VL of m971- L7 connected by a 3xG4S linker. In other embodiments, the Whitlow linker may be used instead. In some embodiments, the amino acid sequences of the entire m971-L7-derived scFv (also referred to as m971-L7 scFv) and its different portions are provided in Table 14 below. In some embodiments, the CD22-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:99, 100, or 104, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:99, 100, or 104. In some embodiments, the CD22-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 101-103 and 105-107. In some embodiments, the CD22-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 101-103. In some embodiments, the CD22-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 105-107. In any of these embodiments, the CD22-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD22 CAR comprises or consists of the one or more CDRs as described herein.
Table 16. Exemplary sequences of anti-CD22 scFv and components
[0676] In some embodiments, the extracellular binding domain of the CD22 CAR comprises immunotoxins HA22 or BL22. Immunotoxins BL22 and HA22 are therapeutic agents that comprise an scFv specific for CD22 fused to a bacterial toxin, and thus can bind to the surface of the cancer cells that express CD22 and kill the cancer cells. BL22 comprises a dsFv of an anti-CD22 antibody, RFB4, fused to a 38-kDa truncated form of Pseudomonas exotoxin A (Bang et al., Clin. Cancer Res., 11:1545-50 (2005)). HA22 (CAT8015, moxetumomab pasudotox) is a mutated, higher affinity version of BL22 (Ho et al., J. Biol. Chem., 280(1): 607-17 (2005)). Suitable sequences of antigen binding domains of HA22 and BL22 specific to CD22 are disclosed in, for example, U.S. Patent Nos. 7,541,034; 7,355,012; and 7,982,011, which are hereby incorporated by reference in their entirety.
[0677] In some embodiments, the hinge domain of the CD22 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:50 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:50. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:51 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:51. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:53 or SEQ ID NO:54, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:53 or SEQ ID NO:54. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:55 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:55.
[0678] In some embodiments, the transmembrane domain of the CD22 CAR comprises a CD 8 a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the CD 8 a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:56 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:56. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:57 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:57.
[0679] In some embodiments, the intracellular costimulatory domain of the CD22 CAR comprises a 4-1BB costimulatory domain, for example, a human 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:59 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:59. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, for example, a human CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:60 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:60.
[0680] In some embodiments, the intracellular signaling domain of the CD22 CAR comprises a CD3 zeta (Q signaling domain, for example, a human CD3^ signaling domain. In some embodiments, the CD3^ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:61 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:61.
[0681] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:90 or SEQ ID NO:99, the CD8a hinge domain of SEQ ID NO:50, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
[0682] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:90 or SEQ ID NO:99, the CD28 hinge domain of SEQ ID NO:51, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
[0683] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:90 or SEQ ID NO:99, the IgG4 hinge domain of SEQ ID NO:53 or SEQ ID NO:54, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
[0684] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:90 or SEQ ID NO:99, the CD8a hinge domain of SEQ ID NO:50, the CD28 transmembrane domain of SEQ ID NO:57, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
[0685] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:90 or SEQ ID NO:99, the CD28 hinge domain of SEQ ID NO:51, the CD28 transmembrane domain of SEQ ID NO:57, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.
[0686] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:90 or SEQ ID NO:99, the IgG4 hinge domain of SEQ ID NO:53 or SEQ ID NO:54, the CD28 transmembrane domain of SEQ ID NO:57, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof. d. BCMA CARs
[0687] In some embodiments, the CAR is a BCMA CAR (“BCMA-CAR”). BCMA is a tumor necrosis family receptor (TNFR) member expressed on cells of the B cell lineage, with the highest expression on terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating the survival of plasma cells for maintaining long-term humoral immunity. The expression of BCMA has been recently linked to a number of cancers, such as multiple myeloma, Hodgkin's and nonHodgkin's lymphoma, various leukemias, and glioblastoma. In some embodiments, the BCMA CAR may comprise a signal peptide, an extracellular binding domain that specifically binds BCMA, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and/or an intracellular signaling domain in tandem.
[0688] In some embodiments, the signal peptide of the BCMA CAR comprises a CD8a signal peptide. In some embodiments, the CD8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:47 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 47. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:48 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:48. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide. In some embodiments, the GMCSFR-a or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:49 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 49.
[0689] In some embodiments, the extracellular binding domain of the BCMA CAR is specific to BCMA, for example, human BCMA. The extracellular binding domain of the BCMA CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain.
[0690] In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv. In some embodiments, the extracellular binding domain of the BCMA CAR is derived from an antibody specific to BCMA, including, for example, belantamab, erlanatamab, teclistamab, LCAR-B38M, and ciltacabtagene. In any of these embodiments, the extracellular binding domain of the BCMA CAR can comprise or consist of the VH, the VL, and/or one or more CDRs of any of the antibodies.
[0691] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from Cl 1D5.3, a murine monoclonal antibody as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013). See also PCT Application Publication No. WO2010/104949. The Cl 1D5.3 -derived scFv may comprise the heavy chain variable region (VH) and the light chain variable region (VL) of Cl 1D5.3 connected by the Whitlow linker, the amino acid sequences of which is provided in Table 17 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 108, 109, or 113, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 108, 109, or 113. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 110- 112 and 114-116. In some embodiments, the BCMA-specific extracellular binding domain may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 110-112. In some embodiments, the BCMA-specific extracellular binding domain may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 114-116. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of the one or more CDRs as described herein.
[0692] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from another murine monoclonal antibody, C12A3.2, as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013) and PCT Application Publication No. WO2010/104949, the amino acid sequence of which is also provided in Table 17 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:117, 118, or 122, or an amino acid sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:117, 118, or 122. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 119-121 and 123-125. In some embodiments, the BCMA-specific extracellular binding domain may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 119-121. In some embodiments, the BCMA-specific extracellular binding domain may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 123-135. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of the one or more CDRs as described herein.
[0693] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a murine monoclonal antibody with high specificity to human BCMA, referred to as BB2121 in Friedman et al., Hum. Gene Ther. 29(5):585-601 (2018)). See also, PCT Application Publication No. WO2012163805.
[0694] In some embodiments, the extracellular binding domain of the BCM A CAR comprises single variable fragments of two heavy chains (VHH) that can bind to two epitopes of BCMA as described in Zhao et al., J. Hematol. Oncol. 11(1): 141 (2018), also referred to as LCAR-B38M. See also, PCT Application Publication No. WO2018/028647.
[0695] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a fully human heavy-chain variable domain (FHVH) as described in Lam et al., Nat. Commun. 11(1):283 (2020), also referred to as FHVH33. See also, PCT Application Publication No. WO2019/006072. The amino acid sequences of FHVH33 and its CDRs are provided in Table 17 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 126 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 126. In some embodiments, the BCMA- specific extracellular binding domain may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 127-129. In any of these embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of the one or more CDRs as described herein.
[0696] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from CT103A (or CAR0085) as described in U.S. Patent No. 11,026,975 B2, the amino acid sequence of which is provided in Table 17 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:130, 131, or 135, or an amino acid sequence that is at least 80% identical e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 130, 131, or 135. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 132-134 and 136-138. In some embodiments, the BCMA-specific extracellular binding domain may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 132-134. In some embodiments, the BCMA-specific extracellular binding domain may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 136-138. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of the one or more CDRs as described herein.
[0697] Additionally, CARs and binders directed to BCMA have been described in U.S. Application Publication Nos. 2020/0246381 Al and 2020/0339699 Al, the entire contents of each of which are incorporated by reference herein.
Table 17. Exemplary sequences of anti-BCMA binder and components
[0698] In some embodiments, the hinge domain of the BCMA CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:50 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:50. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:51 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:51. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:53 or SEQ ID NO:54, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:53 or SEQ ID NO:54. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:55 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:55.
[0699] In some embodiments, the transmembrane domain of the BCM A CAR comprises a CD 8 a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the CD 8 a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:56 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:56. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:57 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:57.
[0700] In some embodiments, the intracellular costimulatory domain of the BCMA CAR comprises a 4-1BB costimulatory domain, for example, a human 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:59 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:59. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, for example, a human CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:60 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:60.
[0701] In some embodiments, the intracellular signaling domain of the BCMA CAR comprises a CD3 zeta (Q signaling domain, for example, a human CD3^ signaling domain. In some embodiments, the CD3^ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:61 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:61.
[0702] In some embodiments, the CAR is a BCMA CAR, including, for example, a BCMA CAR comprising any of the BCMA-specific extracellular binding domains as described, the CD8a hinge domain of SEQ ID NO:50, the CD8a transmembrane domain of SEQ ID NO:56, the 4-1BB costimulatory domain of SEQ ID NO:59, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof. In any of these embodiments, the BCMA CAR may additionally comprise a signal peptide (e.g., a CD 8 a signal peptide) as described.
[0703] In some embodiments, the CAR is a BCMA CAR, including, for example, a BCMA CAR comprising any of the BCMA-specific extracellular binding domains as described, the CD8a hinge domain of SEQ ID NO:50, the CD8a transmembrane domain of SEQ ID NO:56, the CD28 costimulatory domain of SEQ ID NO:60, the CD3^ signaling domain of SEQ ID NO:61, and/or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof. In any of these embodiments, the BCMA CAR may additionally comprise a signal peptide as described.
[0704] In some embodiments, the CAR is a BCMA CAR as set forth in SEQ ID NO: 139 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 139 (see Table 18). The encoded BCMA CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 140 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 140, with the following components: CD8a signal peptide, CT103A scFv (VL- Whitlow linker-Vn), CD8a hinge domain, CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3^ signaling domain.
[0705] In some embodiments, the CAR is a commercially available embodiment of BCMA CAR, including, for example, idecabtagene vicleucel (ide-cel, also called bb2121). In some embodiments, the CAR is idecabtagene vicleucel or portions thereof. Idecabtagene vicleucel comprises a BCMA CAR with the following components: the BB2121 binder, CD8a hinge domain, CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3^ signaling domain.
Table 18. Exemplary sequences of BCMA CARs
B. Gene-Editing Agents
[0706] In some embodiments, the payload agent is associated with a gene editing technology. Any of a variety of agents associated with gene editing technologies can be included as the payload agent, such as for delivery of gene editing machinery to a cell. In some embodiments, the gene editing technology can include systems involving nucleases, integrases, transposases, recombinases. In some embodiments, the gene editing technologies can be used for knock-out or knock-down of genes. In some embodiments, the gene-editing technologies can be used for knock-in or integration of DNA into a region of the genome. In some embodiments, the payload agent mediates double-strand breaks (DSB), including in connection with non-homologous end-joining (NHEJ) or homology-directed repair (HDR). In some embodiments, the payload agent does not mediate DSB. In some embodiments, the payload agent can be used for DNA-based editing or prime-editing. In some embodiments, the payload agent can be used for Programmable Addition via Site-specific Targeting Elements (PASTE). In some embodiments, the payload agent is a programmable dna-binding polypeptide.
[0707] In some embodiments, the pay load agent is a programmable dna-binding polypeptide and/or a nuclease for use in gene editing methods. In some embodiments, the nuclease is a zinc-finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs), or a CRISPR-associated protein- nuclease (Cas). In some embodiments, the programmable dna-binding polypeptide is a CRISPR- associated protein- nuclease (Cas). In some embodiments, the Cas protein is selected from the group consisting of Cas3, Cas9, CaslO, Casl2, and Casl3. In some embodiments, the Cas is a Casl2a (also known as cpfl) from a Prevotella, Francisella novicida, Acidaminococcus sp., Lachnospiraceae bacterium, or Francisella bacteria. In some embodiments, the Cas is Cas9 from Streptococcus pyogenes. In some embodiments, the Cas is Cas9 from Streptococcus pyogenes (SpCas). In some embodiments, the Cas9 is from Staphylococcus aureus (SaCas9). In some embodiments, the Cas9 is from Neisseria meningitidis (NmeCas9). In some embodiments, the Cas9 is from Campylobacter jejuni (CjCas9). In some embodiments, the Cas9 is from Streptococcus thermophilis (StCas9). In some embodiments, the Cas is a Cas 12a (also known as Cpfl) from a Prevotella or Francisella bacteria, or the Cas is a Cas 12b from a Bacillus, optionally Bacillus hisashii. In some embodiments, the Cas is a Casl2a (also known as cpfl) from a Prevotella, Francisella novicida, Acidaminococcus sp., Eachnospiraceae bacterium, or Francisella bacteria. In some embodiments, the nuclease is MAD7 or CasX. In some of any embodiments, the Cas is a Cas3, Casl3, CasMini, or any other Cas protein known in the art. See for example, Wang et al., Biosensors and Bioelectronics (165) 1: 2020, and Wu et al. Nature Reviews Chemistry (4) 441: 2020). The Cas9 nuclease can, in some embodiments, be a Cas9 or functional fragment thereof from any bacterial species. See, e.g., Makarova et al. Nature Reviews, Microbiology, 9: 467-477 (2011), including supplemental information, hereby incorporated by reference in its entirety.
[0708] In some embodiments, delivery of the nuclease is by a provided particle encoding the programmable dna-binding polypeptide and/or nuclease (e.g. Cas, Cas nickase, catalytically inactive Cas). In some embodiments, delivery of the programmable dna-binding polypeptide and/or nuclease is by a provided vector comprising an RNA encoding the programmable dna-binding polypeptide and/or nuclease (e.g. Cas, , Cas nickase, catalytically inactive Cas). In some embodiments, delivery of the programmable dna-binding polypeptide and/or nuclease is by a provided VEP comprising an RNA encoding the programmable dna-binding polypeptide and/or nuclease (e.g. Cas, , Cas nickase, catalytically inactive Cas). In some embodiments, the Cas is a catalytically active Cas. In some embodiments, the Cas is a catalytically inactive Cas (also known as a dead Cas, dCas) that is a Cas that contains one or more mutations that inactivates the catalytic activity of the domain. In some embodiments, the Cas is a Cas nickase. [0709] In some embodiments, the particle contains a programmable dna-binding protein and/or nuclease protein and the programmable dna-binding protein and/or nuclease protein is directly delivered to a target cell. Methods of delivering a programmable dna-binding protein and/or nuclease protein include those as described, for example, in Cai et al. Elife, 2014, 3:e01911 and International patent publication No. W02017068077. For instance, in some embodiments, the particle comprises one or more Cas protein(s), such as Cas9. In some embodiments, the programmable dna-binding protein and/or nuclease protein (e.g. Cas, such as Cas 9) is engineered as a chimeric nuclease protein with a viral structural protein (e.g. GAG) for packaging into the viral vector particle (e.g. lentiviral vector particle). For instance, a chimeric Cas9-protein fusion with the structural GAG protein can be packaged inside a lentiviral particle. In some embodiments, the fusion protein is a cleavable fusion protein between (i) a viral structural protein (e.g. GAG) and (ii) a nuclease protein (e.g. Cas protein, such as Cas 9).
[0710] In some cases, the cleavable fusion protein comprising a viral structural protein (e.g. a GAG protein (e.g. an MEV-gag or an HIV-gag)) and (ii) a programmable dna-binding protein and/or nuclease protein (e.g. Cas protein (e.g. any of the Cas proteins described herein)) includes a nuclear export sequence (NES). In some embodiments, the NES facilitates localization of the fusion protein to the cytosol.
[0711] In some embodiments, the cleavable fusion protein includes at least one NES sequences (e.g. 2 or more, 3 or more, 4 or more, or 5 or more NES sequences). In some embodiments, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and/or the C- terminus of the cleavable fusion protein. In one embodiment, the cleavable fusion protein includes an NES sequence is positioned at the N-terminus and an NES sequence is positioned at the C-terminus of the fusion protein.
[0712] In some embodiments, the cleavable fusion protein includes at least one NES sequences (e.g. 2 or more, 3 or more, 4 or more, or 5 or more NES sequences) positioned at or near (e.g. within 50 amino acids of) the N-terminus and/or the C- terminus of the programmable dna-binding protein and/or nuclease protein. In one embodiment, the cleavable fusion protein includes an NES sequence is positioned at the N-terminus and/or an NES sequence is positioned at the C-terminus of the programmable dna-binding protein and/or nuclease protein.
[0713] In some cases, the cleavable fusion protein comprising a viral structural protein (e.g. a GAG protein (e.g. an MLV-gag or an HIV-gag)) and (ii) a programmable dna-binding protein and/or nuclease protein (e.g. Cas protein (e.g. any of the Cas proteins described herein)) includes a nuclear localization signal (NLS). In some embodiments, the NLS facilitates delivery of the fusion protein, or a therapeutic polypeptide (or polynucleotide encoding the therapeutic polypeptide) released from the fusion protein (for instance, a polypeptide (or polynucleotide encoding a polypeptide) released from the fusion protein following cleavage of the cleavable linker), into the nucleus of a target cell. [0714] In some embodiments, the cleavable fusion protein includes at least one NLS sequences (e.g. 2 or more, 3 or more, 4 or more, or 5 or more NLS sequences). In some embodiments, one or more NLS sequences (2 or more, 3 or more, 4 or more, or 5 or more NLS sequences) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and/or the C- terminus of the cleavable fusion protein. In one embodiment, the cleavable fusion protein includes an NLS sequence is positioned at the N-terminus and an NLS sequence is positioned at the C-terminus of the fusion protein.
[0715] In some embodiments, the cleavable fusion protein includes at least one NLS sequences (e.g. 2 or more, 3 or more, 4 or more, or 5 or more NLS sequences) positioned at or near (e.g., within 50 amino acids of) the N-terminus and/or the C- terminus of the programmable dna-binding protein and/or nuclease protein. In one embodiment, the cleavable fusion protein includes an NLS sequence is positioned at the N-terminus and/or an NLS sequence is positioned at the C-terminus of the the nuclease protein.
[0716] In some embodiments, the cleavable fusion protein comprises one NES sequence and one NLS sequences. In some embodiments, the viral structural protein, the NES sequence, NLS sequence, and the therapeutic polypeptide (e.g. the programmable dna-binding protein and/or nuclease protein) sequence are positioned from N-terminus to C-terminus as follows: viral structural protein-NES -NLS - therapeutic polypeptide or viral structural protein-NES -therapeutic polypeptide-NLS. In some embodiments, the viral structural protein, the NES sequences, NLS sequence, and the therapeutic polypeptide sequence are positioned from N- terminus to C-terminus as follows: viral structural protein- NESn-NLS -therapeutic polypeptide or viral structural protein-NESn-therapeutic polypeptide-NLS, where n is equal to or greater than 2. In such embodiments, the cleavable linker is located before the therapeutic polypeptide (e.g., before an NLS that immediately precedes the therapeutic polypeptide).
[0717] In some embodiments, the cleavable fusion protein comprises one NES sequence and two NLS sequences. In some embodiments, the viral structural protein, the NES sequence, NLS sequences, and the therapeutic polypeptide (e.g. the programmable dna-binding protein and/or nuclease protein) sequences are positioned from N-terminus to C-terminus as follows: viral structural protein-NES -NLS - therapeutic polypeptide-NLS. In some embodiments, the viral structural protein, the NES sequences, NLS sequences, and the therapeutic polypeptide sequence are positioned from N- terminus to C-terminus as follows: viral structural protein-NESn-NLS-therapeutic polypeptide-NLS, where n is equal to or greater than 2. In such embodiments, the cleavable linker is located before the therapeutic polypeptide (e.g., before an NLS that immediately precedes the therapeutic polypeptide). In one embodiment, the cleavable fusion protein comprises from N-terminus to C-terminus: viral structural protein-NES- cleavable linker-NLS -therapeutic polypeptide-NLS.
[0718] In some embodiments, the cleavable fusion protein has a configuration selected from: gagcleavage site-NLS-therapuetic polypeptide (e.g. a Cas protein)-NLS; gag-NES(3x)-cleavage site-NLS- therapuetic polypeptide (e.g. a Cas protein)-NLS ; and gag-cleavage site-NLS-therapuetic polypeptide (e.g. a Cas protein)-NLS -cleavage site-NES.
[0719] In some embodiments, the Cas is wild-type Cas9, which can site-specifically cleave doublestranded DNA, resulting in the activation of the double-strand break (DSB) repair machinery. DSBs can be repaired by the cellular Non-Homologous End Joining (NHEJ) pathway (Overballe-Petersen et al., 2013, Proc Natl Acad Sci USA, Vol. 110: 19860-19865), resulting in insertions and/or deletions (indels) which disrupt the targeted locus. Alternatively, if a donor template with homology to the targeted locus is supplied, the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise replacement mutations to be made (Overballe- Petersen et al., 2013, Proc Natl Acad Sci USA, Vol. 110: 19860-19865; Gong et al., 2005, Nat. Struct Mol Biol, Vol. 12: 304-312). In some embodiments, the Cas is mutant form, known as Cas9 D10A, with only nickase activity. This means that Cas9D10A cleaves only one DNA strand, and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only, resulting in reduced indel mutations (Cong et al., 2013, Science, Vol. 339: 819-823; Jinek et al., 2012, Science, Vol.337: 816-821; Qi et al., 2013 Cell, Vol. 152: 1173-1183). Cas9D10A is even more appealing in terms of target specificity when loci are targeted by paired Cas9 complexes designed to generate adjacent DNA nicks (Ran et al., 2013, Cell, Vol. 154: 1380-1389). In some embodiments, the Cas is a nuclease-deficient Cas9 (Qi et al., 2013 Cell, Vol. 152: 1173-1183). For instance, mutations H840A in the HNH domain and D10A in the RuvC domain inactivate cleavage activity, but do not prevent DNA binding. Therefore, this variant can be used to target in a sequence-specific manner any region of the genome without cleavage. Instead, by fusing with various effector domains, dCas9 can be used either as a gene silencing or activation tools. Furthermore, it can be used as a visualization tool by coupling the guide RNA or the Cas9 protein to a fluorophore or a fluorescent protein.
[0720] In some embodiments, the Cas protein comprises one or more mutations such that the Cas protein is converted into a nickase that lacks the ability to cleave both strands of a double stranded DNA molecule. In some embodiments, the Cas protein comprises one or more mutations such that the Cas protein is converted into a nickase that is able to cleave only one strand of a double stranded DNA molecule (e.g., a SSB). For example, Cas9, which is normally capable of inducing a double strand break, can be converted into a Cas9 nickase, which is capable of inducing a single strand break, by mutating one of two Cas9 catalytic domains: the RuvC domain, which comprises the RuvC I, RuvC II, and RuvC III motifs, or the NHN domain. In some embodiments, the Cas protein comprises one or more mutations in the RuvC catalytic domain or the HNH catalytic domain. In some embodiments, the genome -modifying protein is a recombinant nuclease that has been modified to have nickase activity. In some embodiments, the recombinant nuclease cleaves the strand to which the guide RNA, e.g., sgRNA, hybridizes, but does not cleave the strand that is complementary to the strand to which the guide RNA, e.g., sgRNA, hybridizes. In some embodiments, the recombinant nuclease does not cleave the strand to which the guide RNA, e.g., sgRNA, hybridizes, but does cleave the strand that is complementary to the strand to which the guide RNA, e.g., sgRNA, hybridizes.
[0721] In some embodiments, the Cas protein is selected from the group consisting of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxl 1, Csyl, Csy2, Csy3, and Mad7. In some embodiments, the Cas protein is Cas9. In some embodiments, the Cas9 is from a bacteria selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitides, Campylobacter jejuni, and Streptococcus thermophilis. In some embodiments, the Cas9 is from Streptococcus pyogenes. In some embodiments, the Cas9 is from Streptococcus pyogenes and comprises one or more mutations in the RuvC I, RuvC II, or RuvC III motifs. In some embodiments, the Cas9 is from Streptococcus pyogenes and comprises a D10A mutation in the RuvC I motif. In some embodiments, the Cas9 is from Streptococcus pyogenes and comprises one or more mutations in the HNH catalytic domain. In some embodiments, the Cas9 is from Streptococcus pyogenes and comprises one or more mutations in the HNH catalytic domain selected from the group consisting of H840A, H854A, and H863A. In some embodiments, the Cas9 is from Streptococcus pyogenes and comprises a H840A mutation in the HNH catalytic domain. In some embodiments, the Cas9 is from Streptococcus pyogenes and comprises a mutation selected from the group consisting of D10A, H840A, H854A, and H863A.
[0722] In some embodiments, the one or more agent(s) (e.g., the heterologous protein) capable of inducing a DSB comprise Cas9 or a functional fragment thereof, and a first guide RNA, e.g., a first sgRNA, and a second guide RNA, e.g., a second sgRNA. The guide RNA, e.g., the first guide RNA or the second guide RNA, in some embodiments, binds to the recombinant nuclease and targets the recombinant nuclease to a specific location within the target gene such as at a location within the sense strand or the antisense strand of the target gene that is or includes the cleavage site. In some embodiments, the recombinant nuclease is a Cas protein from any bacterial species, or is a functional fragment thereof. In some embodiments, the Cas protein is Cas9 nuclease. Cas9 can, in some embodiments, be a Cas9 or functional fragment thereof from any bacterial species. See, e.g., Makarova et al. Nature Reviews, Microbiology, 9: 467-477 (2011), including supplemental information, hereby incorporated by reference in its entirety. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 is from Staphylococcus aureus (SaCas9). In some embodiments, the Cas9 is from Neisseria meningitidis (NmeCas9). In some embodiments, the Cas9 is from Campylobacter jejuni (CjCas9). In some embodiments, the Cas9 is from Streptococcus thermophilis (StCas9).
[0723] In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations in the RuvC catalytic domain or the HNH catalytic domain. In some embodiments, the one or more mutations in the RuvC catalytic domain or the HNH catalytic domain inactivates the catalytic activity of the domain. In some embodiments, the recombinant nuclease has RuvC activity but does not have HNH activity. In some embodiments, the recombinant nuclease does not have RuvC activity but does have HNH activity. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of D10A, H840A, H854A, and H863A. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations in the RuvC I, RuvC II, or RuvC III motifs. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises a mutation in the RuvC I motif. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises a D10A mutation in the RuvC I motif. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations in the HNH catalytic domain. In some embodiments, the one or more mutations in the HNH catalytic domain is selected from the group consisting of H840A, H854A, and H863A. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises a H840A mutation in the HNH catalytic domain. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises a H840A mutation. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises a D10A mutation. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of N497A, R661 A, Q695A, and Q926A. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of R780A, K810A, K855A, H982A, K1003A, R1060A, and K848A. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of N692A, M694A, Q695A, and H698A. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of M495V, Y515N, K526E, and R661Q. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of F539S, M763I, and K890N. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of E480K, E543D, E1219V, A262T, S409I, M694I, E108G, S217A.
[0724] In some embodiments, the Cas9 is from Streptococcus pyogenes (SaCas9). In some embodiments, the SaCas9 is wild type SaCas9. In some embodiments, the SaCas9 comprises one or more mutations in REC3 domain. In some embodiments, the SaCas9 comprises one or more mutations in RECI domain. In some embodiments, the SaCas9 comprises one or more mutations selected from the group consisting of N260D, N260Q, N260E, Q414A, Q414L. In some embodiments, the SaCas9 comprises one or more mutations in the recognition lobe. In some embodiments, the SaCas9 comprises one or more mutations selected from the group consisting of R245A, N413A, N419A. In some embodiments, the SaCas9 comprises one or more mutations in the RuvC-III domain. In some embodiments, the SaCas9 comprises a R654A mutation.
[0725] In some embodiments, the Cas protein is Cast 2. In some embodiments, the Cas protein is Casl2a (i.e. cpfl). In some embodiments, the Casl2a is from the group consisting of Francisella novicida U112 (FnCasl2a), Acidaminococcus sp. BV3L6 (AsCasl2a), Moraxella bovoculi AAXl l_00205 (Mb3Casl2a), Lachnospiraceae bacterium ND2006 (LbCasl2a), Thiomicrospira sp. Xs5 (TsCasl2a), Moraxella bovoculi AAX08_00205 (Mb2Casl2a), and Butyrivibrio sp. NC3005 (BsCasl2a). In some embodiments, the Casl2a recognizes a T-rich 5’ protospacer adjacent motif (PAM). In some embodiments, the Casl2a processes its own crRNA without requiring a transactivating crRNA (tracrRNA). In some embodiments, the Casl2a processes both RNase and DNase activity. In some embodiments, the Casl2a is a split Casl2a platform, consisting of N-terminal and C-terminal fragments of Casl2a. In some embodiments, the split Casl2a platform is from Lachnospiraceae bacterium.
[0726] In some embodiments, the particles containing a Cas nuclease (e.g., Cas9) further comprise one or more CRISPR-Cas system guide RNA(s) for targeting a desired target gene. In some embodiments, the CRISPR guide RNAs are efficiently encapsulated in the CAS -containing particles. In some embodiments, the provided particles (e.g., lentiviral particles) further comprise a targeting nucleic acid.
[0727] In some embodiments, the lipid particle further comprises a polynucleotide per se, i.e. a polynucleotide that does not encode for a heterologous protein. In some embodiments, the polynucleotide per se is associated with a gene editing system. For example, a lipid particle may comprise a guide RNA (gRNA), such as a single guide RNA (sgRNA).
[0728] In some embodiments, the one or more agent(s) (e.g., one or more exogenous agent and/or heterologous protein) comprise, or are used in combination with, a guide RNA, e.g., single guide RNA (sgRNA), for inducing a DSB at the cleavage site. In some embodiments, the one or more agent(s) comprise, or are used in combination with, more than one guide RNA, e.g., a first sgRNA and a second sgRNA, for inducing a DSB at the cleavage site through a SSB on each strand. In some embodiments, the one or more agent(s) (e.g., the heterologous protein) can be used in combination with a donor template, e.g., a single-stranded DNA oligonucleotide (ssODN), for HDR-mediated integration of the donor template into the target gene, such as at the targeting sequence. In some embodiments, the one or more agent(s) (e.g., one or more exogenous agent and/or heterologous protein) can be used in combination with a donor template, e.g., an ssODN, and a guide RNA, e.g., a sgRNA, for HDR-mediated integration of the donor template into the target gene, such as at the targeting sequence. In some embodiments, the one or more agent(s) (e.g., one or more exogenous agent and/or heterologous protein) can be used in combination with a donor template, e.g., an ssODN, and a first guide RNA, e.g., a first sgRNA, and a second guide RNA, e.g., a second sgRNA, for HDR-mediated integration of the donor template into the target gene, such as at the targeting sequence. [0729] In some embodiments, the genome-modifying protein, e.g., Cas9, is targeted to the cleavage site by interacting with a guide RNA, e.g., a first guide RNA, such as a first sgRNA, or a second guide RNA, such as a second sgRNA, that hybridizes to a DNA sequence on the sense strand or the antisense strand that immediately precedes a Protospacer Adjacent Motif (PAM) sequence.
[0730] In some embodiments, the genome-modifying agent, e.g., Cas9, is targeted to the cleavage site on the sense strand by interacting with a first guide RNA, e.g., first sgRNA, that hybridizes to a sequence on the sense strand that immediately precedes a PAM sequence. In some embodiments, the genome-modifying agent, e.g., Cas9, is targeted to the cleavage site on the antisense strand by interacting with a second guide RNA, e.g., second sgRNA, that hybridizes to a sequence on the antisense strand that immediately precedes a PAM sequence.
[0731] In some embodiments, the first guide RNA, e.g., first sgNA, that is specific to the sense strand of a target gene of interest is used to target the recombinant nuclease, e.g., Cas9, to induce a SSB at a cleavage site within the sense strand of the target gene. In some embodiments, the first guide RNA, e.g., first sgNA, that is specific to the antisense strand of a target gene of interest is used to target the recombinant nuclease, e.g., Cas9, to induce a SSB at a cleavage site within the antisense strand of the target gene.
[0732] In some embodiments, the second guide RNA, e.g., second sgNA, that is specific to the sense strand of a target gene of interest used to target the recombinant nuclease, e.g., Cas9, to induce a SSB at a cleavage site within the sense strand of the target gene. In some embodiments, the second guide RNA, e.g., second sgNA, that is specific to the antisense strand of a target gene of interest is used to target the recombinant nuclease, e.g., Cas9, to induce a SSB at a cleavage site within the antisense strand of the target gene.
[0733] In some embodiments, the first guide RNA, e.g., first sgNA, that is specific to the sense strand of a target gene of interest is used to target the recombinant nuclease, e.g., Cas9, to induce a SSB at a cleavage site within the sense strand of the target gene; and the second guide RNA, e.g., second sgNA, that is specific to the antisense strand of a target gene of interest is used to target the recombinant nuclease, e.g., Cas9, to induce a SSB at a cleavage site within the antisense strand of the target gene.
[0734] In some embodiments, the first guide RNA, e.g., first sgNA, that is specific to the antisense strand of a target gene of interest is used to target the recombinant nuclease, e.g., Cas9, to induce a SSB at a cleavage site within the antisense strand of the target gene; and the second guide RNA, e.g., second sgNA, that is specific to the sense strand of a target gene of interest is used to target the recombinant nuclease, e.g., Cas9, to induce a SSB at a cleavage site within the sense strand of the target gene. In general, a guide RNA, e.g., a first guide RNA, such as a first sgRNA, or a second guide RNA, such as a second sgRNA, is any nucleotide sequence comprising a sequence, e.g., a crRNA sequence, that has sufficient complementarity with a target gene sequence to hybridize with the target gene sequence at the cleavage site and direct sequence-specific binding of the recombinant nuclease to a portion of the target gene that includes the cleavage site. Full complementarity (100%) is not necessarily required, so long as there is sufficient complementarity to cause hybridization and promote formation of a complex, e.g., CRISPR complex, that includes the recombinant nuclease, e.g., Cas9, and the guide RNA, e.g., the first guide RNA, such as the first sgRNA, or the second guide RNA, such as the second sgRNA.
[0735] In some embodiments, the cleavage site is situated at a site within the target gene that is homologous to a sequence comprised within the guide RNA, e.g., sgRNA. In some embodiments, the cleavage site of the sense strand is situated at a site within the sense strand of the target gene that is homologous to a sequence comprised within the first guide RNA, e.g., the first sgRNA. In some embodiments, the cleavage site of the antisense strand is situated at a site within the antisense strand of the target gene that is homologous to a sequence comprised within the first guide RNA, e.g., the first sgRNA. In some embodiments, the cleavage site of the sense strand is situated at a site within the sense strand of the target gene that is homologous to a sequence comprised within the second guide RNA, e.g., the second sgRNA. In some embodiments, the cleavage site of the antisense strand is situated at a site within the antisense strand of the target gene that is homologous to a sequence comprised within the second guide RNA, e.g., the second sgRNA. In some embodiments, the cleavage site of the sense strand is situated at a site within the sense strand of the target gene that is homologous to a sequence comprised within the first guide RNA, e.g., the first sgRNA; and the cleavage site of the antisense strand is situated at a site within the antisense strand of the target gene that is homologous to a sequence comprised within the second guide RNA, e.g., the second sgRNA. In some embodiments, the cleavage site of the antisense strand is situated at a site within the antisense strand of the target gene that is homologous to a sequence comprised within the first guide RNA, e.g., the first sgRNA; and the cleavage site of the sense strand is situated at a site within the sense strand of the target gene that is homologous to a sequence comprised within the second guide RNA, e.g., the second sgRNA. In some embodiments, the cleavage site of the antisense strand is situated at a site within the antisense strand of the target gene that is homologous to a sequence comprised within the second guide RNA, e.g., the second sgRNA; and the cleavage site of the sense strand is situated at a site within the sense strand of the target gene that is homologous to a sequence comprised within the first guide RNA, e.g., the first sgRNA.
[0736] In some embodiments, the sense strand comprises the targeting sequence, and the targeting sequence includes the SNP and a protospacer adjacent motif (PAM) sequence. In some embodiments, the sense strand comprises the targeting sequence, and the targeting sequence includes the SNP and a protospacer adjacent motif (PAM) sequence; and the antisense strand comprises a sequence that is complementary to the targeting sequence and includes a PAM sequence. In some embodiments, the antisense strand comprises the targeting sequence, and the targeting sequence includes the SNP and a protospacer adjacent motif (PAM) sequence. In some embodiments, the antisense strand comprises the targeting sequence, and the targeting sequence includes the SNP and a protospacer adjacent motif (PAM) sequence; and the sense strand comprises a sequence that is complementary to the targeting sequence and includes a PAM sequence.
[0737] In some embodiments, the cleavage site on the sense strand and/or the antisense strand is situated approximately 3 nucleotides upstream of the PAM sequence. In some embodiments, the cleavage site on the sense strand and/or the antisense strand is situated approximately 3 nucleotides upstream of the juncture between the guide RNA and the PAM sequence. In some embodiments, the cleavage site on the sense strand and/or the antisense strand is situated 3 nucleotides upstream of the PAM sequence. In some embodiments, the cleavage site on the sense strand and/or the antisense strand is situated 4 nucleotides upstream of the PAM sequence.
[0738] In some embodiments, the PAM sequence that is recognized by a recombinant nuclease is in the sense strand. In some embodiments, the PAM sequence that is recognized by a recombinant nuclease is in the antisense strand. In some embodiments, the PAM sequence that is recognized by a recombinant nuclease is in the sense strand and is in the antisense strand. In some embodiments, the PAM sequence on the sense strand and the PAM sequence on the antisense strand are outwardly facing. In some embodiments, the PAM sequence on the sense strand and the PAM sequence on the antisense strand comprise the same nucleic acid sequence, which can be any PAM sequence disclosed herein. In some embodiments, the PAM sequence on the sense strand and the PAM sequence on the antisense strand each comprise a different nucleic acid sequence, each of which can be any of the PAM sequences disclosed herein.
[0739] In some embodiments, the PAM sequence that is recognized by a recombinant nuclease, e.g., Cas9, differs depending on the particular recombinant nuclease and the bacterial species it is from
[0740] Methods for designing guide RNAs, e.g., sgRNAs, and their exemplary targeting sequences, e.g., crRNA sequences, can include those described in, e.g., International PCT Pub. Nos. WO2015/161276, W02017/193107, and WO2017/093969. Exemplary guide RNA structures, including particular domains, are described in WO2015/161276, e.g., in FIGS. 1A-1G therein. Since guide RNA is an RNA molecule, it will comprise the base uracil (U), while any DNA encoding the guide RNA molecule will comprise the base thymine (T). In some embodiments, the guide RNA, e.g., sgRNA, comprises a CRISPR targeting RNA sequence (crRNA) and a trans-activating crRNA sequence (tracrRNA). In some embodiments, the first guide RNA, e.g., the first sgRNA, and the second guide RNA, e.g., the second sgRNA, each comprise a crRNA and a tracrRNA. In some embodiments, the guide RNA, e.g., sgRNA, is an RNA comprising, from 5’ to 3’: a crRNA sequence and a tracrRNA sequence. In some embodiments, each of the first guide RNA, e.g., first sgRNA, and the second guide RNA, e.g., second sgRNA, is an RNA comprising, from 5’ to 3’ : a crRNA sequence and a tracrRNA sequence. In some embodiments, the crRNA and tracrRNA do not naturally occur together in the same sequence. [0741] In some embodiments, the crRNA comprises a nucleotide sequence that is homologous, e.g., is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous, or is 100% homologous, to a portion of the target gene that includes the cleavage site. In some embodiments, the crRNA comprises a nucleotide sequence that is 100% homologous to a portion of the target gene that includes the cleavage site. In some embodiments, the portion of the target gene that includes the cleavage site is a portion of the sense strand of the target gene that includes the cleavage site. In some embodiments, the portion of the target gene that includes the cleavage site is a portion of the antisense strand of the target gene that includes the cleavage site.
[0742] In some embodiments, the sgRNA comprises a crRNA sequence that is homologous to a sequence in the target gene that includes the cleavage site. In some embodiments, the first sgRNA comprises a crRNA sequence that is homologous to a sequence in the sense strand of the target gene that includes the cleavage site; and/or the second sgRNA comprises a crRNA sequence that is homologous to a sequence in the antisense strand of the target gene that includes the cleavage site. In some embodiments, the first sgRNA comprises a crRNA sequence that is homologous to a sequence in the antisense strand of the target gene that includes the cleavage site; and/or the second sgRNA comprises a crRNA sequence that is homologous to a sequence in the sense strand of the target gene that includes the cleavage site.
[0743] In some embodiments, the crRNA sequence has 100% sequence identity to a sequence in the target gene that includes the cleavage site. In some embodiments, the crRNA sequence of the first sgRNA has 100% sequence identity to a sequence in the sense strand of the target gene that includes the cleavage site; and/or the crRNA sequence of the second sgRNA has 100% sequence identity to a sequence in the antisense strand of the target gene that includes the cleavage site. In some embodiments, the crRNA sequence of the first sgRNA has 100% sequence identity to a sequence in the antisense strand of the target gene that includes the cleavage site; and/or the crRNA sequence of the second sgRNA has 100% sequence identity to a sequence in the sense strand of the target gene that includes the cleavage site.
[0744] Guidance on the selection of crRNA sequences can be found, e.g., in Fu Y et al., Nat Biotechnol 2014 (doi: 10.1038/nbt.2808) and Sternberg SH et al., Nature 2014 (doi: 10.1038/naturel3011). Examples of the placement of crRNA sequences within the guide RNA, e.g., sgRNA, structure include those described in WO2015/161276, e.g., in FIGS. 1A-1G therein.
[0745] Reference to “the crRNA” is to be understood as also including reference to the crRNA of the first sgRNA and the crRNA of the second sgRNA, each independently. Thus, embodiments referring to “the crRNA” is to be understood as independently referring to embodiments of (i) the crRNA, (ii) the crRNA of the first sgRNA, and (iii) the crRNA of the second sgRNA. In some embodiments, the crRNA is 15-27 nucleotides in length, i.e., the crRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length. In some embodiments, the crRNA is 18-22 nucleotides in length. In some embodiments, the crRNA is 19-21 nucleotides in length. In some embodiments, the crRNA is 20 nucleotides in length.
[0746] In some embodiments, the crRNA is homologous to a portion of a target gene that includes the cleavage site. In some embodiments, the crRNA is homologous to a portion of the sense strand of the target gene that includes the cleavage site. In some embodiments, the crRNA is homologous to a portion of the antisense strand of the target gene that includes the cleavage site. In some embodiments, the crRNA of the first sgRNA is homologous to a portion of the sense strand of the target gene that includes the cleavage site; and the crRNA of the second sgRNA is homologous to a portion of the antisense strand of the target gene that includes the cleavage site.
[0747] In some embodiments, the crRNA is homologous to a portion of the antisense strand of a target gene that includes the cleavage site. In some embodiments, the crRNA is homologous to a portion of the sense strand of the target gene that includes the cleavage site. In some embodiments, the crRNA of the first sgRNA is homologous to a portion of the antisense strand of the target gene that includes the cleavage site; and the crRNA of the second sgRNA is homologous to a portion of the sense strand of the target gene that includes the cleavage site.
[0748] In some embodiments, the crRNA is homologous to a portion of a target gene that includes the cleavage site, and is 15-27 nucleotides in length, i.e., the crRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length. In some embodiments, the portion of the target gene that includes the cleavage site is on the sense strand. In some embodiments, the portion of the target gene that includes the cleavage site is on the antisense strand.
[0749] In some embodiments, the crRNA is homologous to a portion, i.e., sequence, in the sense strand or the antisense strand of the target gene that includes the cleavage site and is immediately upstream of the PAM sequence.
[0750] In some embodiments, the tracrRNA sequence may be or comprise any sequence for tracrRNA that is used in any CRISPR/Cas9 system known in the art. Reference to “the tracrRNA” is to be understood as also including reference to the tracrRNA of the first sgRNA and the tracrRNA of the second sgRNA, each independently. Thus, embodiments referring to “the tracrRNA” is to be understood as independently referring to embodiments of (i) the tracrRNA, (ii) the tracrRNA of the first sgRNA, and (iii) the tracrRNA of the second sgRNA. Exemplary CRISPR/Cas9 systems, sgRNA, crRNA, and tracrRNA, and their manufacturing process and use include those described in, e.g., International PCT Pub. Nos. WO2015/161276, W02017/193107 and WO2017/093969, and those described in, e.g., U.S. Patent Application Publication Nos. 20150232882, 20150203872, 20150184139, 20150079681, 20150073041, 20150056705, 20150031134, 20150020223, 20140357530, 20140335620, 20140310830, 20140273234, 20140273232, 20140273231, 20140256046, 20140248702, 20140242700, 20140242699, 20140242664, 20140234972, 20140227787, 20140189896, 20140186958, 20140186919, 20140186843, 20140179770, 20140179006, 20140170753, 20140093913, and 20140080216. [0751] In some embodiments, the Cas protein is selected from the group consisting of Cas3, Cas9, CaslO, Casl2, and Casl3. In particular embodiments, the nuclease is a Cas nuclease, such as Cas9. In some embodiments, delivery of the CRISPR/Cas can be used to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. In some embodiments, a dCas9 version of the CRISPR/Cas9 system can be used to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.
[0752] In some embodiments, the payload agent is an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is TnpB (see, e.g., W02018035250 and WO2022159892).
[0753] In some embodiments, the pay load agent is one for use in target-primed reverse transcription (TPRT) or “prime editing”. In some embodiments, prime editing mediates targeted insertions, deletions, all 12 possible base-to-base conversions, and combinations thereof in human cells without requiring DSBs or donor DNA templates.
[0754] Prime editing is a genome editing method that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein (“napDNAbp”) working in association with a polymerase (e.g., in the form of a fusion protein or otherwise provided in trans with the napDNAbp), wherein the prime editing system is programmed with a prime editing (PE) guide RNA (“PEgRNA”) that both specifies the target site and templates the synthesis of the desired edit in the form of a replacement DNA strand by way of an extension (either DNA or RNA) engineered onto a guide RNA (e.g., at the 5' or 3' end, or at an internal portion of a guide RNA). The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the endogenous strand of the target site to be edited (with the exception that it includes the desired edit). Through DNA repair and/or replication machinery, the endogenous strand of the target site is replaced by the newly synthesized replacement strand containing the desired edit. In some cases, prime editing may be thought of as a “search-and- replace” genome editing technology since the prime editors search and locate the desired target site to be edited, and encode a replacement strand containing a desired edit which is installed in place of the corresponding target site endogenous DNA strand at the same time. For example, prime editing can be adapted for conducting precision CRISPR/Cas-based genome editing in order to bypass double stranded breaks. In some embodiments, the payload agent is a Cas protein-reverse transcriptase fusions or related systems to target a specific DNA sequence with a guide RNA, generate a single strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered reverse transcriptase template that is integrated with the guide RNA
[0755] In some embodiments, the pay load agent is a primer editor that is a reverse transcriptase, or any DNA polymerase known in the art. Thus, in one aspect, the prime editor may comprise Cas9 (or an equivalent napDNAbp) which is programmed to target a DNA sequence by associating it with a specialized guide RNA (i.e., PEgRNA) containing a spacer sequence that anneals to a complementary protospacer in the target DNA. Such methods include any disclosed in Anzalone et al., (https://doi.org/10.1038/s41586-019-1711-4), or in PCT publication Nos. WO2020191248, WO2021226558, or W02022067130, which are hereby incorporated in their entirety.
[0756] In some embodiments, the payload agent is for use in Programmable Addition via Sitespecific Targeting Elements (PASTE). In some aspects, PASTE is platform in which genomic insertion is directed via a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. As described in loannidi et al. (doi: https://doi.org/10.1101/2021.l l.01.466786), PASTE does not generate double stranded breaks, but allowed for integration of sequences as large as ~36 kb. In some embodiments, the serine integrase can be any known in the art. In some embodiments, the serine integrase has sufficient orthogonality such that PASTE can be use for multiplexed gene integration, simultaneously integrating at least two different genes at at least two genomic loci. In some embodiments, PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in nondividing cells and fewer detectable off-target events.
[0757] In some embodiments, the payload agent is associate with base editing. Base editors (BEs) are typically fusions of a Cas (“CRISPR-associated”) domaindomain and a nucleobase modification domaindomain (e.g., a natural or evolved deaminase, such as a cytidine deaminase that include APOBEC1 (“apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1”), CDA (“cytidine deaminase”), and AID (“activation-induced cytidine deaminase”)) domains. In some cases, base editors may also include proteins or domains that alter cellular DNA repair processes to increase the efficiency and/or stability of the resulting single-nucleotide change.
[0758] In some aspects, currently available base editors include cytidine base editors (e.g., BE4) that convert target OG to T*A and adenine base editors (e.g., ABE7.10) that convert target A*T to G*C. In some aspects, Cas9-targeted deamination was first demonstrated in connection with a Base Editor (BE) system designed to induce base changes without introducing double-stranded DNA breaks. Further Rat deaminase APOB EC 1 (rAPOBECl) fused to deactivated Cas9 (dCas9) was used to successfully convert cytidines to thymidines upstream of the PAM of the sgRNA. In some aspects, this first BE system was optimized by changing the dCas9 to a “nickase” Cas9 D10A, which nicks the strand opposite the deaminated cytidine. Without being bound by theory, this is expected to initiate long-patch base excision repair (BER), where the deaminated strand is preferentially used to template the repair to produce a U:A base pair, which is then converted to T:A during DNA replication.
[0759] In some embodiments, the payload agent is a base editor (e.g., a nucleobase editor). In some embodiments, the payload agent is a nucleobase editor containing a first DNA binding protein domain that is catalytically inactive, a domain having base editing activity, and a second DNA binding protein domain having nickase activity, where the DNA binding protein domains are expressed on a single fusion protein or are expressed separately (e.g., on separate expression vectors). In some embodiments, the base editor is a fusion protein comprising a domain having base editing activity (e.g., cytidine deaminase or adenosine deaminase), and two nucleic acid programmable DNA binding protein domains (napDNAbp), a first comprising nickase activity and a second napDNAbp that is catalytically inactive, wherein at least the two napDNAbp are joined by a linker. In some embodiments, the base editor is a fusion protein that comprises a DNA domain of a CRISPR-Cas (e.g., Cas9) having nickase activity (nCas; nCas9), a catalytically inactive domain of a CRISPR-Cas protein (e.g., Cas9) having nucleic acid programmable DNA binding activity (dCas; e.g., dCas9), and a deaminase domain, wherein the dCas is joined to the nCas by a linker, and the dCas is immediately adjacent to the deaminase domain. In some embodiments, the base editor is a adenine-to-thymine or “ATBE” (or thymine-to-adenine or “TABE”) transversion base editors. Exemplary base editor and base editor systems include any as described in patent publication Nos. US20220127622, US20210079366, US20200248169, US20210093667, US20210071163, W02020181202, WO2021158921, WO2019126709, W02020181178, W02020181195, WO2020214842, W02020181193, which are hereby incorporated in their entirety.
[0760] In some embodiments, the one or more agent(s) (e.g., one or more exogenous agent and/or heterologous protein) capable of inducing a DSB comprise a fusion protein comprising a DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA cleavage domain is or comprises a recombinant nuclease. In some embodiments, the fusion protein is a TALEN comprising a DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA binding domain is a transcription activator-like (TAL) effector DNA binding domain. In some embodiments, the TAL effector DNA binding domain is from Xanthomonas bacteria. In some embodiments, the DNA cleavage domain is a Fokl nuclease domain. In some embodiments, the TAL effector DNA binding domain is engineered to target a specific target sequence, e.g., a portion of a target gene that includes a cleavage site.
[0761] In some embodiments, the fusion protein is a zinc finger nuclease (ZFN) comprising a zinc finger DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA cleavage domain is a Fokl nuclease domain. In some embodiments, the zinc finger DNA binding domain is engineered to target a specific target sequence, e.g., a portion of a target gene, that includes a cleavage site, such as the targeting sequence.
[0762] In some embodiments, the pay load agent has one or more polypeptides having an activity selected from the group consisting of: nuclease activity (e.g., programmable nuclease activity); nickase activity (e.g., programmable nickase activity); homing activity (e.g., programmable DNA binding activity); nucleic acid polymerase activity (e.g., DNA polymerase or RNA polymerase activity); integrase activity; recombinase activity; or base editing activity (e.g., cytidine deaminase or adenosine deaminase activity). IV. POLYNUCLEOTIDES, CELLS, AND METHODS OF MAKING
[0763] Also provided herein in some embodiments are polynucleotides encoding both the fusogen and targeting agent of any of the provided particles. Also provided herein in some embodiments are cells containing polynucleotides encoding one or both of the fusogen and targeting agent of any of the provided particles, for instance any of the provided polynucleotides. Also provided herein in some embodiments are methods of making a targeted lipid particle, such as any described in Section II, using polynucleotides encoding one or both of the fusogen and targeting agent of any of the provided particles, for instance any of the provided polynucleotides. In some embodiments, the fusogen is any as described in Section II-B. In some embodiments, the targeting agent is any as described in Section II-C. In some embodiments, the provided methods are for making any of the provided particles containing an exogenous agent, such as any described in Section II-D.
A. Polynucleotides
[0764] In some embodiments, the polynucleotide can be a synthetic nucleic acid. Also provided are expression vectors containing any of the provided polynucleotides.
[0765] In some of any embodiments, expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid encoding the gene of interest to a promoter and incorporating the construct into an expression vector. In some embodiments, vectors can be suitable for replication and integration in eukaryotes. In some embodiments, cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for expression of the desired nucleic acid sequence. In some of any embodiments, a plasmid comprises a promoter suitable for expression in a cell.
[0766] In some embodiments, the polynucleotides contain at least one promoter that is operatively linked to control expression of the fusogen and/or targeting agent. For expression, at least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 genes, a discrete element overlying the start site itself helps to fix the place of initiation.
[0767] In some embodiments, additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. In some embodiments, additional promoter elements are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. In some embodiments, spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In some embodiments, the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. In some embodiments, depending on the promoter, individual elements can function either cooperatively or independently to activate transcription. [0768] A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR, in connection with the compositions disclosed herein (U.S. Pat. Nos. 4,683,202 and 5,928,906).
[0769] In some embodiments, a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. In some embodiments, the promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. In some embodiments, a suitable promoter is Elongation Growth Factor- la (EF-1 a). In some embodiments, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
[0770] In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. In some embodiments, inducible promoters comprise metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
[0771] In some embodiments, exogenously controlled inducible promoters can be used to regulate expression of the fusogen and/or targeting agent. For example, radiation-inducible promoters, heatinducible promoters, and/or drug-inducible promoters can be used to selectively drive transgene expression in, for example, targeted regions. In such embodiments, the location, duration, and level of transgene expression can be regulated by the administration of the exogenous source of induction. [0772] In some embodiments, expression of the fusogen and/or targeting agent is regulated using a drug-inducible promoter. For example, in some cases, the promoter, enhancer, or transactivator comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, a doxycycline operator sequence, a rapamycin operator sequence, a tamoxifen operator sequence, or a hormone-responsive operator sequence, or an analog thereof. In some instances, the inducible promoter comprises a tetracycline response element (TRE). In some embodiments, the inducible promoter comprises an estrogen response element (ERE), which can activate gene expression in the presence of tamoxifen. In some instances, a drug-inducible element, such as a TRE, can be combined with a selected promoter to enhance transcription in the presence of drug, such as doxycycline. In some embodiments, the drug-inducible promoter is a small molecule-inducible promoter.
[0773] Any of the provided polynucleotides can be modified to remove CpG motifs and/or to optimize codons for translation in a particular species, such as human, canine, feline, equine, ovine, bovine, etc. species. In some embodiments, the polynucleotides are optimized for human codon usage (i.e., human codon-optimized). In some embodiments, the polynucleotides are modified to remove CpG motifs. In other embodiments, the provided polynucleotides are modified to remove CpG motifs and are codon-optimized, such as human codon-optimized. Methods of codon optimization and CpG motif detection and modification are well-known. Typically, polynucleotide optimization enhances transgene expression, increases transgene stability and preserves the amino acid sequence of the encoded polypeptide.
[0774] In order to assess the expression of the fusogen and/or targeting agent, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing particles, e.g. viral particles. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic -resistance genes, such as neo and the like.
[0775] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. Reporter genes that encode for easily assayable proteins are well known. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
[0776] Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (see, e.g., Ui-Tei et al., 2000, FEBS Lett. 479:79-82). Suitable expression systems are well known and may be prepared using well known techniques or obtained commercially. Internal deletion constructs may be generated using unique internal restriction sites or by partial digestion of non-unique restriction sites. Constructs may then be transfected into cells that display high levels of the desired polynucleotide and/or polypeptide expression. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
B. Cells and Methods
[0777] Large scale viral particle production is often useful to achieve a desired viral titer. Viral particles can be produced by transfecting a transfer vector into a packaging cell line that comprises viral structural and/or accessory genes, e.g., gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes.
[0778] In some embodiments, viral particles may be produced in multiple cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast and plant cells. Exemplary methods for producing viral particles are described.
[0779] In some embodiments, elements for the production of a viral particle, e.g., a recombinant viral particle such as a replication incompetent lentiviral vector, are included in a packaging cell line or are present on a packaging vector. In some embodiments, viral particles can include packaging elements, rev, gag, and pol, delivered to the packaging cells line via one or more packaging vectors.
[0780] In embodiments, the packaging vector is an expression vector or viral vector that lacks a packaging signal and comprises a polynucleotide encoding one, two, three, four or more viral structural and/or accessory genes. Typically, the packaging vectors are included in a packaging cell, and are introduced into the cell via transfection, transduction or infection. A retroviral, e.g., lentiviral, transfer vector can be introduced into a packaging cell line, via transfection, transduction or infection, to generate a source cell or cell line. The packaging vectors can be introduced into human cells or cell lines by standard methods including, e.g., calcium phosphate transfection, lipofection or electroporation. In some embodiments, the packaging vectors are introduced into the cells together with a dominant selectable marker, such as neomycin, hygromycin, puromycin, blastocidin, zeocin, thymidine kinase, DHFR, Gin synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. A selectable marker gene can be linked physically to genes encoding by the packaging vector, e.g., by IRES or self cleaving viral peptides. In some embodiments, the packaging vector is a packaging plasmid.
[0781] Producer cell lines (also called packaging cell lines) include cell lines that do not contain a packaging signal, but do stably or transiently express viral structural proteins and replication enzymes (e.g., gag, pol and env) which can package viral particles. Any suitable cell line can be employed, e.g., mammalian cells, e.g., human cells. Suitable cell lines which can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos- 2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells. In embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells.
[0782] In some embodiments, a producer cell (i.e., a source cell line) includes a cell line which is capable of producing recombinant retroviral particles, comprising a packaging cell line and a transfer vector construct comprising a packaging signal. Methods of preparing viral stock solutions are illustrated by, e.g., Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and N. R. Landau et al. (1992) J. Virol. 66:5110-5113, which are incorporated herein by reference. Infectious virus particles may be collected from the packaging cells, e.g., by cell lysis, or collection of the supernatant of the cell culture. Optionally, the collected virus particles may be enriched or purified.
[0783] In some embodiments, the source cell comprises one or more plasmids coding for viral structural proteins and replication enzymes (e.g., gag, pol and env) which can package viral particles (i.e, a packaging plasmid). In some embodiments, the sequences coding for at least two of the gag, pol, and env precursors are on the same plasmid. In some embodiments, the sequences coding for the gag, pol, and env precursors are on different plasmids. In some embodiments, the sequences coding for the gag, pol, and env precursors have the same expression signal, e.g., promoter. In some embodiments, the sequences coding for the gag, pol, and env precursors have a different expression signal, e.g., different promoters. In some embodiments, expression of the gag, pol, and env precursors is inducible. In some embodiments, the plasmids coding for viral structural proteins and replication enzymes are transfected at the same time or at different times. In some embodiments, the plasmids coding for viral structural proteins and replication enzymes are transfected at the same time or at a different time from the packaging vector.
[0784] In some embodiments, the source cell line comprises one or more stably integrated viral structural genes. In some embodiments expression of the stably integrated viral structural genes is inducible.
[0785] In some embodiments, expression of the viral structural genes is regulated at the transcriptional level. In some embodiments, expression of the viral structural genes is regulated at the translational level. In some embodiments, expression of the viral structural genes is regulated at the post- translational level.
[0786] In some embodiments, expression of the viral structural genes is regulated by a tetracycline (Tet) -dependent system, in which a Tet-regulated transcriptional repressor (Tet-R) binds to DNA sequences included in a promoter and represses transcription by steric hindrance (Yao et al, 1998; Jones et al, 2005). Upon addition of doxycycline (dox), Tet-R is released, allowing transcription. Multiple other suitable transcriptional regulatory promoters, transcription factors, and small molecule inducers are suitable to regulate transcription of viral structural genes. [0787] In some embodiments, the third-generation lenti virus components, human immunodeficiency virus type 1 (HIV) Rev, Gag/Pol, and an envelope under the control of Tet- regulated promoters and coupled with antibiotic resistance cassettes are separately integrated into the source cell genome. In some embodiments the source cell only has one copy of each of Rev, Gag/Pol, and an envelope protein integrated into the genome.
[0788] In some embodiments a nucleic acid encoding the exogenous agent (e.g., a retroviral nucleic acid encoding the exogenous agent) is also integrated into the source cell genome. In some embodiments a nucleic acid encoding the exogenous agent is maintained episomally. In some embodiments a nucleic acid encoding the exogenous agent is transfected into the source cell that has stably integrated Rev, Gag/Pol, and an envelope protein in the genome. See, e.g., Milani et al. EMBO Molecular Medicine , 2017, which is herein incorporated by reference in its entirety.
[0789] In some embodiments, a retroviral nucleic acid described herein is unable to undergo reverse transcription. Such a nucleic acid, in embodiments, is able to transiently express an exogenous agent. The retrovirus or VLP, may comprise a disabled reverse transcriptase protein, or may not comprise a reverse transcriptase protein. In embodiments, the retroviral nucleic acid comprises a disabled primer binding site (PBS) and/or att site. In embodiments, one or more viral accessory genes, including rev, tat, vif, nef, vpr, vpu, vpx and S2 or functional equivalents thereof, are disabled or absent from the retroviral nucleic acid. In embodiments, one or more accessory genes selected from S2, rev and tat are disabled or absent from the retroviral nucleic acid.
[0790] Typically, modern retroviral vector systems include viral genomes bearing cis-acting vector sequences for transcription, reverse-transcription, integration, translation and packaging of viral RNA into the viral particles, and (2) producer cells lines which express the trans-acting retroviral gene sequences (e.g., gag, pol and env) needed for production of virus particles. By separating the cis-and trans-acting vector sequences completely, the virus is unable to maintain replication for more than one cycle of infection. Generation of live virus can be avoided by a number of strategies, e.g., by minimizing the overlap between the cis-and trans-acting sequences to avoid recombination.
[0791] A virus-like particle (VLP) which comprises a sequence that is devoid of or lacking viral genetic material may be the result of removing or eliminating the viral RNA from the sequence. Similar to viral particles, VLPs contain a viral outer envelope made from the host cell (i.e., producer cell or source cell) lipid-bi layer as well as at least one viral structural protein. In some embodiments, a viral structural protein refers to any viral protein or fragment thereof which contributes to the structure of the viral core or capsid.
[0792] Generally, for viral particles, expression of the gag precursor protein alone mediates vector assembly and release. In some aspects, gag proteins or fragments thereof have been demonstrated to assemble into structures analogous to viral cores. In one embodiment this may be achieved by using an endogenous packaging signal binding site on gag. Alternatively, the endogenous packaging signal binding site is on pol. In this embodiment, the RNA which is to be delivered will contain a cognate packaging signal. In another embodiment, a heterologous binding domain (which is heterologous to gag) located on the RNA to be delivered, and a cognate binding site located on gag or pol, can be used to ensure packaging of the RNA to be delivered. The heterologous sequence could be non-viral or it could be viral, in which case it may be derived from a different virus. The VLP could be used to deliver therapeutic RNA, in which case functional integrase and/or reverse transcriptase is not required. These VLPs could also be used to deliver a therapeutic gene of interest, in which case pol is typically included.
[0793] In an embodiment, gag-pol are altered, and the packaging signal is replaced with a corresponding packaging signal. In this embodiment, the particle can package the RNA with the new packaging signal. The advantage of this approach is that it is possible to package an RNA sequence which is devoid of viral sequence for example, RNAi.
[0794] An alternative approach is to rely on over-expression of the RNA to be packaged. In one embodiment the RNA to be packaged is over-expressed in the absence of any RNA containing a packaging signal. This may result in a significant level of therapeutic RNA being packaged, and that this amount is sufficient to transduce a cell and have a biological effect.
[0795] In some embodiments, a polynucleotide comprises a nucleotide sequence encoding a viral gag protein or retroviral gag and pol proteins, wherein the gag protein or pol protein comprises a heterologous RNA binding domain capable of recognizing a corresponding sequence in an RNA sequence to facilitate packaging of the RNA sequence into a viral vector particle. In some embodiments, the heterologous RNA binding domain comprises an RNA binding domain derived from a bacteriophage coat protein, a Rev protein, a protein of the U 1 small nuclear ribonucleoprotein particle, a Nova protein, a TF111 A protein, a TIS 11 protein, a trp RNA-binding attenuation protein (TRAP) or a pseudouridine synthase.
[0796] In some embodiments, the assembly of a viral based vector vehicle particle (i.e., a VLP) is initiated by binding of the core protein to a unique encapsidation sequence within the viral genome (e.g. UTR with stem-loop structure). In some embodiments, the interaction of the core with the encapsidation sequence facilitates oligomerization.
[0797] In some embodiments, the source cell for VLP production comprises one or more plasmids coding for viral structural proteins (e.g., gag, pol) which can package viral particles (i.e., a packaging plasmid). In some embodiments, the sequences coding for at least two of the gag and pol precursors are on the same plasmid. In some embodiments, the sequences coding for the gag and pol precursors are on different plasmids. In some embodiments, the sequences coding for the gag and pol precursors have the same expression signal, e.g., promoter. In some embodiments, the sequences coding for the gag and pol precursors have a different expression signal, e.g., different promoters. In some embodiments, expression of the gag and pol precursors is inducible. [0798] In some embodiments, formation of VLPs or any viral-based particle can be detected by any suitable technique known in the art. Examples of such techniques include, e.g., electron microscopy, dynamic light scattering, selective chromatographic separation and/or density gradient centrifugation.
V. COMPOSITIONS
[0799] Also provided herein in some embodiments are compositions containing a plurality of any of the provided particles, such as any described in Section II. In some embodiments, the provided composition contains a plurality of any of the provided lenti viral particles, such as any described in Section II.
[0800] In some embodiments, a particle or composition of particles can be assessed for one or more features related to their size, including diameter, average (e.g., mean or median) diameter, range of variation thereof above and below an average (e.g., mean or median) value of the diameter, coefficient of variation, polydispersity index, or other measure of size of particles. Various methods for particle characterization can be used, including laser diffraction, dynamic light scattering (DLS; also known as photon correlation spectroscopy) or image analysis, such as microscopy or automated image analysis.
[0801] In some embodiments, the plurality of particles has an average (e.g., mean) diameter of less than about 3 pm, less than about 2 pm, less than about 1 pm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 m, less than about 400 nm, less than about 300, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 50 nm, or less than about 20 nm. In some embodiments, the plurality of particles has an average (e.g., mean) diameter of less than about 400 nm. In another embodiment, the plurality of particles has an average (e.g., mean) diameter of less than about 150 nm. In some embodiments, the plurality of particles has an average (e.g., mean) diameter of between at or about 2 pm and at or about 1 pm, between at or about 1 pm and at or about 900 nm, between at or about 900 nm and at or about 800 nm, between at or about 800 and at or about 700 nm, between at or about 700 nm and at or about 600 nm, between at or about 600 nm and at or about 500 nm, between at or about 500 nm and at or about 400 nm, between at or about 400 nm and at or about 300 nm, between at or about 300 nm and at or about 200 nm, between at or about 200 and at or about 100 nm, between at or about 100 and at or about 50 nm, or between at or about 20 nm and at or about 50 nm.
[0802] In some embodiments, the plurality of particles has an average (e.g., mean) diameter of between at or about 10 nm and at or about 1000 nM, between at or about 25 nm and at or about 500 nm, between at or about 40 nm and at or about 300 nm, between at or about 50 nm and at or about 250 nm, between at or about 60 nm and at or about 225 nm, between at or about 70 nm and at or about 200 nm, between at or about 80 nm and at or about 175 nm, or between at or about 90 nm and at or about 150 nm.
[0803] In some embodiments, 90% of the particles in a composition fall within 50% of the average diameter of the particles. In some embodiments, 90% of the particles in a composition fall within 25% of the average diameter of the particles. In some embodiments, 90% of the particles in a composition fall within 20% of the average diameter of the particles. In some embodiments, 90% of the particles in a composition fall within 15% of the average diameter of the particles. In some embodiments, 90% of the particles in a composition fall within 10% of the average diameter of the particles.
[0804] In some embodiments, 75% of the particles in a composition fall within +/- 2 or +/- 1 St Dev standard deviations (St Dev) of the average diameter of the particles. In some embodiments, 80% of the particles in a composition fall within +/- 2 St Dev or +/- 1 St Dev of the average diameter of the particles. In some embodiments, 85% of the particles in a composition fall within +/- 2 St Dev or +/- 1 St Dev of the average diameter of the particles. In some embodiments, 90% of the particles in a composition fall within +/- 2 St Dev or +/- 1 St Dev of the average diameter of the particles. In some embodiments, 95% of the particles in a composition fall within +/- 2 St Dev or +/- 1 St Dev of the average diameter of the particles.
[0805] In some embodiments, the particles have an average hydrodynamic radius, e.g. as determined by DLS, of about 100 nm to about two microns. In some embodiments, the lipid particles have an average hydrodynamic radius between at or about 2 pm and at or about 1 pm, between at or about 1 pm and at or about 900 nm, between at or about 900 nm and at or about 800 nm, between at or about 800 and at or about 700 nm, between at or about 700 nm and at or about 600 nm, between at or about 600 nm and at or about 500 nm, between at or about 500 nm and at or about 400 nm, between at or about 400 nm and at or about 300 nm, between at or about 300 nm and at or about 200 nm, between at or about 200 and at or about 100 nm, between at or about 100 and at or about 50 nm, or between at or about 20 nm and at or about 50 nm.
[0806] In some embodiments, the particles have an average geometric radius, e.g., as determined by a multi-angle light scattering, of about 100 nm to about two microns. In some embodiments, the particles have an average geometric radius between at or about 2 pm and at or about 1 pm, between at or about 1 pm and at or about 900 nm, between at or about 900 nm and at or about 800 nm, between at or about 800 and at or about 700 nm, between at or about 700 nm and at or about 600 nm, between at or about 600 nm and at or about 500 nm, between at or about 500 nm and at or about 400 nm, between at or about 400 nm and at or about 300 nm, between at or about 300 nm and at or about 200 nm, between at or about 200 and at or about 100 nm, between at or about 100 and at or about 50 nm, or between at or about 20 nm and at or about 50 nm.
[0807] In some embodiments, the coefficient of variation (COV) (i.e., standard deviation divided by the mean) of a radius of a composition of particles is less than at or about 30%, less than at or about 25%, less than at or about 20%, less than at or about 15%, less than at or about 10% or less than at or about 5%.
[0808] In some embodiment, the compositions are characterized by their polydispersity index, which is a measure of the size distribution of the particles wherein values between 1 (maximum dispersion) and 0 (identical size of all of the particles) are possible. In some embodiments, the composition has a polydispersity index of between at or about 0.05 and at or about 0.7, between at or about 0.05 and at or about 0.6, between at or about 0.05 and at or about 0.5, between at or about 0.05 and at or about 0.4, between at or about 0.05 and at or about 0.3, between at or about 0.05 and at or about 0.2, between at or about 0.05 and at or about 0.1, between at or about 0.1 and at or about 0.7, between at or about 0.1 and at or about 0.6, between at or about 0.1 and at or about 0.5, between at or about 0.1 and at or about 0.4, between at or about 0.1 and at or about 0.3, between at or about 0.1 and at or about 0.2, between at or about 0.2 and at or about 0.7, between at or about 0.2 and at or about 0.6, between at or about 0.2 and at or about 0.5, between at or about 0.2 and at or about 0.4 between at or about 0.2 and at or about 0.3, between at or about 0.3 and at or about 0.7, between at or about 0.3 and at or about 0.6, between at or about 0.3 and at or about 0.5, between at or about 0.3 and at or about 0.4, between at or about 0.4 and at or about 0.7, between at or about 0.4 and at or about 0.6, between at or about 0.4 and at or about 0.5, between at or about 0.5 and at or about 0.7, between at or about 0.5 and at or about 0.6, or between at or about 0.6 and at or about 0.7. In some embodiments, the polydispersity index is less than at or about 0.05, less than at or about 0.1, less than at or about 0.15, less than at or about 0.2, less than at or about 0.25, less than at or about 0.3, less than at or about 0.4, less than at or about 0.5, less than at or about 0.6 or less than at or about 0.7.
[0809] In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
[0810] In some aspects, the choice of carrier is determined in part by the particular lipid particle and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
[0811] In some embodiments, the composition meets a pharmaceutical or good manufacturing practices (GMP) standard. In some embodiments, the composition is made according to good manufacturing practices (GMP). In some embodiments, the composition has a pathogen level below a predetermined reference value, e.g., is substantially free of pathogens. In some embodiments, the composition has a contaminant level below a predetermined reference value, e.g., is substantially free of contaminants. In some embodiments, the composition has low immunogenicity.
[0812] In some embodiments, formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In some embodiments, preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
[0813] In some embodiments, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. In some embodiments, the amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. In some embodiments, the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). In some embodiments, when multiple daily doses are used, the unit dosage form may be the same or different for each dose.
[0814] In some embodiments, the compositions provided herein can be formulated in dosage units of genome copies (GC). Suitable method for determining GC have been described and include, e.g., qPCR or digital droplet PCR (ddPCR) as described in, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods 25(2): 115-25. 2014, which is incorporated herein by reference.
[0815] In some embodiments, the compositions provided herein can be formulated in dosage units of infectious units. The techniques available for quantifying infectious units are routine in the art and include viral particle number determination, fluorescence microscopy, and titer by plaque assay. For example, the number of adenovirus particles can be determined by measuring the absorbance at A260. Similarly, infectious units can also be determined by quantitative immunofluorescence of vector specific proteins using monoclonal antibodies or by plaque assay. [0816] In some embodiments, methods that calculate the infectious units include the plaque assay, in which titrations of the virus are grown on cell monolayers and the number of plaques is counted after several days to several weeks. For example, the infectious titer is determined, such as by plaque assay, for example an assay to assess cytopathic effects (CPE). In some embodiments, a CPE assay is performed by serially diluting virus on monolayers of cells, such as HFF cells, that are overlaid with agarose. After incubation for a time period to achieve a cytopathic effect, such as for about 3 to 28 days, generally 7 to 10 days, the cells can be fixed and foci of absent cells visualized as plaques are determined. In some embodiments, infectious units can be determined using an endpoint dilution (TCID50) method, which determines the dilution of virus at which 50% of the cell cultures are infected and hence, generally, can determine the titer within a certain range, such as one log.
[0817] Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
[0818] Sterile injectable solutions can be prepared by incorporating the particles in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
[0819] Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. As used herein, "parenteral administration" includes intradermal, intranasal, subcutaneous, intramuscular, intraperitoneal, intravenous and intratracheal routes, as well as a slow release or sustained release system such that a constant dosage is maintained.
[0820] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
[0821] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
[0822] In some embodiments, vehicle formulations may comprise cryoprotectants. As used herein, there term “cryoprotectant” refers to one or more agent that when combined with a given substance, helps to reduce or eliminate damage to that substance that occurs upon freezing. In some embodiments, cryoprotectants are combined with vector vehicles in order to stabilize them during freezing. In some aspects, Frozen storage of RNA between -20° C. and -80° C. may be advantageous for long term (e.g. 36 months) stability of polynucleotide. In some embodiments, the RNA species is mRNA. In some embodiments, cryoprotectants are included in vehicle formulations to stabilize polynucleotide through freeze/thaw cycles and under frozen storage conditions. Cryoprotectants of the provided embodiments may include, but are not limited to sucrose, trehalose, lactose, glycerol, dextrose, raffinose and/or mannitol. Trehalose is listed by the Food and Drug Administration as being generally regarded as safe (GRAS) and is commonly used in commercial pharmaceutical formulations.
[0823] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
[0824] In some of any embodiments, suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like.
[0825] In some of any embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of the particle, alone or in combination with a second pharmaceutical agent; and instructions for using the particle to treat, prevent, or reduce one or more symptoms of a disease in a subject.
[0826] In some embodiments, the term “container” includes any receptacle for holding the pharmaceutical composition. In some embodiments, the container is the packaging that contains the pharmaceutical composition. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. In some embodiments, instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., treating or preventing a disease in a subject, or delivering an imaging or diagnostic agent to a subject.
VI. METHODS OF USE
[0827] Also provided herein in some embodiments are methods of using any of the provided particles or compositions, such as any described in Section II or V. In some embodiments, the method involves contacting a target cell with the particle or composition, such as any of the target cells described in Section II-C. In some embodiments, the particle contains an exogenous agent, such as any described in Section II-D. In some embodiments, the method is for delivering the exogenous agent to the target cell. In some embodiments, the method is for transducing the target cell with a lenti viral particle or composition. In some embodiments, the transduction introduces the exogenous agent into the target cell.
[0828] In some embodiments, the provided method involves administering the particle or composition to a subject.
[0829] In some embodiments, the provided method further involves mobilizing hematopoietic cells, such as hematopoietic stem cells (HSCs).
[0830] In some embodiments, also provided herein are methods of mobilizing hematopoietic cells, such as hematopoietic stem cells (HSCs), in connection with the provided methods of delivering a particle (e.g., lentiviral particle), including a particle containing an exogenous agent for a target cell. In some embodiments, the method of delivery includes administering a mobilization agent, e.g., a mobilization regimen, to the subject and administering the particle (e.g., lentiviral particle) to the subject in accordance with the provided methods. In some embodiments, the mobilization agent is administered to the subject prior to introducing or administering the particles (e.g., lentiviral particle) to the subject. In some embodiments, the mobilization agent includes a mobilization regimen that cause therapeutically inaccessible hematopoietic cells to become therapeutically accessible. In some embodiments, a mobilization agent increases the number of hematopoietic cells in peripheral blood, thus allowing for a more accessible source of hematopoietic cells for targeting by the particles (e.g., lentiviral particles) in accordance with the methods as described. In some embodiments, the agent stimulates mobilization of bone marrow cells from the bone marrow to the peripheral blood in the subject.
[0831] Also provided herein in some embodiments are methods involving administering to a subject (i) an agent that stimulates mobilization of bone marrow cells from the bone marrow to the peripheral blood in the subject and (ii) a particle or composition, such as any described in Section II or V, either with or without any of the targeting agents described herein. In some embodiments, the particle contains an exogenous agent, such as any described in Section II-D. In some embodiments, the exogenous is for delivering to a target cell in a subject, such as any of the target cells described in Section II-C.
[0832] In some embodiments, the exogenous agent is for treating a disease or condition in the subject. In some embodiments, the particle contains an exogenous agent to deliver to a target cell in the subject. In some embodiments, the particle contains an exogenous agent to deliver to a target cell for treating a disease or condition in the subject.
[0833] In some embodiments, the method further involves administering to the subject a transduction adjuvant. In some embodiments, the transduction adjuvant is administered to the subject on the day of the administration of at least one dose of the particle. In some embodiments, the transduction adjuvant is administered to the subject on the days of the administration of at least two doses of the particle. In some embodiments, the transduction adjuvant is a cationic peptide. In some embodiments, the cationic peptide is a histidine -rich cationic peptide. In some embodiments, the cationic peptide is a cationic amphipathic peptide. In some embodiments, the cationic peptide is derived from LAH4 peptide family. In some embodiments, the transduction adjuvant is Vectofusin-1 (Fenard et al. Mol Ther Nucleic Acids. 2013 May 7;2(5):e90).
[0834] In some embodiments, the particle is capable of delivering (e.g., delivers) an exogenous agent (e.g., payload gene) to a target cell for treating a disease or condition in a subject. In some embodiments, the exogenous agent is an agent that is entirely heterologous or not produced or normally expressed by the target cell. In some embodiments, delivery of the payload agent to the target cell can provide a therapeutic effect to treat a disease or condition in the subject. The therapeutic effect may be by providing a payload agent in which the exogenous agent is a protein (or a nucleic acid encoding the protein, e.g., an mRNA encoding the protein) which is then expressed on or in the target cell to thereby mediate the therapeutic effect. In some aspects, the payload agent is a engineered receptor, such as a chimeric antigen receptor, that is delivered to target cells for expression on the surface, in which the engineered receptor is able to bind or recognize an antigen on a cells associated with a disease or condition. In some embodiments, targeting of such cells stimulates the engineered receptor (e.g., CAR) to induce cytotoxicity of the cells associated with the disease or condition which, in some aspects, removes or kills the cells and thereby treats the disease or condition. In some embodiments, cells associated with the disease or condition are tumor cells.
[0835] In some embodiments, the particles can be administered to a subject, e.g., a mammal, e.g., a human. In some embodiments, the administration delivers the particles to a target cell in the subject. In such embodiments, the subject may be at risk of, may have a symptom of, or may be diagnosed with or identified as having, a particular disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject. In one embodiment, the subject has cancer. In one embodiment, the subject has an infectious disease. In some embodiments, the particle contains nucleic acid sequences encoding a therapeutic agent for treating the disease or condition in the subject. For example, in some embodiments, the exogenous agent is one that targets or is specific for a protein of neoplastic cells, and the particle is administered to a subject for treating a tumor or cancer in the subject. In another example, the exogenous agent is an inflammatory mediator or immune molecule, such as a cytokine, and the particle is administered to a subject for treating any condition in which it is desired to modulate (e.g., increase) the immune response, such as a cancer or infectious disease.
[0836] In some embodiments, the particle composition described herein may be administered to a subject having a cancer, an autoimmune disease, an infectious disease, a metabolic disease, a neurodegenerative disease, or a genetic disease (e.g., enzyme deficiency).
[0837] In some embodiments, delivery of the exogenous agent to the target cell can provide a therapeutic effect to treat a disease or condition in the subject. The therapeutic effect may be by targeting, modulating or altering an antigen or protein present or expressed by the target cell that is associated with or involved in a disease or condition. The therapeutic effect may be by providing an exogenous agent in which the exogenous agent is a protein (or a nucleic acid encoding the protein, e.g., an mRNA encoding the protein) which is absent, mutant, or at a lower level than wild-type in the target cell. In some embodiments, the target cell is from a subject having a genetic disease, e.g., a monogenic disease, e.g., a monogenic intracellular protein disease.
[0838] In some embodiments, the target cell is from a subject having a hematopoietic disease or disorder. In some embodiments, the provided methods and uses include methods and uses for treating a hematopoietic disease or disorder. In some embodiments, the hematopoietic disorder may be due to a blood disease, in particular disease involving hematopoietic cells. In some embodiments, the hematopoietic disorder is a monogenic hematopoietic disease, such as due to mutation of a single gene. In some embodiments, the hematopoietic disorder is myelodysplasia, aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria, Sickle cell disease, Diamond Blackfan anemia, Schachman Diamond disorder, Kostmann's syndrome, chronic granulomatous disease, adrenoleukodystrophy, leukocyte adhesion deficiency, hemophilia, thalassemia, beta-thalassemia, leukaemia such as acute lymphocytic leukemia (ALL), acute myelogenous (myeloid) leukemia (AML), adult lymphoblastic leukaemia, chronic lymphocytic leukemia (CLL), B-cell chronic lymphocytic leukemia (B-CLL), chronic myeloid leukemia (CML), juvenile chronic myelogenous leukemia (CML), and juvenile myelomonocytic leukemia (JMML), severe combined immunodeficiency disease (SCID), X-linked severe combined immunodeficiency, Wiskott-Aldrich syndrome (WAS), adenosine-deaminase (ADA) deficiency, chronic granulomatous disease, Chediak-Higashi syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL) or AIDS. In some embodiemnts, the hematopoietic disorder is ADA, SCID or a hemoglobinopathies .
[0839] In some embodiments, the target cell is from a subject having an autoimmune disease. In some embodiments, the provided methods and uses include methods and uses for treating the autoimmune disease. In some embodiments, the autoimmune disease is acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison's disease, Agammaglobulinemia, Alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic allergy, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Balo disease, Balo concentric sclerosis, Bechets syndrome, Berger's disease, Bickerstaffs encephalitis, Blau syndrome, bullous pemphigoid, cancer, Castleman's disease, celiac disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1, diffuse cutaneous systemic sclerosis, Dressier's syndrome, discoid lupus erythematosus, eczema, enthesitis-related arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa acquisita, erythema nodosum, essential mixed cryoglobulinemia, Evan's syndrome, firodysplasia ossificans progressiva, fibrosing aveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome (GBS), Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anaemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, inflammatory demyelinating polyneuropathy, interstitial cystitis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Kawasaki's disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), Lou Gehrig's disease, lupoid hepatitis, lupus erythematosus, Majeed syndrome, Meniere's disease, microscopic polyangiitis, Miller- Fisher syndrome, mixed connective tissue disease, morphea, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, neuropyelitis optica, neuromyotonia, ocular cicatricial pemphigoid, opsoclonus myoclonus syndrome, ord thyroiditis, palindromic rheumatism, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, pars planitis, pemphigus, pemphigus vulgaris, permicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, relapsing polychondritis, Reiter's syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatoid fever, sarcoidosis, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjogren's syndrome, spondylarthropathy, Still's disease, stiff person syndrome, subacute bacterial endocarditis, Susac's syndrome, Sweet's syndrome, Sydenham chorea, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondylarthropathy, vasculitis, vitiligo or Wegener's granulomatosis.
[0840] In some embodiments, the target cell is from a subject having a cancer. In some embodiments, the cancer is leukemia. In some embodiments, the leukemia is B-CLL, CML or T cell based leukemia such as ALT. In some embodiments, the cancer is a melanoma.
[0841] In some embodiments, the target cell is from a subject having a demyelinating disease of the central nervous system.
[0842] In some embodiments, the particle delivers the exogenous agent to at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the number of cells in the target cell population. In some embodiments, the particle delivers at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the exogenous agent to the target cell population.
[0843] In some embodiments, the particle delivers at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% more of the exogenous agent to the target cell population compared to a non-target cell population. In some embodiments, when a plurality of particles are contacted with a cell population comprising target cells and non-target cells, the exogenous agent is present in at least 10-fold more target cells than non-target cells. In some embodiments, when the plurality of particles are contacted with a cell population comprising target cells and non-target cells, the exogenous agent is present at least 2-fold, 5-fold, 10-fold, 20-fold, or 50-fold higher in target cells than non-target cells.
[0844] In some embodiments, the particle fuses at a higher rate with a target cell than with a non- target cell. In some embodiments, the particle fuses at a higher rate with a target cell than with a non- target cell, e.g., by at least at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold. In some embodiments, the particle fuses at a higher rate with a target cell than with other viral vectors, e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the particle fuses with target cells at a rate such that an exogenous agent is delivered to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, of target cells after 24, 48, or 72 hours. In embodiments, the amount of targeted fusion is about 30%-70%, 35%-65%, 40%-60%, 45%-55%, or 45%-50%. In embodiments, the amount of targeted fusion is about 20%-40%, 25%-35%, or 30%-35%.
[0845] In some of any embodiments, the particle composition mediates an effect on a target cell, and the effect lasts for at least 1, 2, 3, 4, 5, 6, or 7 days, 2, 3, or 4 weeks, or 1, 2, 3, 6, or 12 months. In some embodiments, the effect lasts for less than 1, 2, 3, 4, 5, 6, or 7 days, 2, 3, or 4 weeks, or 1, 2, 3, 6, or 12 months. [0846] In some embodiments, the particle is administered in an effective amount or dose to effect treatment of the disease, condition or disorder. In some embodiments, one or more transducing units of viral particles or virus-like particles, e.g., retroviral particles or retroviral-like particles, are administered to the subject. In some embodiments, at least 1, 10, 100, 1000, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, or 1014, transducing units per kg are administered to the subject. In some embodiments at least 1, 10, 100, 1000, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, or 1014, transducing units per target cell per ml of blood are administered to the subject.
[0847] In some embodiments, delivery of an exogenous agent by administration of a particle composition described herein may modify cellular protein expression levels. In certain embodiments, the administered composition directs upregulation of (via expression in the cell, delivery in the cell, or induction within the cell) of one or more exogenous agents (e.g., a polypeptide or mRNA) that provide a functional activity which is substantially absent or reduced in the cell in which the polypeptide is delivered. In some embodiments, the missing functional activity may be enzymatic, structural, or regulatory in nature. In some embodiments, the administered composition directs up-regulation of one or more polypeptides that increases (e.g., synergistically) a functional activity which is present but substantially deficient in the cell in which the polypeptide is upregulated. In some of any embodiments, the administered composition directs downregulation (via expression in the cell, delivery in the cell, or induction within the cell) of one or more exogenous agents (e.g., a polypeptide, siRNA, or miRNA) that repress a functional activity which is present or upregulated in the cell in which the polypeptide, siRNA, or miRNA is delivered. In some of any embodiments, the upregulated functional activity may be enzymatic, structural, or regulatory in nature. In some embodiments, the administered composition directs down-regulation of one or more polypeptides that decreases (e.g., synergistically) a functional activity which is present or upregulated in the cell in which the polypeptide is downregulated. In some embodiments, the administered composition directs upregulation of certain functional activities and downregulation of other functional activities.
[0848] In some embodiments, the particle is capable of delivering (e.g., delivers) a nucleic acid to a target cell, e.g., to stably modify the genome of the target cell, e.g., for gene therapy. Similarly, in some embodiments, a method herein comprises delivering a nucleic acid to a target cell.
[0849] In some embodiments, a method herein comprises causing ligand presentation on the surface of a target cell by presenting cell surface ligands on the particle. In some embodiments, the particle is capable of causing cell death of the target cell. In some embodiments, the viral vector is from an NK source cell.
[0850] In some embodiments, a particle or target cell is capable of phagocytosis (e.g., of a pathogen). Similarly, in some embodiments, a method herein comprises causing phagocytosis.
[0851] In some embodiments, the particle comprises (e.g., is capable of delivering to the target cell) a membrane protein or a nucleic acid encoding the membrane protein. [0852] In some embodiments, the particle is capable of causing (e.g., causes) a target cell to secrete a protein, e.g., a therapeutic protein. In some embodiments, the particle is capable of delivering (e.g., delivers) a secreted exogenous agent, e.g., a secreted protein to a target site (e.g., an extracellular region), e.g., by delivering a nucleic acid (e.g., mRNA) encoding the protein to the target cell under conditions that allow the target cell to produce and secrete the protein. Similarly, in some embodiments, a method herein comprises delivering a secreted exogenous agent as described herein. In embodiments, the secreted protein comprises a protein therapeutic, e.g., an antibody molecule, a cytokine, or an enzyme. In embodiments, the secreted protein comprises an autocrine signaling molecule or a paracrine signaling molecule. In embodiments, the secreted exogenous agent comprises a secretory granule.
[0853] In some embodiments, the particle is capable of secreting (e.g., secretes) an exogenous agent, e.g., a protein. In some embodiments, the exogenous agent, e.g., secreted agent, is delivered to a target site in a subject. In some embodiments, the exogenous agent is a protein that cannot be made recombinantly or is difficult to make recombinantly. In some embodiments, the particle that secretes a protein is from a source cell selected from an MSC or a chondrocyte.
[0854] In some embodiments, the particle is capable of reprogramming (e.g., reprograms) a target cell (e.g., an immune cell), e.g., by delivering an exogenous agent selected from a transcription factor, a nucleic acid encoding a transcription factor, mRNA, or a plurality of said exogenous agents. Similarly, in some embodiments, a method herein comprises reprogramming a target cell. In embodiments, reprogramming comprises inducing an exhausted T cell to take on one or more characteristics of a nonexhausted T cell, e.g., a killer T cell. In some embodiments, the exogenous agent comprises an antigen. In some embodiments, the particle comprises a first exogenous agent comprising an antigen and a second exogenous agent comprising an antigen presenting protein.
[0855] In some embodiments, a particle is capable of modifying, e.g., modifies, a target tumor cell, for instance by delivering an exogenous agent (protein or nucleic acid). Similarly, in some embodiments, a method herein comprises modifying a target tumor cell. In embodiments, the particle delivers an mRNA encoding an immunostimulatory ligand, an antigen presenting protein, a tumor suppressor protein, or a pro-apoptotic protein. In some embodiments, the particle delivers an miRNA capable of reducing levels in a target cell of an immunosuppressive ligand, a mitogenic signal, or a growth factor.
[0856] In some embodiments, a particle delivers an exogenous agent that is immunomodulatory, e.g., immunostimulatory.
[0857] In some embodiments, a particle is capable of causing (e.g., causes) the target cell to present an antigen, for instance by delivering an exogenous agent comprising an antigen or a nucleic acid encoding the antigen. Similarly, in some embodiments, a method herein comprises presenting an antigen on a target cell. In some embodiments, the particle promotes regeneration in a target tissue. Similarly, in some embodiments, a method herein comprises promoting regeneration in a target tissue. [0858] In some embodiments, the particle is capable of delivering (e.g., delivers) a nucleic acid to a target cell, e.g., to stably modify the genome of the target cell, e.g., for gene therapy. Similarly, in some embodiments, a method herein comprises delivering a nucleic acid to a target cell. In some embodiments, the target cell has an enzyme deficiency, e.g., comprises a mutation in an enzyme leading to reduced activity (e.g., no activity) of the enzyme.
[0859] In some embodiments, the particle is capable of delivering (e.g., delivers) a reagent that mediates a sequence specific modification to DNA (e.g., Cas9, ZFN, or TALEN) in the target cell. Similarly, in some embodiments, a method herein comprises delivering the reagent to the target cell. In embodiments, the target cell is a CNS cell.
[0860] In some embodiments, the particle is capable of delivering (e.g., delivers) a nucleic acid to a target cell, e.g., to transiently modify gene expression in the target cell.
[0861] In some embodiments, the particle is capable of delivering (e.g., delivers) a protein to a target cell, e.g., to transiently rescue a protein deficiency. Similarly, in some embodiments, a method herein comprises delivering a protein to a target cell. In embodiments, the protein is a membrane protein (e.g., a membrane transporter protein), a cytoplasmic protein (e.g., an enzyme), or a secreted protein (e.g., an immunosuppressive protein).
[0862] In some embodiments, the particle is capable of intracellular molecular delivery, e.g., delivers a protein exogenous agent to a target cell. Similarly, in some embodiments, a method herein comprises delivering a molecule to an intracellular region of a target cell. In embodiments, the protein exogenous agent is an inhibitor. In some embodiments, the protein exogenous agent comprises a nanobody, scFv, camelid antibody, peptide, macrocycle, or small molecule.
[0863] In some embodiments, the particle comprises on its membrane one or more cell surface ligands (e.g., 1, 2, 3, 4, 5, 10, 20, 50, or more cell surface ligands), said cell surface ligands to be presented by the particle to a target cell. Similarly, in some embodiments, a method herein comprises presenting one or more cell surface ligands to a target cell. In some embodiments, the particle having a cell surface ligand is from a source cell chosen from a neutrophil (e.g., and the target cell is a tumorinfiltrating lymphocyte), dendritic cell (e.g., and the target cell is a naive T cell), or neutrophil (e.g., and the target is a tumor cell or virus-infected cell). In some embodiments the particle comprises a membrane complex, e.g., a complex comprising at least 2, 3, 4, or 5 proteins, e.g., a homodimer, heterodimer, homotrimer, heterotrimer, homotetramer, or heterotetramer. In some embodiments, the particle comprises an antibody, e.g., a toxic antibody, e.g., the particle is capable of delivering the antibody to the target site, e.g., by homing to a target site. In some embodiments, the source cell is an NK cell or neutrophil.
[0864] In some embodiments, a method herein comprises causing ligand presentation on the surface of a target cell by presenting cell surface ligands on the particle. In some embodiments, the particle is capable of causing cell death of the target cell. In some embodiments, the particle is from a NK source cell. In some embodiments, a particle or target cell is capable of phagocytosis (e.g., of a pathogen). Similarly, in some embodiments, a method herein comprises causing phagocytosis. In some embodiments, a particle senses and responds to its local environment. In some embodiments, the particle is capable of sensing level of a metabolite, interleukin, or antigen.
[0865] In embodiments, a particle is capable of chemotaxis, extravasation, or one or more metabolic activities. In embodiments, the metabolic activity is selected from kyneurinine, gluconeogenesis, prostaglandin fatty acid oxidation, adenosine metabolism, urea cycle, and thermogenic respiration. In some embodiments, the source cell is a neutrophil and the particle is capable of homing to a site of injury. In some embodiments, the source cell is a macrophage and the particle is capable of phagocytosis. In some embodiments, the source cell is a brown adipose tissue cell and the particle is capable of lipolysis.
[0866] In some embodiments, the particle comprises (e.g., is capable of delivering to the target cell) a plurality of exogenous agents (e.g., at least 2, 3, 4, 5, 10, 20, or 50 exogenous agents). In embodiments, the particle comprises an inhibitory nucleic acid (e.g., siRNA or miRNA) and an mRNA.
[0867] In some embodiments, the particle comprises (e.g., is capable of delivering to the target cell) a membrane protein or a nucleic acid encoding the membrane protein. In embodiments, the particle is capable of reprogramming or transdifferentiating a target cell, e.g., the particle comprises one or more agents that induce reprogramming or transdifferentiation of a target cell.
[0868] In some embodiments, the provided methods involve administration of a pharmaceutical composition comprising oral, inhaled, transdermal or parenteral (including intravenous, intratumoral, intraperitoneal, intramuscular, intracavity, and subcutaneous) administration. In some embodiments, the particles may be administered alone or formulated as a pharmaceutical composition. In some embodiments, the particles may be administered in the form of a unit-dose composition, such as a unit dose oral, parenteral, transdermal or inhaled composition. In some embodiments, the particles are prepared by admixture and are adapted for oral, inhaled, transdermal or parenteral administration, and as such may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable and infusable solutions or suspensions or suppositories or aerosols.
[0869] In some embodiments, the regimen of administration may affect what constitutes an effective amount. In some embodiments, the therapeutic formulations may be administered to the subject either prior to or after a diagnosis of disease. In some embodiments, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. In some embodiments, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
[0870] In some embodiments, the administration of the particles to a subject, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to prevent or treat disease. In some embodiments, an effective amount of the particles necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular particles employed; the time of administration; the rate of excretion of the particles; the duration of the treatment; other drugs, compounds or materials used in combination with the particles; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts. In some embodiments, the dosage regimens may be adjusted to provide the optimum therapeutic response. In some embodiments, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the particles without undue experimentation.
[0871] In some embodiments, the composition may be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. In some embodiments, the amount of a composition may be administered every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as the type and severity of the disease being treated, the type and age of the animal, etc.
[0872] In some embodiments, dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
[0873] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. In some embodiments, the physician or veterinarian could start doses of the particles employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
[0874] In some embodiments, it is especially advantageous to formulate the particles in dosage unit form for ease of administration and uniformity of dosage. In some embodiments, dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of particles calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. In some embodiments, the dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such particles for the treatment of a disease in a subject. [0875] In some embodiments, the particles can be formulated in dosage units of genome copies (GC). Suitable method for determining GC have been described and include, e.g., qPCR or digital droplet PCR (ddPCR) as described in, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods 25(2): 115-25. 2014, which is incorporated herein by reference. In some embodiments, the dosage of administration of a particle is from about 104 to about IO10 GC units, inclusive. In some embodiments, the dosage of administration of a particle is from about 109 to about 1015 GC units, inclusive. In some embodiments, the dosage of administration of a particle is from about 105 to about 109 GC units, inclusive. In some embodiments, the dosage of administration of a particle is from about 106 to about 109 GC units, inclusive. In some embodiments, the dosage of administration of a particle is from about 109 to about 1012 GC units, inclusive. In some embodiments, the dosage of administration of a particle is from about 1012 to about 1014GC units, inclusive. In some embodiments, the dosage of administration is l.OxlO9 GC units, 5.0xl09 GC units, l.OxlO10 GC units, 5.0xl010 GC units, l.OxlO11 GC units, 5.0x10" GC units, l.OxlO12 GC units, 5.0xl012 GC units, or l.OxlO13 GC units, 5.0xl013 GC units, l.OxlO14 GC units, 5.0xl014 GC units, or l.OxlO15 GC units.
[0876] In some embodiments, the dosage of administration of a particle is from about 104 to about 1010 infectious units, inclusive. In some embodiments, the dosage of administration of a particle is from about 109 to about 1015 infectious units, inclusive. In some embodiments, the dosage of administration of a particle is from about 105 to about 109 infectious units. In some embodiments, the dosage of administration of a particle is from about 106 to about 109 infectious units. In some embodiments, the dosage of administration of a particle is from about 109 to about 1012 infectious units, inclusive. In some embodiments, the dosage of administration of a particle is from about 1012 to about 1014 infectious units, inclusive. In some embodiments, the dosage of administration is l.OxlO9 infectious units, 5.0xl09 infectious units, l.OxlO10 infectious units, 5.0xl010 infectious units, 1.0x10" infectious units, 5.0x10" infectious units, l.OxlO12 infectious units, 5.0xl012 infectious units, or l.OxlO13 infectious units, 5.0xl013 infectious units, l.OxlO14 infectious units, 5.0xl014 infectious units, or l.OxlO15 infectious units. The techniques available for quantifying infectious units are routine in the art and include viral particle number determination, fluorescence microscopy, and titer by plaque assay. For example, the number of adenovirus particles can be determined by measuring the absorbance at A260. Similarly, infectious units can also be determined by quantitative immunofluorescence of vector specific proteins using monoclonal antibodies or by plaque assay.
[0877] In some embodiments, methods that calculate the infectious units include the plaque assay, in which titrations of the virus are grown on cell monolayers and the number of plaques is counted after several days to several weeks. For example, the infectious titer is determined, such as by plaque assay, for example an assay to assess cytopathic effects (CPE). In some embodiments, a CPE assay is performed by serially diluting virus on monolayers of cells, such as HFF cells, that are overlaid with agarose. After incubation for a time period to achieve a cytopathic effect, such as for about 3 to 28 days, generally 7 to 10 days, the cells can be fixed and foci of absent cells visualized as plaques are determined. In some embodiments, infectious units can be determined using an endpoint dilution (TCID50) method, which determines the dilution of virus at which 50% of the cell cultures are infected and hence, generally, can determine the titer within a certain range, such as one log.
[0878] In some embodiments, the dosage of administration of a particle is from about 104 to about IO10 plaque forming units (pfu), inclusive. In some embodiments, the dosage of administration of a particle is from about 109 to about 1015pfu, inclusive. In some embodiments, the dosage of administration of a particle is from about 105 to about 109pfu. In some embodiments, the dosage of administration of a particle is from about 106 to about 109 pfu. In some embodiments, the dosage of administration of a particle is from about 109 to about 1012pfu, inclusive. In some embodiments, the dosage of administration of a particle is from about 1012 to about 1014pfu, inclusive. In some embodiments, the dosage of administration is 1.0xl09 pfu, 5.0xl09 pfu, 1.0xl010 pfu, 5.0xl010 pfu, l.OxlO11 pfu, 5.0xl0n pfu, l.OxlO12 pfu, 5.0xl012 pfu, or l.OxlO13 pfu, 5.0xl013 pfu, l.OxlO14 pfu, 5.0xl014 pfu, or l.OxlO15 pfu.
[0879] In some embodiments, the subject will receive a single injection. In some embodiments, administration can be repeated at daily/weekly/monthly intervals for an indefinite period and/or until the efficacy of the treatment has been established. As set forth herein, the efficacy of treatment can be determined by evaluating the symptoms and clinical parameters described herein and/or by detecting a desired response.
[0880] The exact amount of particle required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular polynucleic acid, polypeptide, or vector used, its mode of administration, etc. An appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
[0881] In some aspects, the dosage of administration of a vehicle within the pharmaceutical compositions provided herein varies depending on a subject’s body weight. For example, a composition may be formulated as GC/kg, infectious units/kg, pfu/kg, etc. In some aspects, the dosage at which a therapeutic effect is obtained is from at or about 108 GC/kg to at or about 1014 GC/kg of the subject’s body weight, inclusive. In some aspects, the dosage at which a therapeutic effect is obtained is at or about 108 GC/kg of the subject’s body weight (GC/kg). In some aspects, the dosage is from at or about 108 infectious units/kg to at or about 1014 infectious units/kg of the subject’s body weight, inclusive.
[0882] In some of any embodiments, the compositions are administered to the subject in dosages that range from one to five times per day or more. In another embodiment, the compositions are administered to the subject in range of dosages that include once every day, every two, days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions will vary from subject to subject depending on many factors including age, disease or disorder to be treated, gender, overall health, and other factors. [0883] In some of any of the provided methods, the methods further include administering a cytokine receptor agonist to a subject as described.
[0884] In some embodiments, routes of administration of any of the compositions disclosed herein include oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intratumoral intrabronchial, inhalation, and topical administration.
[0885] In some of any embodiments, suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like.
[0886] In some of any embodiments, the particle composition described herein is delivered ex-vivo to a cell or tissue, e.g., a human cell or tissue. In embodiments, the composition improves function of a cell or tissue ex-vivo, e.g., improves cell viability, respiration, or other function (e.g., another function described herein).
[0887] In some embodiments, the composition is delivered to an ex vivo tissue that is in an injured state (e.g., from trauma, disease, hypoxia, ischemia or other damage).
[0888] In some embodiments, the composition is delivered to an ex-vivo transplant (e.g., a tissue explant or tissue for transplantation, e.g., a human vein, a musculoskeletal graft such as bone or tendon, cornea, skin, heart valves, nerves; or an isolated or cultured organ, e.g., an organ to be transplanted into a human, e.g., a human heart, liver, lung, kidney, pancreas, intestine, thymus, eye). In some embodiments, the composition is delivered to the tissue or organ before, during and/or after transplantation.
[0889] In some embodiments, the composition is delivered, administered or contacted with a cell, e.g., a cell preparation. In some embodiments, the cell preparation may be a cell therapy preparation (a cell preparation intended for administration to a human subject). In embodiments, the cell preparation comprises cells expressing a chimeric antigen receptor (CAR), e.g., expressing a recombinant CAR. The cells expressing the CAR may be, e.g., T cells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells. In embodiments, the cell preparation is a neural stem cell preparation. In embodiments, the cell preparation is a mesenchymal stem cell (MSC) preparation. In embodiments, the cell preparation is a hematopoietic stem cell (HSC) preparation. In embodiments, the cell preparation is an islet cell preparation.
[0890] In some embodiments, the source of particles are from the same subject that is administered a particle composition. In other embodiments, they are different. In some embodiments, the source of particles and recipient tissue may be autologous (from the same subject) or heterologous (from different subjects). In some embodiments, the donor tissue for particle compositions described herein may be a different tissue type than the recipient tissue. In some embodiments, the donor tissue may be muscular tissue and the recipient tissue may be connective tissue (e.g., adipose tissue). In other embodiments, the donor tissue and recipient tissue may be of the same or different type, but from different organ systems.
A. Mobilization
[0891] In some embodiments, also provided herein are methods of mobilizing hematopoietic cells, such as hematopoietic stem cells (HSCs), in connection with the provided methods of delivering a particle (e.g., lentiviral particle), including a particle containing an exogenous agent for a target cell. In some embodiments, the method of delivery includes administering a mobilization agent, e.g., a mobilization regimen, to the subject and administering the particle (e.g., lentiviral particle) to the subject in accordance with the provided methods. In some embodiments, the mobilization agent is administered to the subject prior to introducing or administering the particles (e.g., lentiviral particle) to the subject. In some embodiments, the mobilization agent includes a mobilization regimen that cause therapeutically inaccessible hematopoietic cells to become therapeutically accessible. In some embodiments, a mobilization agent increases the number of hematopoietic cells in peripheral blood, thus allowing for a more accessible source of hematopoietic cells for targeting by the particles (e.g., lentiviral particles) in accordance with the methods as described. In some embodiments, the mobilization agent stimulates mobilization of bone marrow cells from the bone marrow to the peripheral blood in the subject.
[0892] Also provided herein in some embodiments are methods involving administering to a subject (i) an agent that stimulates mobilization of bone marrow cells from the bone marrow to the peripheral blood in the subject and (ii) a particle, such as any described in Section II, either with or without any of the targeting agents described herein.
[0893] In some embodiments, the mobilization agent increases the number of stem cells in peripheral blood, thus allowing for a more accessible source of stem cells for use in the method as described. In some embodiments, the mobilization agent increases the number of hematopoietic cells in circulation in the subject. In some embodiments, the mobilization agent is a mobilizer of hematopoietic stem cells or progenitor cells. In some embodiments, the mobilization agent induces the exit of hematopoietic cells from bone marrow.
[0894] In some embodiments, the hematopoietic cells are CD34+ and may include CD34+ progenitor cells. In some embodiments, the hematopoietic cells are HSCs. In some aspects, the mobilization agent is a stem cell mobilization agent.
[0895] As used herein, “mobilizing” and “mobilizing hematopoietic cells” are used interchangeably to refer to the act of inducing the migration of hematopoietic cells, such as CD34+ cells, including progenitor cells and/or hematopoietic stem cells, from a first location (e.g., stem cell niche, e.g., bone marrow) into a second location (e.g., tissue (e.g., peripheral blood) or organ (e.g., spleen). In some embodiments, the process of mobilizing hematopoietic cells involves the recruitment of stem cells from their tissue or organ of residence to peripheral blood following treatment with a mobilization agent, such as using a mobilization agent known to a skilled artisan, including any cytokine and chemotherapeutic drugs known in the art for this purpose (e.g., G-CSF). In some aspects, this process mimics the enhancement of the physiological release of stem cells from tissues or organs in response to stress signals during injury and inflammation. In some embodiments, the mobilization agent or agents act as agonists or antagonists that prevent the attachment of hematopoietic cells to cells or tissues of their microenvironment. In some embodiments, the mobilization agent or agents induce the release of proteases that cleave the adhesion molecules or support structures between hematopoietic cells and their sites of attachment. In some embodiments, the mobilization agent is capable of mobilizing any hematopoietic cell, such as stem cells and/or progenitor cells, in which heparan sulfate proteoglycans are responsible for maintaining adhesion of the cells in their cell niche. In an aspect, a method of mobilizing hematopoietic cells in a subject comprises administering to a subject an effective amount of an agent that inhibits the level or activity of heparan sulfate proteoglycans, thereby mobilizing hematopoietic cells in the subject.
[0896] In some embodiments, a mobilization agent increases the circulation of hematopoietic cells and/or mobilizes hematopoietic cells sequestered in bone marrow to exit bone marrow into compartments where they are accessible, e.g., accessible for transduction by the lipid particle and/or viral vector. For example, administration to a subject of a mobilization therapy can increase the circulation of hematopoietic cells and/or mobilize hematopoietic cells sequestered in bone marrow to exit bone marrow into compartments where they are accessible, such as the peripheral blood.
[0897] In some embodiments, the mobilization agent is administered prior to the particle (e.g., lentiviral particle or other viral particle). In some embodiments, the mobilization agent is administered to the subject within 7 days prior to administering the particle (e.g., lentiviral particle), such as within 6 days, 5 days, 4 days, 3 days, 2 days or 1 day prior to administering the particle (e.g., lentiviral particle). In some embodiments, the mobilization agent is administered twice a day, once a day or two or three times within a week. In some embodiments, the mobilization agent is administered once daily for consecutive days prior to administering the particle (e.g., lentiviral particle). In some embodiments, at least one dose of the mobilization agent is administered to the subject on the same day as the provided methods of contacting target cells with a particle (e.g., lentiviral particle). In some embodiments, at least one dose of the mobilization agent is administered to the subject within 12 hours prior to administering the lipid particle (e.g. lentiviral vector), such as within 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours or 1 hour prior to administering the particle (e.g., lentiviral particle).
[0898] Exemplary mobilization agents include stem cell factor (SCF), small molecule VLA-4 inhibitor BI05192, BOP (N-(henzenesulfonyl)-L-prolyl-L-0-(l-pyrrolidinylcarbonyl)tyrosine), heparin, granulocyte colony-stimulating factor (G-CSF), and plerixafor (also known as AMD3100). In some embodiments, the mobilization agent of the method is selected from the group consisting of granulocytemacrophage colony-stimulating factor (GM-CSF), Fms-related tyrosine kinase 3 (flt-3) ligand, stromal cell-derived factor 1 (SDF-1), agonists of the chemokine (C — C motif) receptor 1 (CCR1), such as chemokine (C — C motif) ligand 3 (CCL3, also known as macrophage inflammatory protein- la (Mip- la)), agonists of the chemokine (C — X — C motif) receptor 1 (CXCR1) and CXCR2, such as chemokine (C — X — C motif) ligand (CXCL1), CXCL2 (also known as growth-related oncogene protein-P (Gro- )), and CXCL8 (also known as interleukin-8 (IL-8)), agonists of CXCR4, such as CTCE-002, ATI-2341, and Met-SDF-1, Very Late Antigen (VLA)-4 inhibitor, TG-0054, AMD3465, and any combination thereof.
[0899] In some embodiments, the mobilization agent is stem cell factor (SCF), small molecule VLA-4 inhibitor BI05192, BOP (N-(henzenesulfonyl)-L-prolyl-L-0-(l-pyrrolidinylcarbonyl)tyrosine), heparin, granulocyte colony-stimulating factor (G-CSF), or plerixafor (AMD3100). In some embodiments, the mobilization agent includes the combination of G-CSF and plerixafor (AMD3100). In some of any embodiments, G-CSF is filgrastim (e.g. Neupogen® or Zarzio®). In some embodiments, the G-CSF is a pegylated G-CSF, such as pegfilgrastim (e.g. Neulasta®).
[0900] Any of various known methods for mobilizing hematopoietic cells using a mobilization agent can be used, including but not limited to, any as described in PCT publication No. W02021211450; U.S. publication Nos. US20200268850 and US20170106021; and U.S. Patent No. 7,939,057 and 10,907,177.
[0901] In some embodiments, the mobilization regimen includes administration of at least one mobilizing agent. In various embodiments, at least one mobilizing agent is administered to a subject (i) daily on the four days prior to administration of a first dose of the particle (e.g., lentiviral particle); (ii) on the day of administration of a first dose of the particle (e.g., lentiviral particle); and (iii) on the day of administration of one or more subsequent doses of the particle (e.g., lentiviral particle). In some embodiments, at least one mobilizing agent is administered to a subject (i) on the day prior to administration of a first dose of the particle (e.g., lentiviral particle) and (ii) on the day of administration of a first dose of the particle (e.g., lentiviral particle).
[0902] In some embodiments, the mobilization regimen includes administration of one or both of G- CSF and plerixafor/AMD3100. In various embodiments G-CSF is administered to a subject (i) daily on the four days prior to administration of a first dose of the particle (e.g., lentiviral particle); (ii) on the day of administration of a first dose of the particle (e.g., lentiviral particle); and (iii) on the day of administration of one or more subsequent doses of the particle (e.g., lentiviral particle). In various embodiments plerixafor/AMD3100 is administered to a subject (i) on the day prior to administration of a first dose of the particle (e.g., lentiviral particle) and (ii) on the day of administration of a first dose of the particle (e.g., lentiviral particle). [0903] In some embodiments, the at least one mobilizing agent is administered once daily at a dose that is, or is at least, 0.1, 1.0, 10, 20, 30, 40, 50, 75, 100, 150, or 200 pg/kg. In various embodiments, a daily dose of the at least one mobilizing agent has a range having a lower bound of 0.1 pg/kg/day, 1.0 pg/kg/day, 10 pg/kg/day, 20 pg/kg/day, 30 pg/kg/day, 40 pg/kg/day, 50 pg/kg/day, or 75 pg/kg/day and an upper bound of 100 pg/kg/day, 150 pg/kg/day, or 200 ug/kg/day. In various embodiments the at least one mobilizing agent is administered once daily at a dose that is, or is at least, 1 mg/kg , 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg. In various embodiments, a daily dose of the at least one stem cell mobilizing agent has a range having a lower bound of 1 mg/kg/day, 2 mg/kg/day, 3 mg/kg/day, 4 mg/kg/day, 5 mg/kg/day, or 7.5 mg/kg/day and an upper bound of 10 mg/kg/day, 15 mg/kg/day, or 20 mg/kg/day.
[0904] In various embodiments G-CSF is administered once daily at a dose that is, or is at least, 10 pg/kg, 20 pg/kg, 30 pg/kg, 40 pg/kg, 50 pg/kg, 75 pg/kg, 100 pg/kg, 150 pg/kg, or 200 pg/kg. In various embodiments, a daily dose of G-CSF has a range having a lower bound of 10 pg/kg/day, 20 pg/kg/day, 30 pg/kg/day, 40 pg/kg/day, 50 pg/kg/day, or 75 pg/kg/day and an upper bound of 100 pg/kg/day, 150 pg/kg/day, or 200 pg/kg/day. In various embodiments plerixafor/AMD3100 is administered once daily at a dose that is, or is at least, 1 mg/kg , 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg. In various embodiments, a daily dose of G-CSF has a range having a lower bound of 1 mg/kg/day, 2 mg/kg/day, 3 mg/kg/day, 4 mg/kg/day, 5 mg/kg/day, or 7.5 mg/kg/day and an upper bound of 10 mg/kg/day, 15 mg/kg/day, or 20 mg/kg/day.
[0905] In some embodiments, G-CSF may be administered daily as a dose of 0.5-16 pg/kg (e.g., 5- 16 pg/kg or 10-16 pg/kg) for 1-10 days (e.g., 1-7 days, or particularly, 1-3 days). In another example, G- CSF may be administered to a healthy donor at a dosage of 10-16 pg/kg daily for up to seven days. Three or four days of treatment may be sufficient when the peripheral blood collections are combined with apheresis starting on, e.g., day 4. In another example, G-CSF may be administered at a dosage of 10 pg/kg daily for four days with apheresis starting on, e.g., day 4. The most commonly used dosage of G- CSF in healthy donors is 10 pg/kg body weight daily with leukapheresis starting on day 5 onward until collection of an adequate number of stem cells (e.g., collections can be taken once or twice daily for 1 to 4 days, such as 1 or 2 days). G-CSF may be administered subcutaneously or intravenously. Methods of G-CSF administration and dosage are described in Juttner et al. (Blood 89:2233-2258, 1997), Kroschinsky et al. (Haematologica 90:1556-1671, 2005), U.S. Pat. No. 6,162,427, 2005/0186182, WO 2010051335, and WO 2005014023, all of which are incorporated herein by reference in their entireties. If cyclophosphamide is administered for mobilization, G-CSF is usually started 2-5 days after completion of cyclophosphamide infusion. Methods of administering combined mobilization agents of G-CSF and one or more chemotherapeutic agents are described in Andre et al. (Transfusion 43:50-57, 2003), Ataergin et al. (Am. J. Hematol. 83:644-648, 2008), and Demirer et al. (Br. J. Haematol. 116:468-474, 2002), all of which are incorporated herein by reference in their entireties. [0906] Plerixafor may be administered at a dosage of 1-300 pg/kg. At 240 pg/kg, the number of mobilized stem cells peak at around 4-10 hours after plerixafor administration. In some examples, plerixafor may be administered once or twice daily at a dose of ~l-300 pg/kg (e.g., 100-300 pg/kg or 200- 300 pg/kg) for 1-10 days (e.g., 1-5 days).
[0907] The combination of plerixafor and G-CSF for stem cell mobilization was approved by the FDA in 2008 for use in patients with non-Hodgkin's lymphoma and multiple myeloma. This combination therapy may also be used to mobilize stem cells. A typical combination therapy may include, e.g., the administration of G-CSF at ~0.5-16 pg/kg (e.g., 10 pg/kg) daily with plerixafor at 1-300 pg/kg (e.g., 240 pg/kg) given a few days (e.g., 1-3 days) after G-CSF administration. Both agents may be given together for about 2-10 days (e.g., 4 days) or until adequate hematopoietic cells are collected. Plerixafor, either alone or in combination with G-CSF, may be administered subcutaneously or intravenously.
[0908] In some embodiments, the method of mobilizing hematopoietic cells in a subject includes administering to the subject at least one mobilization agent. In some embodiments, the method of mobilizing hematopoietic cells includes administering to the subject at least one mobilization agent comprising (i) at least one heparan sulfate inhibitor and (ii) at least one of a CXCR2 agonist and a CXCR4 antagonist.
[0909] In some embodiments, the mobilized hematopoietic cells comprise KLS-CD150+CD48- cells. In some embodiments, the mobilized hematopoietic cells comprise CD34- CD 133+ cells. In some embodiments, the mobilized hematopoietic cells and/or progenitor cells comprise common myeloid progenitor cells. In some embodiments, the mobilized hematopoietic cells and/or progenitor cells comprise granulocyte/monocyte progenitor cells. In some embodiments, the mobilized hematopoietic cells and/or progenitor cells comprise megakaryocyte/erythroid progenitor cells. In some embodiments, the mobilized hematopoietic cells and/or progenitor cells comprise committed lymphoid progenitor cells. In some embodiments, the mobilized hematopoietic cells and/or progenitor cells comprise a combination of common myeloid progenitor cells, granulocyte/monocyte progenitor cells, megakaryocyte/erythroid progenitor cells. In some embodiments, the hematopoietic progenitor cells comprise CD150- CD48- CD244+ cells. In some embodiments, the hematopoietic progenitor cells comprise CD150- CD48+CD244+ cells. In some embodiments, the hematopoietic progenitor cells comprise Lin- SCA- l-c-Kit+CD34+CD16/32mid cells. In some embodiments, the hematopoietic progenitor cells comprise lin-SCA-1- c-kit+CD34- CD16/321ow cells. In some embodiments, the mobilized hematopoietic cells comprise CD34+ peripheral blood stem cells.
VII. EXEMPLARY EMBODIMENTS
[0910] Among the provided embodiments are:
[0911] 1. A targeted lipid particle, comprising a lipid bilayer, a fusogen, and a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3, wherein the fusogen and the targeting agent are exposed on the surface of the lipid bilayer.
[0912] 2. The particle of embodiment 1 , wherein the targeting agent is fused to a transmembrane domain incorporated into the lipid bilayer.
[0913] 3. The particle of embodiment 1 , wherein the targeting agent is fused to the fusogen.
[0914] 4. The particle of any of embodiments 1-3, wherein the particle is a viral particle or viruslike particle.
[0915] 5. The particle of any of embodiments 1-4, wherein the lipid bilayer is derived from a membrane of a producer cell used for producing a viral particle or virus-like particle.
[0916] 6. The particle of embodiment 4 or embodiment 5, wherein the viral particle or viruslike particle is a retroviral particle or retrovirus -like particle.
[0917] 7. The particle of any of embodiments 4-6, wherein the viral particle or virus-like particle is a lentivirus-like particle.
[0918] 8. The particle of any of embodiments 4-6, wherein the viral particle or virus-like particle is a lentiviral particle.
[0919] 9. A targeted lentiviral particle, comprising a fusogen and a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3, wherein the fusogen and the targeting agent are exposed on the surface of the lentiviral particle.
[0920] 10. The particle of any of embodiments 4-9, wherein the particle is pseudotyped with the fusogen.
[0921] 11. The particle of embodiment 9 or embodiment 10, wherein the targeting agent is fused to the fusogen.
[0922] 12. The particle of any of embodiments 1 and 3-11, wherein the targeting agent is fused to the fusogen directly.
[0923] 13. The particle of any of embodiments 1 and 3-11, wherein the targeting agent is fused to the fusogen via a linker.
[0924] 14. The particle of any of embodiments 4-10, wherein the targeting agent is fused to a transmembrane domain incorporated into the viral envelope.
[0925] 15. The particle of any of embodiments 1-14, wherein the particle is prepared by a method comprising transducing a producer cell with plasmids encoding the fusogen and targeting agent and a Gag-pol and Rev.
[0926] 16. The particle of any of embodiments 5-8 and 10-15, wherein the producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells.
[0927] 17. The particle of any of embodiments 5-8 and 10-16, wherein the producer cell is a 293T cell.
[0928] 18. The particle of any of embodiments 4-17, wherein the particle is replication defective.
[0929] 19. The particle of any of embodiments 1-6 and 8-18, wherein the particle comprises a viral nucleic acid.
[0930] 20. The particle of embodiment 19, wherein the viral nucleic acid is a retroviral nucleic acid, optionally wherein the viral nucleic acid is a lentiviral nucleic acid.
[0931] 21. The particle of any of embodiments 19-20, wherein the viral nucleic acid comprises one or more, optionally all, of the following nucleic acid sequences: 5’ LTR (e.g., comprising U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT)/central termination sequence (CTS) (e.g., DNA flap), Poly A tail sequence, a posttranscriptional regulatory element (e.g., WPRE), a Rev response element (RRE), and 3’ LTR (e.g., comprising U5 and lacking a functional U3).
[0932] 22. The particle of any of embodiments 1-7 and 10-18, wherein the particle is devoid of viral genomic nucleic acids.
[0933] 23. The particle of any of embodiments 1-22, wherein the target molecule is ASCT2.
[0934] 24. The particle of any of embodiments 1-22, wherein the target molecule is CD117.
[0935] 25. The particle of any of embodiments 1-24, wherein the fusogen is a viral fusion protein.
[0936] 26. The particle of any of embodiments 1-25, wherein the fusogen is a viral envelope protein.
[0937] 27. The particle of any of embodiments 1-26, wherein the fusogen is a VSV-G protein or a functional variant thereof.
[0938] 28. The particle of any of embodiments 1-26, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
[0939] 29. A targeted lipid particle, comprising a lipid bilayer, a fusogen, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof and is exposed on the surface of the lipid bilayer.
[0940] 30. The particle of embodiment 28 or 29, wherein the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild-type BaEV inhibitory R peptide.
[0941] 31. The particle of embodiment 28, 29 or 30, wherein the fusogen is set forth in any of SEQ ID NO:254-260. [0942] 32. The particle of embodiment 28 or 29, wherein the fusogen is set forth in SEQ ID NO:261 (BaEVRLess).
[0943] 33. The particle of embodiment 29, wherein the fusogen is set forth in SEQ ID NO:262 (BaEVTR).
[0944] 34. The particle of any of embodiments 1-26, wherein the fusogen is a Cocal virus G protein or a functional variant thereof.
[0945] 35. The particle of any of embodiments 1-26, wherein the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
[0946] 36. The particle of any of embodiments 1-26, wherein the fusogen is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof.
[0947] 37. The particle of any of embodiments 1-26 and 36, wherein the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des- petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
[0948] 38. The particle of any of embodiments 1-26 and 36, wherein the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
[0949] 39. The particle of any of embodiments 1-26, 36, and 38, wherein the fusogen is a Nipah virus fusion protein or a functional variant thereof.
[0950] 40. The particle of any of embodiments 1-26, 36, 38, and 39, wherein the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
[0951] 41. The particle of embodiment 40, wherein the targeting agent is fused to the NiV-G protein or biologically active portion thereof.
[0952] 42. The particle of embodiment 40 or embodiment 41, wherein the targeting agent is fused to the C-terminus of the NiV-G protein or biologically active portion thereof.
[0953] 43. The particle of any of embodiments 40-42, wherein the NiV-G or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof.
[0954] 44. The particle of any of embodiments 40-43, wherein the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4.
[0955] 45. The particle of any of embodiments 40-44, wherein the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4. [0956] 46. The particle of any of embodiments 40-45, wherein the NiV-G protein or biologically active portion thereof does not include an initial methionine.
[0957] 47. The particle of any of embodiments 40-46, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42.
[0958] 48. The particle of any of embodiments 40-47, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
[0959] 49. The particle of any of embodiments 1-48, wherein the fusogen comprises one or more modifications to reduce binding to its native receptor.
[0960] 50. The particle of any of embodiments 40-46 and 49, wherein the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
[0961] 51. The particle of any of embodiments 40-46, 49, and 50, wherein the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
[0962] 52. The particle of any of embodiments 40-46 and 49-51, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17.
[0963] 53. The particle of any of embodiments 40-46 and 49-52, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17.
[0964] 54. The particle of any of embodiments 40-46 and 49-51, wherein the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18.
[0965] 55. The particle of any of embodiments 40-46, 49-51, and 54, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
[0966] 56. The particle of any of embodiments 40-55, wherein the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.
[0967] 57. The particle of any of embodiments 40-56, wherein the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30. [0968] 58. The particle of any of embodiments 40-57, wherein the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV- F protein set forth in SEQ ID NO:30.
[0969] 59. The particle of any of embodiments 40-58, wherein the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30.
[0970] 60. The particle of any of embodiments 40-59, wherein the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16.
[0971] 61. The particle of any of embodiments 40-60, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16.
[0972] 62. The particle of any of embodiments 40-59, wherein the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21.
[0973] 63. The particle of any of embodiments 40-59 and 62, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
[0974] 64. The particle of any of embodiments 40-46, 49-51, 54-59, 62, and 63, wherein the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
[0975] 65. The particle of any of embodiments 1-64, wherein the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule.
[0976] 66. The particle of any of embodiments 1-65, wherein the targeting agent is a single domain antibody that binds to the target molecule.
[0977] 67. The particle of any of embodiments 1-66, wherein the targeting agent is a VHH that binds to the target molecule.
[0978] 68. The particle of any of embodiments 1-66, wherein the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
[0979] 69. The particle of any of embodiments 1-68, wherein the target molecule is expressed on a target cell.
[0980] 70. The particle of any of embodiments 1-69, wherein the particle comprises an exogenous agent for delivery to a target cell. [0981] 71. The particle of any one of embodiments 1-69, wherein the particle comprises a fusion protein between a viral structural protein and an exogenous agent.
[0982] 72. The particle of embodiment 71, wherein the fusion protein is a cleavable fusion protein comprising a cleavable linker positioned between the viral structural protein and the exogenous agent.
[0983] 73. The particle of embodiment 71 or 72, wherein the fusion protein comprises one or more nuclear localization sequences.
[0984] 74. The particle of embodiment 71 or 72, wherein the fusion protein comprises one or more nuclear export sequences.
[0985] 75. The particle of embodiment 70 or embodiment 71, wherein the target molecule is expressed on the target cell.
[0986] 76. The particle of any of embodiments 70-75, wherein the exogenous agent is present in the lumen.
[0987] 77. The particle of any of embodiments 70-76, wherein the exogenous agent is a protein or a nucleic acid.
[0988] 78. The particle of any of embodiments 70-77, wherein the exogenous agent is or encodes a therapeutic agent or a diagnostic agent.
[0989] 79. The particle of any of embodiments 70-78, wherein the exogenous agent is or encodes a factor associated with gene editing.
[0990] 80. The particle of any of embodiments 70-79, wherein the exogenous agent is or encodes a genome-modifying protein for gene editing a target gene encoding an endogenous protein.
[0991] 81. The particle of embodiment 80, wherein the genome-modifying protein is associated with gene editing by a sequence-specific nuclease, a CRISPR-associated transposase (CAST), prime editing, or Programmable Addition via Site-specific Targeting Elements (PASTE).
[0992] 82. The particle of embodiment 80 or embodiment 81, wherein the genome-modifying protein is a sequence-specific nuclease.
[0993] 83. The particle of embodiment 81 or embodiment 82, wherein the sequence-specific nuclease is selected from the group consisting of an RNA-guided nuclease, a meganuclease, a transcription activator-like effector nuclease (TALEN), and a zinc-finger nuclease (ZFN).
[0994] 84. The particle of any of embodiments 81-83, wherein the sequence-specific nuclease is an RNA-guided nuclease.
[0995] 85. The particle of embodiment 83 or embodiment 84, wherein the RNA-guided nuclease is TnpB.
[0996] 86. The particle of embodiment 83 or embodiment 84, wherein the RNA-guided nuclease comprises a Cas nuclease and a guide RNA (CRISPR-Cas combination). [0997] 87. The particle of embodiment 86, wherein the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease.
[0998] 88. The particle of embodiment 86 or embodiment 87, wherein the Cas nuclease is a Type II or Type V Cas protein.
[0999] 89. The particle of any of embodiments 80-84 and 86-88, wherein the genome-modifying protein is selected from the group consisting of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxl l, Csyl, Csy2, Csy3, Mad7.
[1000] 90. The particle of any of embodiments 86-89, wherein the Cas is a Cas9 or a Casl2.
[1001] 91. The particle of any of embodiments 70-81, wherein the exogenous agent is or encodes a factor associated with base editing or prime editing (e.g., target-primed reverse transcription (TPRT)).
[1002] 92. The particle of any of embodiments 70-81 and 91, wherein the exogenous agent is or encodes a transposase, integrase, or recombinase.
[1003] 93. The particle of any of embodiments 70-81 and 91, wherein the exogenous agent is or encodes a DNA polymerase, RNA polymerase, or reverse-transcriptase.
[1004] 94. The particle of any of embodiments 70-93, wherein the exogenous agent is for use in gene therapy to correct a genetic deficiency in the target cell.
[1005] 95. The particle of embodiment 94, wherein the exogenous agent is a nucleic acid comprising a payload gene for correcting the genetic deficiency.
[1006] 96. The particle of any of embodiments 70-78, wherein the exogenous agent is or encodes a membrane protein.
[1007] 97. The particle of embodiment 96, wherein the membrane protein is an antigen receptor.
[1008] 98. The particle of embodiment 97, wherein the antigen receptor binds to an antigen expressed on cells associated with a disease or condition.
[1009] 99. The particle of embodiment 98, wherein the disease or condition is a cancer.
[1010] 100. The particle of any of embodiments 97-99, wherein the antigen receptor binds to an antigen expressed on tumor cells.
[1011] 101. The particle of any of embodiments 97-100, wherein the antigen receptor is a chimeric antigen receptor (CAR).
[1012] 102. The particle of any of embodiments 97-100, wherein the antigen receptor is an engineered T cell receptor (TCR).
[1013] 103. The particle of any of embodiments 69-102, wherein the target cell is a hematopoietic cell.
[1014] 104. The particle of any of embodiments 69-103, wherein the target cell is CD34+. [1015] 105. The particle of any of embodiments 69-104, wherein the target cell is a CD34+ progenitor cell.
[1016] 106. The particle of any of embodiments 69-105, wherein the target cell is a hematopoietic stem cell.
[1017] 107. The particle of any of embodiments 70-106, wherein the delivery to target cells expressing the target molecule is increased by or by greater than 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5- fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5- fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, or more compared to delivery to non-target cells not expressing the target molecule.
[1018] 108. A polynucleotide comprising a nucleic acid sequence encoding a fusogen and a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3.
[1019] 109. The polynucleotide of embodiment 108, wherein the polynucleotide encodes a transmembrane domain that is fused to the targeting agent.
[1020] 110. The polynucleotide of embodiment 108, wherein the targeting agent is fused to the fusogen.
[1021] 111. The polynucleotide of embodiment 108 or embodiment 110, wherein the targeting agent is fused to the fusogen directly.
[1022] 112. The polynucleotide of any of embodiments 108, 110, and 111, wherein the targeting agent is fused to the fusogen via a linker.
[1023] 113. The polynucleotide of any of embodiments 108-112, wherein the target molecule is ASCT2.
[1024] 114. The polynucleotide of any of embodiments 108-112, wherein the target molecule is CD117.
[1025] 115. The polynucleotide of any of embodiments 108-114, wherein the fusogen is a viral fusion protein.
[1026] 116. The polynucleotide of any of embodiments 108-115, wherein the fusogen is a viral envelope protein.
[1027] 117. The polynucleotide of any of embodiments 108-116, wherein the fusogen is a VSV-G protein or a functional variant thereof.
[1028] 118. The polynucleotide of any of embodiments 108-116, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
[1029] 119. A polynucleotide comprising a nucleic acid sequence encoding a fusogen, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof. [1030] 120. The polynucleotide of embodiment 118 or 119, wherein the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild-type BaEV inhibitory R peptide.
[1031] 121. The particle of any of embodiments 118-120, wherein the fusogen is set forth in any of SEQ ID NO:254-260.
[1032] 122. The particle of embodiment 118 or 119, wherein the fusogen is set forth in SEQ ID NO:261 (BaEVRLess).
[1033] 123. The particle of embodiment 118 or 119, wherein the fusogen is set forth in SEQ ID NO:262 (BaEVTR).
[1034] 124. The polynucleotide of any of embodiments 108-116, wherein the fusogen is a Cocal virus G protein or a functional variant thereof.
[1035] 125. The polynucleotide of any of embodiments 108-116, wherein the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
[1036] 126. The polynucleotide of any of embodiments 108-116, wherein the fusogen is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof.
[1037] 127. The polynucleotide of any of embodiments 108-116 and 126, wherein the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
[1038] 128. The polynucleotide of any of embodiments 108-116 and 126, wherein the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
[1039] 129. The polynucleotide of any of embodiments 108-116, 126, and 128, wherein the fusogen is a Nipah virus fusion protein or a functional variant thereof.
[1040] 130. The polynucleotide of any of embodiments 108-116, 126, 128, and 129, wherein the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
[1041] 131. The polynucleotide of embodiment 130, wherein the targeting agent is fused to the NiV- G protein or biologically active portion thereof.
[1042] 132. The polynucleotide of embodiment 130 or embodiment 131, wherein the targeting agent is fused to the C-terminus of the NiV-G protein or biologically active portion thereof.
[1043] 133. The polynucleotide of any of embodiments 130-132, wherein the NiV-G or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof. [1044] 134. The polynucleotide of any of embodiments 130-133, wherein the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4.
[1045] 135. The polynucleotide of any of embodiments 130-134, wherein the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4
[1046] 136. The polynucleotide of any of embodiments 130-135, wherein the NiV-G protein or biologically active portion thereof does not include an initial methionine.
[1047] 137. The polynucleotide of any of embodiments 130-136, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42.
[1048] 138. The polynucleotide of any of embodiments 130-137, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
[1049] 139. The polynucleotide of any of embodiments 108-138, wherein the fusogen comprises one or more modifications to reduce binding to its native receptor.
[1050] 140. The polynucleotide of any of embodiments 130-136 and 139, wherein the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
[1051] 141. The polynucleotide of any of embodiments 130-136, 139, and 140, wherein the NiV- G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
[1052] 142. The polynucleotide of any of embodiments 130-136 and 139-141, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17.
[1053] 143. The polynucleotide of any of embodiments 130-136 and 139-142, wherein the NiV- G protein or biologically active portion thereof is set forth in SEQ ID NO: 17.
[1054] 144. The polynucleotide of any of embodiments 130-136 and 139-141, wherein the NiV- G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18.
[1055] 145. The polynucleotide of any of embodiments 130-136, 139-141, and 144, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18. [1056] 146. The polynucleotide of any of embodiments 130-145, wherein the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.
[1057] 147. The polynucleotide of any of embodiments 130-146, wherein the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30.
[1058] 148. The polynucleotide of any of embodiments 130-147, wherein the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30.
[1059] 149. The polynucleotide of any of embodiments 130-148, wherein the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30.
[1060] 150. The polynucleotide of any of embodiments 130-149, wherein the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16.
[1061] 151. The polynucleotide of any of embodiments 130-150, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16.
[1062] 152. The polynucleotide of any of embodiments 130-149, wherein the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21.
[1063] 153. The polynucleotide of any of embodiments 130-149 and 152, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
[1064] 154. The polynucleotide of any of embodiments 130-136, 139-141, 144-149, 152, and 153, wherein the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
[1065] 155. The polynucleotide of any of embodiments 108-154, wherein the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule.
[1066] 156. The polynucleotide of any of embodiments 108-155, wherein the targeting agent is a single domain antibody that binds to the target molecule. [1067] 157. The polynucleotide of any of embodiments 108-156, wherein the targeting agent is a VHH that binds to the target molecule.
[1068] 158. The polynucleotide of any of embodiments 108-156, wherein the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
[1069] 159. The polynucleotide of any of embodiments 108-158, wherein the polynucleotide is codon optimized.
[1070] 160. The polynucleotide of any of embodiments 108-159, wherein the polynucleotide comprises at least one promoter that is operatively linked to control expression of the targeting agent and fusogen.
[1071] 161. The polynucleotide of embodiment 160, wherein the promoter is a constitutive promoter.
[1072] 162. The polynucleotide of embodiment 160, wherein the promoter is an inducible promoter.
[1073] 163. A plasmid, comprising the polynucleotide of any of embodiments 108-162 or 420.
[1074] 164. The plasmid of embodiment 163, wherein the plasmid comprises one or more nucleic acids encoding proteins for lentivirus production.
[1075] 165. A vector, comprising the polynucleotide of any of embodiments 108-162 or 420.
[1076] 166. A cell comprising the polynucleotide of any of embodiments 108-162 or 420, the plasmid of embodiment 163 or embodiment 164, or the vector of embodiment 165.
[1077] 167. A cell comprising (i) a first polynucleotide comprising a nucleic acid sequence encoding a fusogen and (ii) a second polynucleotide comprising a nucleic acid sequence encoding a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3.
[1078] 168. The cell of embodiment 167, wherein the second polynucleotide encodes a transmembrane domain that is fused to the targeting agent.
[1079] 169. A cell comprising (i) a first polynucleotide encoding a nucleic acid sequence encoding a fusogen, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
[1080] 170. The cell of any of embodiments 167-169, wherein the first and/or second polynucleotide is a plasmid.
[1081] 171. The cell of embodiment 170, wherein the plasmid of the first and/or second polynucleotide comprises one or more nucleic acids encoding proteins for lentivirus production.
[1082] 172. The cell of any of embodiments 167-169, wherein the first and/or second polynucleotide is a vector.
[1083] 173. The cell of any of embodiments 167-172, wherein the first and/or second polynucleotide is codon optimized. [1084] 174. The cell of any of embodiments 167-173, wherein: the first polynucleotide comprises at least one promoter that is operatively linked to control expression of the fusogen; and/or the second polynucleotide comprises at least one promoter that is operatively linked to control expression of the targeting agent.
[1085] 175. The cell of embodiment 174, wherein the promoter of the first and/or second polynucleotide is a constitutive promoter.
[1086] 176. The cell of embodiment 174, wherein the promoter of the first and/or second polynucleotide is an inducible promoter.
[1087] 177. The cell of any of embodiments 166-176, wherein the cell is a producer cell used for producing a viral particle or virus-like particle.
[1088] 178. The cell of embodiment 177, wherein the viral particle or virus-like particle is a retroviral particle or retrovirus-like particle.
[1089] 179. The cell of embodiment 177 or embodiment 178, wherein the viral particle or viruslike particle is a lentiviral particle or lentivirus-like particle.
[1090] 180. The cell of any of embodiments 166-179, wherein the cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells.
[1091] 181. The cell of any of embodiments 166-180, wherein the cell is a 293T cell.
[1092] 182. The cell of any of embodiments 166-181, wherein the cell comprises a viral nucleic acid.
[1093] 183. The cell of embodiment 182, wherein the viral nucleic acid is a retroviral nucleic acid
[1094] 184. The cell of embodiment 182 or embodiment 183, wherein the viral nucleic acid is a lentiviral nucleic acid.
[1095] 185. The cell of any of embodiments 182-184, wherein the viral nucleic acid lacks one or more genes involved in viral replication.
[1096] 186. The cell of any of embodiments 182-185, wherein the viral nucleic acid comprises a nucleic acid sequence encoding a viral packaging protein selected from one or more of Gag, Pol, and Rev.
[1097] 187. The cell of any of embodiments 182-186, wherein the viral nucleic acid comprises one or more, optionally all, of the following nucleic acid sequences: 5’ LTR (e.g., comprising U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT)/central termination sequence (CTS) (e.g., DNA flap), Poly A tail sequence, a posttranscriptional regulatory element (e.g., WPRE), a Rev response element (RRE), and 3’ LTR (e.g., comprising U5 and lacking a functional U3).
[1098] 188. The cell of any of embodiments 166-187, wherein the target molecule is ASCT2.
[1099] 189. The cell of any of embodiments 166-187, wherein the target molecule is CD117.
[1100] 190. The cell of any of embodiments 166-189, wherein the fusogen is a viral fusion protein.
[1101] 191. The cell of any of embodiments 166-190, wherein the fusogen is a viral envelope protein.
[1102] 192. The cell of any of embodiments 166-191, wherein the fusogen is a VSV-G protein or a functional variant thereof.
[1103] 193. The cell of any of embodiments 166-191, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
[1104] 194. The cell of any of embodiments 166-191 and 193, wherein the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild-type BaEV inhibitory R peptide.
[1105] 195. The cell of embodiment 168 or 169, wherein the fusogen is set forth in any of SEQ ID NO:254-260.
[1106] 196. The cell of embodiment 168, 169, or 195, wherein the fusogen is set forth in SEQ ID NO:261 (BaEVRLess).
[1107] 197. The cell of embodiment 168, 169 or 195, wherein the fusogen is set forth in SEQ ID NO:262 (BaEVTR).
[1108] 198. The cell of any of embodiments 166-191, wherein the fusogen is a Cocal virus G protein or a functional variant thereof.
[1109] 199. The cell of any of embodiments 166-191, wherein the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
[1110] 200. The cell of any of embodiments 166-191, wherein the fusogen is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof.
[1111] 201. The cell of any of embodiments 166-191 and 200, wherein the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
[1112] 202. The cell of any of embodiments 166-191 and 200, wherein the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
[1113] 203. The cell of any of embodiments 166-191, 200, and 202, wherein the fusogen is a Nipah virus fusion protein or a functional variant thereof. [1114] 204. The cell of any of embodiments 166-191, 200, 202, and 203, wherein the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
[1115] 205. The cell of embodiment 204, wherein the NiV-G or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof.
[1116] 206. The cell of embodiment 204 or embodiment 205, wherein the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4.
[1117] 207. The cell of any of embodiments 204-206, wherein the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4.
[1118] 208. The cell of any of embodiments 204-207, wherein the NiV-G protein or biologically active portion thereof does not include an initial methionine.
[1119] 209. The cell of any of embodiments 204-208, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42.
[1120] 210. The cell of any of embodiments 204-209, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
[1121] 211. The cell of any of embodiments 166-210, wherein the fusogen comprises one or more modifications to reduce binding to its native receptor.
[1122] 212. The cell of any of embodiments 204-208 and 211, wherein the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
[1123] 213. The cell of any of embodiments 204-208, 211, and 212, wherein the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
[1124] 214. The cell of any of embodiments 204-208 and 211-213, wherein the NiV-G protein or biologically active portion thereof comprises the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17.
[1125] 215. The cell of any of embodiments 204-208 and 211-214, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17. [1126] 216. The cell of any of embodiments 204-208 and 211-213, wherein the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18.
[1127] 217. The cell of any of embodiments 204-208, 211-213, and 216, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
[1128] 218. The cell of any of embodiments 204-217, wherein the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.
[1129] 219. The cell of any of embodiments 204-218, wherein the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30.
[1130] 220. The cell of any of embodiments 204-219, wherein the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV- F protein set forth in SEQ ID NO:30.
[1131] 221. The cell of any of embodiments 204-220, wherein the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525- 546 of SEQ ID NO:30.
[1132] 222. The cell of any of embodiments 204-221, wherein the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16.
[1133] 223. The cell of any of embodiments 204-222, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16.
[1134] 224. The cell of any of embodiments 204-221, wherein the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21.
[1135] 225. The cell of any of embodiments 204-221 and 224, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
[1136] 226. The cell of any of embodiments 204-208, 211-213, 216-221, 224, and 225, wherein the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21. [1137] 227. The cell of any of embodiments 166-226, wherein the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule.
[1138] 228. The cell of any of embodiments 166-227, wherein the targeting agent is a single domain antibody that binds to the target molecule.
[1139] 229. The cell of any of embodiments 166-228, wherein the targeting agent is a VHH that binds to the target molecule.
[1140] 230. The cell of any of embodiments 166-228, wherein the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
[1141] 231. A method of making a targeted lipid particle, comprising: (a) introducing into a source cell the polynucleotide of any of embodiments 108-162 or 420, the plasmid of embodiment 163 or embodiment 164, or the vector of embodiment 165; (b) culturing the source cell under conditions for producing a lipid particle comprising a lipid bilayer and the targeting agent and fusogen exposed on the surface of the lipid bilayer; and (c) separating, enriching, or purifying the lipid particle from the source cell, thereby making the lipid particle.
[1142] 232. A method of making a targeted lipid particle, comprising: (a) introducing into a source cell (i) a first polynucleotide comprising a nucleic acid sequence encoding a fusogen and (ii) a second polynucleotide comprising a nucleic acid sequence encoding a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3; (b) culturing the source cell under conditions for producing a lipid particle comprising a lipid bilayer and the targeting agent and fusogen exposed on the surface of the lipid bilayer; and (c) separating, enriching, or purifying the lipid particle from the source cell, thereby making the lipid particle.
[1143] 233. The method of embodiment 232, wherein the second polynucleotide encodes a transmembrane domain that is fused to the targeting agent.
[1144] 234. The method of embodiment 232 or embodiment 233, wherein the first and/or second polynucleotide is a plasmid.
[1145] 235. The method of embodiment 234, wherein the plasmid of the first and/or second polynucleotide comprises one or more nucleic acids encoding proteins for lentivirus production.
[1146] 236. The method of embodiment 232 or embodiment 233, wherein the first and/or second polynucleotide is a vector.
[1147] 237. The method of any of embodiments 226-230, wherein the first and/or second polynucleotide is codon optimized.
[1148] 238. The method of any of embodiments 226-231, wherein: the first polynucleotide comprises at least one promoter that is operatively linked to control expression of the fusogen; and/or the second polynucleotide comprises at least one promoter that is operatively linked to control expression of the targeting agent.
[1149] 239. The method of embodiment 232, wherein the promoter of the first and/or second polynucleotide is a constitutive promoter.
[1150] 240. The method of embodiment 232, wherein the promoter of the first and/or second polynucleotide is an inducible promoter.
[1151] 241. The method of any of embodiments 225-234, wherein the source cell is a mammalian cell.
[1152] 242. The method of any of embodiments 225-235, wherein the source cell is a producer cell used for producing a viral particle or virus-like particle.
[1153] 243. The method of embodiment 236, wherein the particle is a viral particle or virus-like particle.
[1154] 244. The method of embodiment 236 or embodiment 237, wherein the viral particle or virus- like particle is a retroviral particle or retrovirus-like particle.
[1155] 245. The method of any of embodiments 236-238, wherein the viral particle or virus-like particle is a lentivirus-like particle.
[1156] 246. The method of any of embodiments 236-238, wherein the viral particle or virus-like particle is a lentiviral particle.
[1157] 247. The method of any of embodiments 225-240, wherein the particle is pseudotyped with the fusogen.
[1158] 248. The method of any of embodiments 236-241, wherein the producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells, optionally wherein the producer cell is a 293T cell.
[1159] 249. The method of any of embodiments 231-248, wherein the method comprises transducing the source cell with packaging plasmids encoding a Gag-pol and Rev.
[1160] 250. The method of any of embodiments 231-249, wherein the target molecule is ASCT2.
[1161] 251. The method of any of embodiments 231-249, wherein the target molecule is CD117.
[1162] 252. The method of any of embodiments 231-251, wherein the fusogen is a viral fusion protein.
[1163] 253. The method of any of embodiments 231-252, wherein the fusogen is a viral envelope protein. [1164] 254. The method of any of embodiments 231-253, wherein the fusogen is a VSV-G protein or a functional variant thereof.
[1165] 255. The method of any of embodiments 231-253, wherein the fusogen is a Cocal virus G protein or a functional variant thereof.
[1166] 256. The method of any of embodiments 231-253, wherein the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
[1167] 257. The method of any of embodiments 231-253, wherein the fusogen is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof.
[1168] 258. The method of any of embodiments 231-253 and 257, wherein the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
[1169] 259. The method of any of embodiments 231-253 and 257, wherein the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
[1170] 260. The method of any of embodiments 231-253, 257, and 259, wherein the fusogen is a Nipah virus fusion protein or a functional variant thereof.
[1171] 261. The method of any of embodiments 231-253, 257, 259, and 260, wherein the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
[1172] 262. The method of embodiment 261, wherein the NiV-G or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof.
[1173] 263. The method of embodiment 261 or embodiment 262, wherein the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4.
[1174] 264. The method of any of embodiments 261-263, wherein the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4.
[1175] 265. The method of any of embodiments 261-264, wherein the NiV-G protein or biologically active portion thereof does not include an initial methionine.
[1176] 266. The method of any of embodiments 261-265, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42. [1177] 267. The method of any of embodiments 261-266, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
[1178] 268. The method of any of embodiments 231-267, wherein the fusogen comprises one or more modifications to reduce binding to its native receptor.
[1179] 269. The method of any of embodiments 261-265 and 268, wherein the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
[1180] 270. The method of any of embodiments 261-265, 268, and 269, wherein the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
[1181] 271. The method of any of embodiments 261-265 and 268-270, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17.
[1182] 272. The method of any of embodiments 261-265 and 268-271, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17.
[1183] 273. The method of any of embodiments 261-265 and 268-270, wherein the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18.
[1184] 274. The method of any of embodiments 261-265, 268-270, and 273, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
[1185] 275. The method of any of embodiments 261-274, wherein the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.
[1186] 276. The method of any of embodiments 261-275, wherein the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30.
[1187] 277. The method of any of embodiments 261-276, wherein the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30. [1188] 278. The method of any of embodiments 261-277, wherein the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30.
[1189] 279. The method of any of embodiments 261-278, wherein the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16.
[1190] 280. The method of any of embodiments 261-279, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16.
[1191] 281. The method of any of embodiments 261-278, wherein the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21.
[1192] 282. The method of any of embodiments 261-278 and 281, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
[1193] 283. The method of any of embodiments 261-265, 268-270, 273-278, 281, and 282, wherein the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
[1194] 284. The method of any of embodiments 231-283, wherein the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule.
[1195] 285. The method of any of embodiments 231-284, wherein the targeting agent is a single domain antibody that binds to the target molecule.
[1196] 286. The method of any of embodiments 231-285, wherein the targeting agent is a VHH that binds to the target molecule.
[1197] 287. The method of any of embodiments 231-285, wherein the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
[1198] 288. A targeted lipid particle produced by the method of any of embodiments 231-287 and 418.
[1199] 289. A targeted lentiviral particle produced by the method of any of embodiments 231, 241- 287 and 418.
[1200] 290. A composition comprising a plurality of the lipid particle of any of embodiments 1-8, 10-107, 288 and 419. [1201] 291. A composition comprising a plurality of the lenti viral particle of any of embodiments 8- 107, 289 and 419.
[1202] 292. The composition of embodiment 290 or embodiment 291, wherein the composition further comprises a pharmaceutically acceptable excipient.
[1203] 293. A method of transducing a target cell, the method comprising contacting a target cell with the lentiviral particle of any of embodiments 8-107, 289, and 419, or the composition of embodiment 291 or embodiment 292.
[1204] 294. The method of embodiment 293, wherein the particle comprises an exogenous agent, and the transduction introduces the exogenous agent into the target cell.
[1205] 295. A method of delivering an exogenous agent to a target cell, the method comprising contacting a target cell with the particle of any of embodiments 70-107, 288, 289, and 419, or the composition of any of embodiments 290-292, the particle or plurality of particles comprising an exogenous agent for delivery to the target cell.
[1206] 296. The method of any of embodiments 293-295, wherein the contacting is in vitro or ex vivo.
[1207] 297. The method of any of embodiments 293-295, wherein the contacting is in vivo in a subject.
[1208] 298. A method of delivering an exogenous agent to a target cell in a subject, comprising administering to a subject the particle of any of embodiments 70-107, 288, 289, and 419, or the composition of any of embodiments 290-292, the particle or plurality of particles comprising an exogenous agent for delivery to a target cell in the subject.
[1209] 299. The method of any of embodiments 294-298, wherein the exogenous agent is or encodes a therapeutic agent or a diagnostic agent.
[1210] 300. The method of any of embodiments 297-299, wherein the exogenous agent is for treating a disease or condition in the subject.
[1211] 301. A method of treating a disease or condition in a subject, comprising administering to a subject the particle of any of embodiments 70-107, 288, 289, or 419, or the composition of any of embodiments 290-292, the particle or plurality of particles comprising an exogenous agent for treating a disease or condition in the subject.
[1212] 302. The method of any of embodiments 297-301, wherein the method comprises administering to the subject one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood.
[1213] 303. The method of any of embodiments 297-301, wherein the subject has previously been administered one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood. [1214] 304. A method of treating a disease or condition in a subject, comprising administering to a subject: (a) one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood; and (b) a lipid particle comprising a lipid bilayer, a fusogen exposed on the surface of the lipid bilayer, and an exogenous agent for treating a disease or condition in the subject.
[1215] 305. The method of embodiment 302 or embodiment 304, wherein the administration of at least one of the one or more agents that stimulate mobilization is initiated prior to the administration of the particle.
[1216] 306. A method of treating a disease or condition in a subject, comprising administering to a subject a lipid particle comprising a lipid bilayer, a fusogen exposed on the surface of the lipid bilayer, and an exogenous agent for treating a disease or condition in the subject, wherein the subject has previously been administered one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood.
[1217] 307. The method of any of embodiments 301-306, wherein the exogenous agent is for delivery to a target cell for treating the disease or condition in the subject.
[1218] 308. The method of any of embodiments 295-307, wherein the particle is a viral particle or virus-like particle.
[1219] 309. The method of any of embodiments 293-308, wherein the lipid bilayer is derived from a membrane of a producer cell used for producing a viral particle or virus-like particle.
[1220] 310. The method of any of embodiments 293-309, wherein the particle is prepared by a method comprising transducing a producer cell with plasmids encoding the fusogen and a Gag-pol and Rev.
[1221] 311. The method of any of embodiments 308-310, wherein the viral particle or virus-like particle is a retroviral particle or retrovirus-like particle.
[1222] 312. The method of any of embodiments 308-311, wherein the viral particle or virus-like particle is a lentivirus-like particle.
[1223] 313. The method of any of embodiments 308-311, wherein the viral particle or virus-like particle is a lentiviral particle.
[1224] 314. The method of any of embodiments 293-303, 305, and 307-313, wherein the particle is pseudotyped with the fusogen.
[1225] 315. The method of any of embodiments 309-314, wherein the producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells. [1226] 316. The method of any of embodiments 309-315, wherein the producer cell is a 293T cell.
[1227] 317. The method of any of embodiments 293-303, 305, and 307-316, wherein the particle is replication defective.
[1228] 318. The method of any of embodiments 293-311 and 313-317, wherein the particle comprises a viral nucleic acid.
[1229] 319. The method of embodiment 318, wherein the viral nucleic acid is a retroviral nucleic acid.
[1230] 320. The method of embodiment 318 or embodiment 319, wherein the viral nucleic acid is a lentiviral nucleic acid.
[1231] 321. The method of any of embodiments 318-320, wherein the viral nucleic acid comprises one or more, optionally all, of the following nucleic acid sequences: 5’ LTR (e.g., comprising U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT)/central termination sequence (CTS) (e.g., DNA flap), Poly A tail sequence, a posttranscriptional regulatory element (e.g., WPRE), a Rev response element (RRE), and 3’ LTR (e.g., comprising U5 and lacking a functional U3).
[1232] 322. The method of any of embodiments 295-312 and 314-317, wherein the particle is devoid of viral genomic nucleic acids.
[1233] 323. The method of any of embodiments 293-322, wherein the fusogen is a viral fusion protein.
[1234] 324. The method of any of embodiments 293-323, wherein the fusogen is a viral envelope protein.
[1235] 325. The method of any of embodiments 293-324, wherein the fusogen is a VSV-G protein or a functional variant thereof.
[1236] 326. The method of any of embodiments 293-324, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
[1237] 327. The method of any of embodiments 293-324 and 326, wherein the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild-type BaEV inhibitory R peptide.
[1238] 328. The method of any of embodiments 293-324, 326, and 326, wherein the fusogen is set forth in any of SEQ ID NO:254-260.
[1239] 329. The method of any of embodiments 293-324 and 326, wherein the fusogen is set forth in SEQ ID NO:261 (BaEVRLess).
[1240] 330. The method of any of embodiments 293-324 and 326, wherein the fusogen is set forth in SEQ ID NO:262 (BaEVTR). [1241] 331. The method of any of embodiments 293-324, wherein the fusogen is a Cocal virus G protein or a functional variant thereof.
[1242] 332. The method of any of embodiments 293-324, wherein the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
[1243] 333. The method of any of embodiments 293-324, wherein the fusogen is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof.
[1244] 334. The method of any of embodiments 293-324 and 333, wherein the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
[1245] 335. The method of any of embodiments 293-324 and 333, wherein the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
[1246] 336. The method of any of embodiments 293-324, 333, and 335, wherein the fusogen is a Nipah virus fusion protein or a functional variant thereof.
[1247] 337. The method of any of embodiments 293-324, 333, 335, and 336, wherein the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
[1248] 338. The method embodiment 337, wherein the NiV-G or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof.
[1249] 339. The method of embodiment 337 or embodiment 338, wherein the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4.
[1250] 340. The method of any of embodiments 337-339, wherein the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4
[1251] 341. The method of any of embodiments 337-340, wherein the NiV-G protein or biologically active portion thereof does not include an initial methionine.
[1252] 342. The method of any of embodiments 337-341, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42.
[1253] 343. The method of any of embodiments 337-342, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42. [1254] 344. The method of any of embodiments 293-343, wherein the fusogen comprises one or more modifications to reduce binding to its native receptor.
[1255] 345. The method of any of embodiments 337-341 and 344, wherein the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
[1256] 346. The method of any of embodiments 337-341, 344, and 345, wherein the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
[1257] 347. The method of any of embodiments 337-341 and 344-346, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17.
[1258] 348. The method of any of embodiments 337-341 and 344-347, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17.
[1259] 349. The method of any of embodiments 337-341 and 344-346, wherein the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18.
[1260] 350. The method of any of embodiments 337-341, 344-346, and 349, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
[1261] 351. The method of any of embodiments 337-350, wherein the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.
[1262] 352. The method of any of embodiments 337-351, wherein the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30.
[1263] 353. The method of any of embodiments 337-352, wherein the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30.
[1264] 354. The method of any of embodiments 337-353, wherein the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30. [1265] 355. The method of any of embodiments 337-354, wherein the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16.
[1266] 356. The method of any of embodiments 337-355, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16.
[1267] 357. The method of any of embodiments 337-354, wherein the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21.
[1268] 358. The method of any of embodiments 337-354 and 357, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
[1269] 359. The method of any of embodiments 337-341, 344-346, 349-354, 357, and 358, wherein the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
[1270] 360. The method of any of embodiments 302-359, wherein the bone marrow cells are CD34+.
[1271] 361. The method of any of embodiments 302-360, wherein the bone marrow cells are CD34+ progenitor cells.
[1272] 362. The method of any of embodiments 302-361, wherein the bone marrow cells are hematopoietic stem cells.
[1273] 363. The method of any of embodiments 302-362, wherein the one or more agents that stimulate mobilization are selected from the group consisting of stem cell factor (SCF), small molecule VLA-4 inhibitor BI05192, BOP (N-(benzenesulfonyl)-L-prolyl-L-0-(l-pyrrolidinylcarbonyl)tyrosine), heparin, granulocyte colony-stimulating factor (G-CSF), MGTA-145, and plerixafor (AMD3100).
[1274] 364. The method of any of embodiments 302-362, wherein the one or more agents that stimulate mobilization comprise G-CSF.
[1275] 365. The method of embodiment 364, wherein the G-CSF is administered to the subject daily on the two days, three days, four days, or five days prior to the administration of the first dose of the particle.
[1276] 366. The method of embodiment 364 or embodiment 365, wherein the G-CSF is administered to the subject on the day of the administration of the first dose of the particle.
[1277] 367. The method of any of embodiments 364-366, wherein the G-CSF is administered to the subject on the day of administration of a second or later dose of the particle. [1278] 368. The method of any of embodiments 302-367, wherein the one or more agents that stimulate mobilization comprise plerixafor.
[1279] 369. The method of embodiment 368, wherein the plerixafor is administered to the subject on the day of the administration of the first dose of the particle.
[1280] 370. The method of embodiment 368 or embodiment 369, wherein the plerixafor is administered to the subject on the day of the administration of a second or later dose of the particle.
[1281] 371. The method of any of embodiments 302-370, wherein the one or more agents that stimulate mobilization are G-CSF and plerixafor.
[1282] 372. The method of embodiment 371, wherein: the G-CSF is administered to the subject daily on the four days prior to the administration of the first dose of the particle; and the plerixafor is administered to the subject on the day of the administration of the first dose of the particle.
[1283] 373. The method of embodiment 371, wherein: the G-CSF is administered to the subject (i) daily on the two days prior to the administration of the particle; (ii) on the day of the administration of the first dose of the particle; and (iii) on the day of administration of a second or later dose of the particle; and the plerixafor is administered to the subject on the day of administration of a second or later dose of the particle.
[1284] 374. The method of any of embodiments 297-373, wherein the method comprises administering to the subject a transduction adjuvant.
[1285] 375. The method of embodiment 374, wherein the transduction adjuvant is administered to the subject on the day of the administration of at least one dose of the particle.
[1286] 376. The method of embodiment 374 or embodiment 375, wherein the transduction adjuvant is administered to the subject on the days of the administration of at least two doses of the particle.
[1287] 377. The method of any of embodiments 374-376, wherein the transduction adjuvant is a cationic peptide.
[1288] 378. The method of any of embodiments 374-377, wherein the transduction adjuvant is a histidine-rich cationic peptide.
[1289] 379. The method of any of embodiments 374-378, wherein the transduction adjuvant is a cationic amphipathic peptide.
[1290] 380. The method of any of embodiments 374-379, wherein the transduction adjuvant is derived from the LAH4 peptide family.
[1291] 381. The method of any of embodiments 374-380, wherein the transduction adjuvant is Vectofusin-1.
[1292] 382. The method of any of embodiments 293-300, 302, 303, 305, and 307-381, wherein the target molecule is expressed on the target cell. [1293] 383. The method of any of embodiments 293-300, 302, 303, 305, and 307-382, wherein the target cell is a hematopoietic cell.
[1294] 384. The method of any of embodiments 293-300, 302, 303, 305, and 307-383, wherein the target cell is CD34+.
[1295] 385. The method of any of embodiments 293-300, 302, 303, 305, and 307-384, wherein the target cell is a CD34+ progenitor cell.
[1296] 386. The method of any of embodiments 293-300, 302, 303, 305, and 307-385, wherein the hematopoietic cell is a hematopoietic stem cell.
[1297] 387. The particle of any of embodiments 294-386, wherein the exogenous agent is present in the lumen.
[1298] 388. The method of any of embodiments 294-387, wherein the exogenous agent is a protein or a nucleic acid.
[1299] 389. The method of any of embodiments 294-388, wherein the exogenous agent is or encodes a factor associated with gene editing.
[1300] 390. The method of any of embodiments 294-389, wherein the exogenous agent is or encodes a genome-modifying protein for gene editing a target gene encoding an endogenous protein.
[1301] 391. The method of any one of embodiments 294-390, wherein the exogenous agent is a fusion protein between a viral structural protein and an exogenous agent.
[1302] 392. The method of embodiment 391, wherein the fusion protein is a cleavable fusion protein comprising a cleavable linker positioned between the viral structural protein and the exogenous agent.
[1303] 393. The method of embodiment 391 or 392, wherein the fusion protein comprises one or more nuclear localization sequences.
[1304] 394. The particle of embodiment 391 or 392, wherein the fusion protein comprises one or more nuclear export sequences.
[1305] 395. The method of any of embodiments 390-394, wherein the genome -modifying protein is associated with gene editing by a sequence-specific nuclease, a CRISPR-associated transposase (CAST), prime editing, or Programmable Addition via Site-specific Targeting Elements (PASTE).
[1306] 396. The method of any of embodiments 390-395, wherein the genome -modifying protein is a sequence-specific nuclease.
[1307] 397. The method of any of embodiments 390-396, wherein the sequence-specific nuclease is selected from the group consisting of an RNA-guided nuclease, a meganuclease, a transcription activatorlike effector nuclease (TALEN), and a zinc-finger nuclease (ZFN).
[1308] 398. The method of any of embodiments 395-397, wherein the sequence-specific nuclease is an RNA-guided nuclease. [1309] 399. The method of embodiment 397 or embodiment 398, wherein the RNA-guided nuclease is TnpB.
[1310] 400. The method of embodiment 397 or embodiment 398, wherein the RNA-guided nuclease comprises a Cas nuclease and a guide RNA (CRISPR-Cas combination).
[1311] 401. The method of embodiment 400, wherein the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease.
[1312] 402. The method of embodiment 400 or embodiment 401, wherein the Cas nuclease is a Type II or Type V Cas protein.
[1313] 403. The method of any of embodiments 390-398 and 400-402, wherein the genomemodifying protein is selected from the group consisting of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxl l, Csyl, Csy2, Csy3, Mad7.
[1314] 404. The method of any of embodiments 400-403, wherein the Cas is a Cas9 or a Casl2.
[1315] 405. The method of any of embodiments 294-395, wherein the exogenous agent is or encodes a factor associated with base editing or prime editing (e.g., target-primed reverse transcription (TPRT)).
[1316] 406. The method of any of embodiments 294-395 and 405, wherein the exogenous agent is or encodes a transposase, integrase, or recombinase.
[1317] 407. The method of any of embodiments 294-395 and 405, wherein the exogenous agent is or encodes a DNA polymerase, RNA polymerase, or reverse-transcriptase.
[1318] 408. The method of any of embodiments 294-407, wherein the exogenous agent is for use in gene therapy to correct a genetic deficiency in the target cell.
[1319] 409. The method of embodiment 408, wherein the exogenous agent is a nucleic acid comprising a payload gene for correcting the genetic deficiency.
[1320] 410. The method of any of embodiments 294-388, wherein the exogenous agent is or encodes a membrane protein.
[1321] 411. The method of embodiment 410, wherein the membrane protein is an antigen receptor.
[1322] 412. The method of embodiment 411, wherein the antigen receptor binds to an antigen expressed on cells associated with a disease or condition in the subject.
[1323] 413. The method of embodiment 412, wherein the disease or condition is a cancer.
[1324] 414. The method of any of embodiments 411-413, wherein the antigen receptor binds to an antigen expressed on tumor cells in the subject.
[1325] 415. The method of any of embodiments 411-414, wherein the antigen receptor is a chimeric antigen receptor (CAR). [1326] 416. The method of any of embodiments 411-414, wherein the antigen receptor is an engineered T cell receptor (TCR).
[1327] 417. The method of any of embodiments 293-300, 302, 303, 305, and 307-416, wherein the delivery to target cells expressing the target molecule is increased by or by greater than 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, or more compared to delivery to non-target cells not expressing the target molecule.
[1328] 418. The method of any of embodiments 297-417, wherein the subject is a human subject.
[1329] 419. The particle of any of embodiments 1-22 and 25-107, wherein the target molecule is CD133.
[1330] 420. The polynucleotide of any of embodiments 108-112 and 115-162, wherein the target molecule is CD 133.
[1331] 421. The cell of any of embodiments 167-187 and 190-230, wherein the target molecule is CD133.
[1332] 422. The method of making a targeted lipid particle of any of embodiments 232-249 and 252-287, wherein the target molecule is CD133.
[1333] 423. The method of any of embodiments 306-418, wherein the fusogen is fused to a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3.
[1334] 424. The method of embodiment 423, wherein the target molecule is ASCT2.
[1335] 425. The method of embodiment 423, wherein the target molecule is CD117.
[1336] 426. The method of embodiment 423, wherein the target molecule is CD133.
[1337] 427. The particle of embodiment 419, the polynucleotide of embodiment 420, the cell of embodiment 421, the method of embodiments 422, 423 or embodiment 426, wherein the targeting agent is a CD 133 binding agent that is an antibody or antigen-binding fragment comprising a variable heavy (VH) chain and a variable light (VL) chain selected from a) a VH chain comprising a CDR-H1, a CDR- H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 271, 272, 273, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 280, 281, 282, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 284, 285, and 286, respectively; (c) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 298, 299, and 300, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively; (d) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 307, 308, and 309, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 311, 312, and 313, respectively; or (e) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 298, 290, and 291, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 293, 294, and 295, respectively.
[1338] 428. The particle of embodiment 419, the polynucleotide of embodiment 420, the cell of embodiment 421, the method of embodiments 422, 423 or embodiment 426, wherein the targeting agent is a CD 133 binding agent that is an antibody or antigen-binding fragment comprising a variable heavy (VH) chain and a variable light (VL) chain selected from a) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 314, 315, and 273, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 316, 317, and 282, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 284, 285, and 286, respectively; (c) a VH comprising a a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 318, 319, and 300, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively; (d) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 320, 321, and 309, respectively, and a VL comprising a CDR- Ll, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 311, 312, and 313, respectively; and (e) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 322, 323, and 291, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 293, 294, and 295, respectively.
[1339] 429. The particle, the polynucleotide, the cell, or the method of embodiment 427 or embodiment 428, wherein the CD 133 binding agent is selected from a) a CD 133 binding agent comprising a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 270, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 274, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; b) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 279, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 283, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; c) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 297, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 301, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; d) a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 306, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 310, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; e) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 288, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 292, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
[1340] 430. The particle, the polynucleotide, the cell, or the method of any of embodiments 427-
429, wherein the CD 133 binding agent is a scFv.
[1341] 431. The particle, the polynucleotide, the cell, or the method of any of embodiments 427-
430, wherein the CD 133 binding agent comprises the amino acid sequence set forth in any one of SEQ ID NOs: 269, 278, 287, 296 or 305, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
[1342] 432. The particle of embodiment 24, the polynucleotide of embodiment 114, the cell of embodiment 189, the method of embodiments 251 or embodiment 425, wherein the targeting agent is a CD117 binding agent that is a VHH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
[1343] 433. The particle, the polynucleotide, the cell or the method of embodiment 432, wherein the CD117 binding agent comprises the amino acid sequence set forth in any one of SEQ ID NOS: 324-374.
[1344] 434. An anti-CD117 binding agent comprising a VHH single domain antibody comprising a CDR-H1, a CDR-H2, and a CDR-H3 contained within an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374.
[1345] 435. An anti-CD117 binding agent comprising a VHH single domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
[1346] 436. The anti-CD117 binding agent of embodiment 434 or embodiment 435, wherein the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOS: 324-374. [1347] 437. A viral fusion protein comprising a viral envelope protein and the anti-CDl 17 binding agent of any one of embodiments 434-436.
[1348] 438. A viral fusion protein comprising a viral envelope protein and an anti-CDl 33 binding agent, wherein the anti-CDl 33 binding agent is an antibody or antigen-binding fragment comprising a variable heavy (VH) chain and a variable light (VL) chain selected from a) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 314, 315, and 273, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 316, 317, and 282, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 284, 285, and 286, respectively; (c) a VH comprising a a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 318, 319, and 300, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively; (d) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 320, 321, and 309, respectively, and a VL comprising a CDR- Ll, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 311, 312, and 313, respectively; and (e) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 322, 323, and 291, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 293, 294, and 295, respectively.
[1349] 439. The viral fusion protein of embodiment 438, wherein the CD133 binding agent is selected from a) a CD 133 binding agent comprising a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 270, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 274, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; b) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 279, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 283, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; c) a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 297, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 301, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; d) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 306, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 310, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; e) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 288, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 292, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
[1350] 440. The viral fusion protein of embodiment 438 or embodiment 439, wherein the CD133 binding agent is a scFv.
[1351] 441. The viral fusion protein of any of embodiments 438-440, wherein the CD133 binding agent comprises the amino acid sequence set forth in any one of SEQ ID NOs: 269, 278, 287, 296 or 305, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
[1352] 442. The viral fusion protein of any of embodiments 437-441, wherein the viral envelope protein is a VSV-G protein or a functional variant thereof.
[1353] 443. The viral fusion protein of any of embodiments 437-441, wherein the viral envelope protein is a Cocal virus G protein or a functional variant thereof.
[1354] 444. The viral fusion protein of any of embodiments 437-441, wherein the viral envelope protein is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
[1355] 445. The viral fusion protein of any of embodiments 437-441, wherein the viral envelope protein is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof.
[1356] 446. The viral fusion protein of any of embodiments 437-441 and 445, wherein the viral envelope protein is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
[1357] 447. The viral fusion protein of any of embodiments 437-441 and 445, wherein the viral envelope protein is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
[1358] 448. The viral fusion protein of any of embodiments 437-441, 445 and 447, wherein the viral envelope protein is a Nipah virus fusion protein or a functional variant thereof.
[1359] 449. The viral fusion protein of any of embodiments 437-441, 445, 447 and 448, wherein the viral envelope protein comprises a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof. [1360] 450. The viral fusion protein of embodiment 449, wherein the binding agent is fused to the C-terminus of the NiV-G protein or biologically active portion thereof.
[1361] 451. The viral fusion protein of embodiment 449 or embodiment 450, wherein the NiV-G or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof.
[1362] 452. The viral fusion protein of any of embodiments 449-451, wherein the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4.
[1363] 453. The viral fusion protein of any of embodiments 449-452, wherein the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4.
[1364] 454. The viral fusion protein of any of embodiments 449-453, wherein the NiV-G protein or biologically active portion thereof does not include an initial methionine.
[1365] 455. The viral fusion protein of any of embodiments 449-454, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42.
[1366] 456. The viral fusion protein of any of embodiments 449-455, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
[1367] 457. The viral fusion protein of any of embodiments 437-456, wherein the viral envelope protein comprises one or more modifications to reduce binding to its native receptor.
[1368] 458. The viral fusion protein of any of embodiments 449-457, wherein the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
[1369] 459. The viral fusion protein of any of embodiments 449-458, wherein the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
[1370] 460. The viral fusion protein of any of embodiments 449-459, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17.
[1371] 461. The viral fusion protein of any of embodiments 449-460, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17. [1372] 462. The viral fusion protein of any of embodiments 449-461, wherein the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18.
[1373] 463. The viral fusion protein of any of embodiments 449-462, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
VIII. EXAMPLES
[1374] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1 VSV-G and BaEVTR vectors for transducing human HSCs, blood lineage cells, and hepatocytes in vitro
[1375] This Example demonstrates the capacity of VSV-G and BaEVTR vectors to transduce human HSCs, blood lineage cells, and hepatocytes in vitro. Lentiviral vectors were pseudotyped either with VSV-G or a BaEVTR fusogen (set forth in SEQ ID NO:262) carrying a green fluorescent protein (GFP) transgene.
[1376] For transduction of bulk CD34+cells, cryopreserved CD34+ cells from G-CSF+AMD3100 mobilized peripheral blood (Allcells) were thawed in STEMSPAN SFEM II media (StemCell, 09065) supplemented with GLUTAMAX (ThermoFisher, 35050061) and Penicillin-Streptomycin (ThermoFisher, 15140122). 100 pl of cells were plated in a round bottom 96 well plate at 2e5 viable cells/mL. After overnight rest in 37 °C incubator, 100 pl of diluted vector + VECTOFUSIN-1 (Miltenyi Biotec, 130-111-163) was added at a final concentration of 12 pg/mL. After 24 hours of transduction, STEMSPAN SFEM II media containing 2x CC110 cytokine cocktail (StemCell, 02697) was added. Following 6-7 days in culture with cytokines, the cells were assessed for degree of transduction by GFP expression on a BD Fortessa instrument.
[1377] For transduction of CD34+ subpopulations, CD34+ cells from G-CSF+AMD3100 mobilized peripheral blood (Allcells)were thawed in STEMSPAN SFEM II media (StemCell, 09065) supplemented with GLUTAMAX (ThermoFisher, 35050061) and Penicillin-Streptomycin (ThermoFisher, 15140122). Approximately 10e6 total cells were stained in an antibody cocktail containing CD34 APC (BD, 340441), Lineage Cocktail Pacific Blue (Biolegend, 348805), CD15 PACIFIC BLUE (Biolegend, 980504), CD38 PE-Cy5 (Biolegend, 303508), CD90 PE-CF594 (BD, 562385), CD45RA BV711 (BD, 740806), CD7 AF700 (BD561603), CD10 BV605 (Biolegend, 312222), CD135 PE (Biolegend, 313306), and DAPI (ThermoFisher, D1306). Cells were sorted into various HSPC subpopulations using a BD Symphony S6 cell sorter. The following combinations of cell surface markers were used to identify and separate the HSPC subsets. Hematopoietic stem cells (HSC): Lin-CD34+CD38-CD90+CD45RA-; multipotent progenitors (MPP): Lin -CD34+CD38-CD90-CD45RA-; multi-lymphoid progenitors (MLP): Lin-CD34+CD38-CD90-CD45RA+; MEP: Lin-CD34+CD38+CD7-
CD10-CD135-CD45RA-; CMP: Lin-CD34+CD38+CD7-CD10-CD135+ CD45RA-; GMP: Lin-CD34+CD38+CD7-CD10-CD135-CD45RA+. Sorted cells were assessed for sort purity post-sort, resuspended at 2e5 viable cells/mL, and seeded into 96 well round bottom plates. Sorted subpopulations were transduced as described above.
[1378] For transduction of hepatocytes, cryopreserved primary human hepatocytes (Lonza, HUCPG) were thawed in IMDM media (ThermoFisher, 12440053), and 5e4 viable cells were plated in 100 ul in a 96 well plate (ThermoFisher, Al 142803). Four hours after plating, media was exchanged and diluted vector with VECTOFUSIN-1 was added. The media was exchanged 24 hours after transduction, and 4-7 days after transduction, the cells were stained with DRAQ7 and degree of transduction by GFP expression was read out using an Agilent Cytation5 plate reader.
[1379] A schematic of the experiments is depicted in FIG. 1A.
[1380] Transduction of cells with VSV-G pseudotyped vector was performed with vector at 2.039e9 TU/ml, for an approximate MOI of 1000. Transduction of cells with BaEVTR (fusogen set forth in SEQ ID NO:262) pseudotyped vectors was performed with vector at 3.86e5 TU/ml, for an approximate MOI of 2. As can be seen in FIG. IB, the VSV-G vector transduced bulk CD34+ cells and different subpopulations at about 50-80% of live cells. The BaEVTR vector transduced the same cell populations at about 20-30% of live cells, though it is important to note that this was observed using about 500-fold less vector than with VSV-G (FIG. IB). At the same amounts of vector, VSV-G vector transduced 80.9% of primary human hepatocytes, whereas BaEVTR vector transduced 0.8% of primary human hepatocytes, indicating that BaEVTR is highly selective for CD34+ cells as compared to hepatocytes.
[1381] In another experiment, transduction of cells with VSV-G pseudotyped vector or BaEVTR (fusogen set forth in SEQ ID NO:262) was performed in serial dilutions. As can be seen in FIG. 1C and FIG. ID, the BaEVTR vector was superior in targeting hematopoietic stem/progenitor cells (HSPCs) (approximately 100% at MOI 66, with Vectofusin-1) compared with VSV-g (40% at MOI 50,750).
[1382] In another experiment, transduction of cells with BaEVTR was performed at two different MOI values, MOI 32 and MOI 158. All sorted resting HSPC sub-populations achieved LV dosedependent transduction using BaEVTR in vitro (FIG. IE).
Example 2 : Ex vivo transduction and engraftment of hematopoietic stem/progenitor cells (HSPCs) with VSV-G and BaEVTR vectors
[1383] To study the ability of VSV-G and BaETVR vectors to target HSPCs, lentivial vectors described in Example 1 were used to transduce human cord-blood derived CD34+ cells. Lentiviral vectors also were produced with Nipah virus (NiV) fusogens retargeted with specific binders against the cellular receptors ASCT2 (one of the receptors for BaEV, along with ASCT1) and CD117, as described in Example 9 below.
[1384] Cord blood CD34+ cells (StemCell, 70008) were thawed and cultured in a 6-well plate for 24h in StemSpan SFEM II media + 1:100 GlutaMax and cytokine cocktail CC110. The next day, a cell count was obtained, and cells were transferred to 96-well plates coated 5ug/cm2 with retronectin (1 mg/mL), plated at 20K cells per well. Freshly-thawed LV was diluted in StemSpan SFEM II media + 1:100 GlutaMax + CC110. This LV was added to the wells of the 96-well plates containing CD34+ cells. Cells+LV were incubated in 37C incubator. 24h after transduction, cells were collected from 96-well plate, wells were pooled injected into NBSGW (Jackson Labs strain Hu-NSG-NBSGW-CD34, 702662) mice I.V. (100K cells per mouse in 200ul sterile PBS). Monthly cheek bleeds were taken and analyzed by flow cytometry.. On week 18, mice were euthanized, and peripheral blood was again collected. Cells from the collected bleeds were monitored for human CD45+, mouse CD45- cells and were analyzed for positive GFP expression using a plate reader. FIG. 2 (top panel) depicts the study design.
[1385] On D2 after transduction, aliquots of the ex vivo cells were measured by plate reader for positive GFP expression. As shown in the bottom, left panel of FIG. 2, both VSV-G, BaETVR, and ASCT2 retargeted vectors resulted in 50-80% successful transduction of the cells ex vivo, whereas CD117 retargeted vectors did not efficiently transduce the cells. As shown in the bottom, right panel of FIG. 2, transduced cells also were detectable in the peripheral blood after engraftment and were positive for the GFP transgene. These results demonstrate that the lentiviral vectors successfully targeted stimulated HSPCs in vitro and maintain high levels of GFP engraftment (50-60%).
Example 3 Phenotypic distribution of cells following humanization of two mouse models
[1386] This Example shows the phenotypic distribution of cells following humanization of two mouse models. NSG (Jackson Labs strain hu-NSG-CD34, 705557) and NBSGW (Jackson Labs strain Hu-NSG-NBSGW-CD34, 702662) mice were engrafted with cord-blood derived CD34+ cells. The mice were included in the study at 19 weeks (NSG) or 12 weeks (NBSGW) after humanization, were sacrificed 24 days later and peripheral blood and bone marrow were processed for analysis of phenotypic markers.
[1387] For peripheral blood, 75-100pl of whole mouse blood was added to a V-bottom 96-well plate. 75-100pl PBS (Gibco 10010-023; 1:1 ratio with the blood) was added to the wells containing blood samples. Samples were pipetted up and down to mix the blood and PBS. The 96-well plate containing the blood/PBS mixture was spun at lOOOx G for 2 minutes and the supernatant was discarded. 200p I of ACK lysis buffer (Gibco A 10492-01) was added to each well that contained a blood pellet. The pellets were mixed well in the ACK buffer by pipetting and incubated at room temperature for 3 minutes. Cells were spun again lOOOx G for 2 minutes and the supernatant was discarded. 200 pl of lx PBS (Gibco 10010- 023) was added to each well. Cells were spun again lOOOx G for 2 minutes and the supernatant was discarded. 200pl of ACK lysis buffer (Gibco A10492-01) was added to each well a second time. The pellets were mixed well in the ACK buffer by pipetting and incubated at room temperature for 3 minutes. Cells were spun again lOOOx G for 2 minutes and the supernatant was discarded. 200 pl of lx PBS (Gibco 10010-023) was added to each well. Cells were spun again lOOOx G for 2 minutes, the supernatant was discarded, cells were resuspended in lx PBS (Gibco 10010-023) and transferred over to a 96-well U-bottom plate for staining for flow cytometry.
[1388] For bone marrow, muscle and residue tissue was removed from the femur and tibia with sterile forceps ant scissors. Bones were transferred into a sterile mortar containing 5ml ice-cold lx PBS (Gibco 10010-023) and smashed with a pestle. The crushed bones were filtered through a 40pm Nylon cell strainer to remove solid fragments, the volume of the filtrate was brought up to 10ml PBS (Gibco 10010-023), and the filtrate was centrifuged at 300x G for 10 minutes. Following centrifugation, the supernatant was removed, and the cell pellet was resuspended in 10ml lx PBS (Gibco 10010-023) and filtered through a 40pm Nylon cell strainer. The filtrate was centrifuged for 5 minutes at 350x G (at 4 degrees C). Following centrifugation, the supernatant was removed and cells were resuspended in lx PBS for staining for flow cytometry.
[1389] Human peripheral blood and bone marrow samples were also obtained and processed similarly for flow cytometry.
[1390] For flow cytometry analysis of a panel of lineage-positive markers, cells were washed in PBS, incubated with Human TruStain FcX receptor block (Biolegend 422302; 1:20 in BSA Stain Buffer BD Biosciences 554657), and stained with an antibody cocktail consisting of CD34 PE (BD 348057), CD15 AF700 (Biolegend 323026), CD14 APC (Biolegend 367118), CD3 BV421 (Biolegend 344834), CD19 PE-Cy7 (Biolegend 302216), CD33 BV650 (Biolegend 30430), CD13 BV650 (BD 740567), huCD45 BV711 (Biolegend 304050), msCD45 BV785 (Biolegend 103149), GlycA APC Vio770 (MACS 130-120-471), and CD41 PE-Dazzle 594 (Biolegend 303732). Immediately prior to running the samples on flow, cells were stained with 7-AAD (Biolegend 420404).
[1391] For flow cytometry analysis of a panel of lineage-positive markers, cells were washed in PBS, stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation (Invitrogen L10119). Subsequently, cells were incubated with Human TruStain FcX receptor block (Biolegend 422302; 1:20 in BSA Stain Buffer BD Biosciences 554657). The Lineage Negative antibody cocktail consists of: huCD45 PE-Cy7 (Biolegend 304016), msCD45 BV785 (Biolegend 103149), CD34 APC (BD 345804), CD38 PE-Cy5 (Biolegend 103149), Pacific Blue anti-human Lineage Cocktail (CD3, CD14, CD16, CD19, CD20, CD56; Biolegend 348805), CD15 Pacific Blue (Biolegend 980504), CD90 PE-CF594 (BD 562385), CD45RA BV711 (BD 740806), CD7 AF700 (BD 561603), CD10 BV605 (Biolegend 312222), CD135 PE (Biolegend 313306), and CD117 BV650 (Biolegend 313222). [1392] As seen in FIG. 3, following long-term humanization the PB and BM composition of the NSG and NGSBW mice was dominated by B lymphoid cells (shown in green), contrary to the normal composition of a human blood where neutrophils and myeloid cells constituted the largest fraction of hematopoietic cells. Also, the primitive HSC and multipotential progenitor (MPP) fractions were less prevalent in these mice as compared to the human samples.
Example 4 : Phenotypic distribution of cells following short-term humanization of a mouse models
[1393] This Example shows the phenotypic distribution of cells following humanization of a the NBSGW (Jackson Labs strain Hu-NSG-NBSGW-CD34, 702662) mouse model after mice were engrafted with human CD34+ cells substantially as described in Example 3 above.Peripheral blood was collected from the mice via a cheek bleed DO. The purpose of this “baseline bleed” was to analyze the composition of the cell types of the humanized mouse blood by flow cytometry. Mouse and human peripheral blood and bone marrow samples were also obtained and processed as described in Example 3 for flow cytometry. A cohort of mice was sacrificed on D7 following humanization, and peripheral blood and bone marrow were processed for analysis of phenotypic markers. An exemplary protocol is shown in FIG. 4A.
[1394] Results in FIG. 4B show cells D7 after humanization when human cells are localized only in the bone marrow (BW) and HSPC composition still resembles closely the infusion material (top panel), which the huCD34+ composition is distinct (bottom panel).
Example 5 : Effect of mobilization on transduction with a BaEVTR vector
[1395] This Example demonstrates the effect of mobilization on transduction with a BaEVTR vector (fusogen set forth in SEQ ID NO:262). NBSGB and NSG mice humanized with human cord-blood derived CD34+ cells (Jackson Labs strain hu-NSG-CD34, 705557; 12 and 19 weeks after humanization, respectively, at study start) were used. Peripheral blood was collected from the mice via a cheek bleed 1 week prior to starting the mobilization regimen. The purpose of this “baseline bleed” was to analyze the composition of the cell types of the humanized mouse blood by flow cytometry (flow method outlined in Flow Cytometry section).
[1396] Mobilization occurred at Days -4 through DO and consisted of two stages: 1) D-4 through D- 1: subcutaneous G-CSF injections (5 ug/day; 24 h between each injection) and 2) DO: subcutaneous AMD3100 (5 mg/kg). G-CSF (Peprotech; Recombinant Human, Cat# 300-23) was resuspended in ultrapure water (Invitrogen 10977-015) and filter-sterilized (0.2 um). G-CSF solution was prepared three days prior to first injection and aliquots were stored at 4 °C for the duration of the study. AMD3100 (Sigma Aldrich Cat# A5602-5MG) was resuspended in ultrapure water (Invitrogen 10977-015) and sterile-filtered shortly prior to in vivo administration. AMD3100 was administered subcutaneously 24h after the last dose of G-CSF. Control groups received equal volume doses of ultrapure water (Invitrogen 10977-015), subcutaneously.
[1397] LV + VECTOFUSIN-1 in vivo administration occurred 75 min after AMD3100 dosing. LV was thawed and kept on ice. LV was pre-mixed with Vectofusin-1 (Miltenyi Biotec, Cat# 130-111-163) at a final Vectofusin-1 concentration of 12 ug/ml) for 10 minutes. Immediately following the 10-minute LV + VECTOFUSIN-1 mixture incubation, the LV + VECTOFUSIN-1 mix was injected into mice intravenously. Two control mouse groups were bled on DO to determine the degree of mobilization at time of LV dosing (1 group underwent mobilization; 1 group underwent mock-regimen).
[1398] On D7, blood was collected from all mice via cheek bleed for transduction analysis by flow cytometry as described above. On D12, mice were anesthetized using CO2 and organs were collected for processing and downstream flow cytometry analysis as described above.
[1399] Mobilization improved transduction in all compartments analyzed, including peripheral blood and BM huCD45+ cells, bone marrow Lin-CD34+ cells, and bone marrow Lin-CD34+ primitive progenitor cells (FIG. 5A-B). Mobilization was important for transduction of bone marrow Lin- CD34+CD38- primitive progenitor cells, including HSCs, MPPs, and MLPs, where transduction of about 2-4% was observed with mobilization and less than 0.2% without mobilization (FIG. 5A-B)
Example 6 : Effect of mobilization on transduction at early stage of humanization
[1400] This Example demonstrates the effect of mobilization on transduction with a BaEVTR vector at an early stage of humanization. NBSGW mice were humanized with human cord-blood derived CD34+ cells.
[1401] Mobilization was achieved through subcutaneous G-CSF injections (5 ug/day; 24 h between each injection) and subcutaneous AMD3100 (5 mg/kg). G-CSF (Peprotech; Recombinant Human, Cat# 300-23) was resuspended in ultrapure water (Invitrogen 10977-015) and filter-sterilized (0.2 um). G-CSF solution was prepared three days prior to first injection and aliquots were stored at 4 °C for the duration of the study. AMD3100 (Sigma Aldrich Cat# A5602-5MG) was resuspended in ultrapure water (Invitrogen 10977-015) and sterile-filtered shortly prior to in vivo administration. AMD3100 was administered subcutaneously 24h after the last dose of G-CSF. Control groups received equal volume doses of ultrapure water (Invitrogen 10977-015), subcutaneously.
[1402] LV + VECTOFUSIN-1 in vivo was administered as described in Example 5.
[1403] On D7, mice were anesthetized using CO2 and peripheral blood and bone marrow were collected for processing and downstream flow cytometry analysis as described above.
[1404] Similar to previous results, mobilization improved transduction in all compartments analyzed, including peripheral blood and BM huCD45+ cells, bone marrow Lin-CD34+ cells, and bone marrow Lin-CD34+ primitive progenitor cells (FIG. 6A). FIG. 6A shows the number of human cells in peripheral blood versus bone marrow at D7 after humanization in mice that did not receive mobilization (-Mob) or did receive mobilization (+Mob). Additional experiments were performed and the data support that mobilization increases the number of BM huCD45+ cells, bone marrow Lin-CD34+ cells, and Lin- CD34+ CD38-CD45Ra-CD90+ cells (FIG. 6B). Mobilization interestingly allowed for BaEVTR LV transduction of naiive HSCs in the bone marrow (approximately 2% transduction), as compared to no transduction in the mobilization control mice (FIG. 6C). Additional experiments were performed showing BaEVTR transduced in vivo up to 6.4% of huCD45+, 5% of huCD34+, and 5.7% of HSCs in the humanized mouse bone marrow (FIG. 6D).
Example 7 : Effect of co-infusion on transduction
[1405] This Example demonstrates the effect of co-infusing human CD34+ cells and BaEVTR LV vector on transduction. To first determine basal peripheral blood access to HSPC, NBSGW mice were humanized with mobilized cord-blood derived CD34+ cells (Jackson Labs strain Hu-NSG-NBSGW- CD34, 702662). Mice were also intravaneously injected with BaEVTR vector further comprising a GFP transgene (lOml/Kg, plus vectofusin) or a control, as described in Example 5. On D12, a cohort of mice were sacrificed and peripheral blood and bone marrow were collected and analyzed as described above. A separate cohort underwent bi-weekly cheek bleeds for an extended 16 weeks. After 16 weeks, this cohort of mice was sacrificed and peripheral blood and bone marrow were collected and analyzed as described above.
[1406] As seen in FIG. 7A, one dose of BaEVTR LV reached approximately 23% transduction of human Lin-Cd34+ cells, and 12% of phenotypic HSC in the bone marrow. As shown in FIG. 7B, freshly infused CD34+ cells have a still physiological composition at the time of vector dosing. Therefore these data demonstrate access to human HSPC in the peripheral blood of humanized animals when they are in a state similar to what is to be expected when in the peripheral blood of a human.
[1407] To futher study the long-term effect of co-infusion of CD34+ cells and LV, cheek bleeds were performed at weeks 2, 4, 6, and 9 to analyze the composition of the cell types of the humanized mouse blood by flow cytometry.
[1408] As seen in FIG. 8A, 21-23% of the human peripheral blood myeloid output expresses GFP over the first 6 weeks following infusion, stabilizing at 7% by week 9. These data suggest the HSCs transduced in the peripheral blood engraft in the bone marrow and appear to maintain their functional characteristics by producing myeloid cells for several weeks after infusion.
[1409] Additional experiments to futher study the long-term effect of co-infusion of CD34+ cells and LV were performed, and cheek bleeds were performed at weeks for an extended 16 weeks to analyze the composition of the cell types of the humanized mouse blood by flow cytometry (FIG. 8B, left panel). [1410] As seen in the right panel of FIG. 8B, 3-7% GFP+ long-term output of HSPCs transduced with BaEVTR on DO in vivo as a co-infusion was observed, indicative of stable output from bone marrow HSPCs. The results of FIG. 8C support the data of FIG. 8A, further supporting that HSCs transduced in the peripheral blood engraft in the bone marrow and maintain their functional characteristics by producing myeloid cells for several weeks after infusion. Further, as seen in the right panel of FIG. 8C, 24.2% of HSCs in the bone marrow expressed GFP in vivo following assessment 16 weeks after transduction using the co-infusion model, supporting there is stable transduction over long-term studies.
Example 8 Effect of different administration schedules on transduction
[1411] This Example demonstrates the effect of different administration schedules on transduction. NBSGW mice were humanized with cord-blood derived CD34+ cells (Jackson Labs strain Hu-NSG- NBSGW-CD34, 702662) and were 12 weeks after humanization at study start. Baseline cheek bleeds were performed at D-19 and D-12 to analyze the composition of the cell types of the humanized mouse blood by flow cytometry.
[1412] The G-CSF and AMD3100 mobilization regimens used were identical to those described in Example 3. The non-mobilized control mouse cohort received a subcutaneous saline injection (Hospira/Pfizer, 00409-4888-20) at the same dosing schedule as the mice that were dosed the G-CSF and AMD3100 mobilizing agents. LV and VECTOFUSIN-1 preparation was also identical to Example 3, except in these experiments, VSV-G LV was diluted 1:10 in sterile PBS (Gibco 10010-023). VECTOFUSIN was not added to the VSV-G LV prior to dosing in vivo. One cohort of mice was dosed at the same schedule as Example 3 - on DO, intravenous at one full dose of 10 mg/ml, 75 min after AMD3100 administration (VSV-G dose 6.09e8 TU/mouse; BaEVTR dose 9.93e8 TU/mouse). The other cohort of mice received the same dose split over three administrations: a dose of LV (3.3ml/Kg) + VECTOFUSIN-1 (12pg/ml) (BaEVTR); the VSV-G LV was diluted 1:10 in sterile PBS (Gibco 10010- 023) and not pre-mixed with VECTOFUSIN-1. On D-2 and D-l, 3.3ml/Kg LV (or LV + VECTOFUSIN -1) was administered intravenously shortly after G-CSF administration. On DO, 3.3ml/Kg LV (or LV + VECTOFUSIN -1) was administered intravenously 75 min after AMD3100 administration. Two control mouse groups were bled on DO to determine the degree of mobilization at time of LV dosing (1 group underwent mobilization; 1 group underwent mock-regimen).
[1413] On D7, blood was collected from all mice via cheek bleed for transduction analysis by flow cytometry as described above. On D12, mice were anesthetized using CO2 and organs were collected for processing and downstream flow cytometry analysis as described above.
[1414] BaEVTR transduction (fusogen set forth in SEQ ID NO:262) was much greater than VSV-G at the doses administered in each of peripheral blood huCD45+ cells, bone marrow huCD45+ cells, and BM Lin-CD34+ cells (FIG. 9). Administration of the LV as a single bolus dose gave about 2-4-fold greater transduction across all groups (FIG. 9).
Example 9 Ability of retargeted fusogens to transduce blood cells and hematopoietic progenitor cells
[1415] This Example demonstrates the ability of lenti viral vectors (LVs) pseudotyped with retargeted fusogens to transduce blood cells and hematopoietic progenitor cells. Lenti viral vectors were produced with Nipah virus (NiV) fusogens retargeted with specific binders against the cellular receptors ASCT2 (one of the receptors for BaEV, along with ASCT1) and CD117. Envelope proteins were provided as plasmids expressing Nipah F protein and the binder-retargeted Nipah G protein (see US 2019/0144885, incorporated by reference herein). The binder-retargeted Nipah G (NiV-G) protein contained an antigen-binding domain against either ASCT2 or CD117 as a fusion with the exemplary NiV-G sequence GcA34 (Bender et al. 2016 PLoS Pathol 12(6):el005641; set forth in SEQ ID NO:17). The Nipah F (NiV-F) protein was the exemplary NiV-F sequence NivFdel22 (SEQ ID NO: 19; or SEQ ID NO:21, which does not have a signal sequence; Bender et al. 2016 PLoS). Following lentiviral vector production, the cell culture was centrifuged to pellet the cells and the supernatant containing crude virus was collected.
[1416] The LVs pseudotyped with retargeted fusogens were administered to humanized NSG and NBSGW mice according to the protocols of Examples 3 and 4, respectively. ASCT2 retargeted LV were administered at a dose of 5.79e8 TU/mouse (Example 3 protocol) and 3.09e8 TU/mouse (Example 4 protocol). CD117 retargeted LV were administered at a dose of 4.4e7 TU/mouse (Example 3 protocol) and 4.87e7 TU/mouse (Example 4 protocol). Both LVs pseudotyped with a retargeted fusogen (ASCT2 retargeted LV and CD117 retargted LV) transduced a lower percentage of huCD45+ cells in the bone marrow of the mice as compared to BaEVTR (FIG. 10A, left panels). However, the CD117 retargeted LV transduced a similar percentage of CD117+ cells as BaEVTR, even at a ten-fold lower dose (FIG. 10A, right panels). Although CD117+ cells were observed to be a small proportion of the human cell population (0.15-0.57%) (FIG. 10B, left panels), a much larger proportion of the transduced (GFP+) cells were CD117+ following transduction with the CD117 fusogen (FIG. 10B, right panels).
[1417] The compositions of GFP+ cells in the peripheral blood and bone marrow of mice administered BaEVTR, ASCT2 retargeted, and CD 117 retargeted LV were analyzed by flow cytometry for phenotypic markers (FIG. 11). CD117 retargeted LV showed a higher transduction of Myeloid cells in the PB and Myeloid/Megakaryo/Erythroid progenitor cells in the BM, whereas BaEVTR GFP+ cells had a more random distribution of phenotypes, and ASCT2 retargeted LV skewed toward B lymphoid transduction. The phenotypes are mapped onto plots in FIG. 12. Example 10 Phenotype of peripheral blood hematopoietic cells following humanization
[1418] Additionally, the phenotype of peripheral blood hematopoietic cells were followed throughout the mouse experiments following humanization. Starting at about 12 weeks after humanization, the phenotype of the NGSBW hu45+ cells shifted from a B cell (CD 19+) to a T cell (CD3+) phenotype, leading to the emergence and expansion of T cell clones (FIG. 13, left panels). A more profound shift in phenotype was observed in the humanized NSG mice during the study as measured at 19 weeks post humanization (Day 0) to 12 days following (FIG. 13, right panel).
Example 11 Assessment of gene editing efficiency of BaEVTR virus-like particle (VLP)
[1419] This Example demonstrates the ability of virus-like particles (VLPs) with a BaEVTR backbone to deliver gene editing cargo to HSPC sub-populations in vitro and in vivo. VLPs were produced with BaEVTR glycoprotein fusogen (set forth in SEQ ID NO:262) expressed on the lipid envelope and packaged with gag-Cas9 cargo and B2M gRNA (FIG. 14, top panel). For the in vitro experiment, VLPs were cultured with bulk CD34+ cells as described in Example 1. Percent B2M knockout (KO) or B2M gene edit was assessed by flow cytometry.
[1420] Serial dilutions were performed with the BaEVTR VLPs. As can be seen in the left panel of FIG. 14, the BaEVTR VLP achieved up to 85% B2M gene editing and 75% B2M gene KO on resting huCD34+ cell in vitro. In another experiment, transduction of cells with BaEVTR VLPs was performed at two different MOI values, MOI 16 and MOI 83. All sorted resting HSPC sub-populations achieved VLP dose-dependent transduction using BaEVTR in vitro, as evidenced by the percent of B2M edited cells (FIG. 14, right panel).
[1421] For the in vivo experiment, BaEVTR B2M VLPs were intravaneously injected into cohorts of mice following protocols described in Example 4 and Example 6. Following sacrifice of the mice according to the appropriate protocol, bone marrow and peripheral blood was collected and processed to assess effective B2M gene editing as described above. A representative flow plot of in vivo B2M gene editing in human Lin- CD34+ HSPC cells is provided in FIG. 15A.
[1422] As seen in FIG. 15B, BaEVTR VLPs performed B2M gene editing in all three humanized mouse models. In the long-term, 11-12 week humanized mouse model described in Example 4, 1.8% of the cells did not express the B2M gene. In the co-infusion model described in Example 6, 10.9% of huCD45+ cells in the peripheral blood did not express the B2M gene (VLP dose 1.7E+09 EU/mouse). In the short-term, 7 day humanized mouse model described in Example 4, 2.1% of the huCD45+ cells did not express the B2M gene (VLP dose 1.7E+09 EU/mouse). Example 12 Selective Modulation of CD34+ Cells in vitro and in vivo
[1423] This Example demonstrates the ability of virus-like particles (VLPs) with a BaEVTR backbone to deliver gene editing cargo to CD34+ cells in vitro and in vivo. VLPs were produced with BaEVTR glycoprotein fusogen (SEQ ID NO:262) expressed on the lipid envelope, and packaged with gag-Cas9 cargo and B2M gRNA.
[1424] CD34+ cells were isolated from cord blood before being contacted with serial dilutions of the BaEVTR VLPs. At 7 days post transduction, cells were analyzed for the percentage of cells knocked out for B2M. As shown for replicate studies in FIG 16A, the BaEVTR VLP efficiently modified CD34+ cells.
[1425] In a complementary set of experiments, the ability of the BaEVTR VLPs to modify cells in vivo were assessed. First, human CD34+ cells (huCD34+) were infused into 8 week old NBSGW mice (N0D.Cg-KitW-41J Tyr + Prkdcscid I12rgtmlWjl/ThomJ). In some aspects, NBSGW mice support engraftment of human hematopoietic stem cells (HSCs) without irradiation. Following 7 days for engraftment, the BaEVTR VLPs carrying the gene editing components as described above were administered to the mice intravenously at one of two doses (IX and 3X). Mice were sacrificed on Day 12, and cells of the bone marrow were analyzed for knock-out of the target B2M gene. Results in FIG. 16B depict percent of edited cells with respect to the entire cell population or only Lin-CD34+ progenitors per flow cytometric analysis. As shown in FIG. 16C, the in vivo knockout of B2M gene (e.g., B2M editing) in different HSPC subpopulations present in the bone marrow was both dose and time dependent.
[1426] Further, the ability of the BaEVTR VLPs to modify cells in vivo were assessed in long term humanized mice. Long term-humanized mice comprise substantially higher numbers of human bone marrow (BM) HSPC and a complete peripheral blood (PB) human blood cell engraftment. As described above, human CD34+ cells (huCD34+) were infused into 8 week old NBSGW mice (NOD.Cg-KitW-41J Tyr + Prkdcscid I12rgtmlWjl/ThomJ) as shown in FIG. 16D. Following 9 weeks for engraftment, the BaEVTR VLPs carrying the gene editing components as described above were administered to the mice intravenously at a single dose. Mice were sacrificed at week 11 , and cells of the bone marrow were analyzed for knock-out of the target B2M gene. Results in FIG. 16E depict a similar level of editing (~ 12%) was obtained upon single VLP dosing in long-term humanized mice.
[1427] In some aspects, flow-based readout of B2M KO (knock-out) is affected by B2M protein turnover, which can be characterized as dependent on the activation state of the target cells. Moreover, it is considered that the conservative gating strategy used to define B2M KO (knock-out) event by flow cytometry could exclude cells with monoallelic edits carrying residual B2M expression. Therefore, genome editing levels were also analyzed by high throughput sequencing of PCR-amplicons at the B2M target locus (OTA). Notably, with this unbiased analysis it was observed that the levels of edited B2M alleles in the total huCD45+ population actually corresponded to roughly 50% and 75%, in regards to administration of 1 dose and 3 doses respectively, in the D7 post-humanization model described above. As shown in FIG. 16F, true in vivo B2M editing measured by OTA is 2x higher than the flow cytometric readout of the same editing metric.
[1428] The data demonstrate that the BaEVTR VLPs can physically access and efficiently edit HSCs in vivo for editing a target gene.
Example 13 In vivo access to HSPCs in long-term humanized NBSGW mice
[1429] To study the ability of VSV-G and BaETVR vectors to target HSPCs in long-term humanized mice, lentiviral vectors described in Example 1 and expressing GFP were used to transduce human cord-blood derived CD34+ cells.
[1430] As described above, human CD34+ cells (huCD34+) were infused into 8 week old NBSGW mice (NOD.Cg-KitW-41J Tyr + Prkdcscid I12rgtmlWjl/ThomJ) as shown in FIG. 17A. . LV was injected 84 days following the cell infusion (e.g., VSV-G LV, BaEVTR LV, or untranduced (UT)). On Day 96, mice were euthanized, and peripheral blood was again collected. Cells from the bleeds were monitored for BM huCD45 GFP+ cells and BM HSPC GFP+ cells using a plate reader. FIG. 17A depicts the study design (top panel) and huCD34+ cell composition at the day of cell infusion and day of LV administration (bottom panel). As shown in FIG. 17B, BaEVTR, LV resulted in successful transduction of BM cells in vivo.
Example 14 Gene Editing in vivo in Non- Human Primate Model
[1431] This example sets forth an exemplary design to assess various fusogen-payload combinations for administration in a Non-Human Primate (NHP). Virus-like particles (VLPs) are produced with Nipah virus (NiV) fusogens retargeted to HSPC with specific binders against CD133 or CD117. For BaEVTR glycoprotein fusogens, VLPs are produced with BaEVTR glycoprotein fusogen (set forth in SEQ ID NO:262) expressed on the lipid envelope and packaged with payload. In some instances, the VLP produced with BaEVTR glycoprotein fusogen (set forth in SEQ ID NO:262) expressed on the lipid envelope also includes a MLV-gag (SEQ ID NO: 504).
[1432] For NiV fusogens, VLPs are produced with the Nipah F protein and the binder-retargeted Nipah G protein (see US 2019/0144885, incorporated by reference herein) expressed on the lipid envelope and packaged with payload.. The binder-retargeted Nipah G (NiV-G) protein contains an antigen-binding domain against either CD133, or CD117 as a fusion with the exemplary NiV-G sequence GcA34 (Bender et al. 2016 PLoS Pathol 12(6):el005641; set forth in SEQ ID NO: 17). The Nipah F (NiV-F) protein was the exemplary NiV-F sequence NivFdel22 (SEQ ID NO:19; or SEQ ID NO:21, which does not have a signal sequence; Bender et al. 2016 PLoS). Following lentiviral vector production, the cell culture is centrifuged to pellet the cells and the supernatant containing crude virus is collected.
[1433] A primary fusogen is selected based in part on overall VLP performance as measured by percent of long-term editing (i.e., edits resulting from the payload) in peripheral blood myeloid cells. A second fusogen is selected based in part on HSPC specific tropism as measured, for example, by percent of editing (i.e., edits resulting from the payload) in the various mature hematopoietic lineages derived from the HSPCs or in other cell lines vs the target HSPC population.
[1434] A payload (e.g., such as nuclease-based editor or base editing-based editor) is selected based on performance in resting NHPCD34+ cells in vitro as per OTA PCR based analysis described above in Example 12. Performance can be either reporter-based or a functional readout (such as change in mRNA or protein levels in a biological sample). A genomic target is selected that is characterized by a “neutral” locus or a “therapeutic” locus homologous to a potential therapeutic target for a disease indication (e.g. BCL11A).
[1435] A model study design is illustrated in FIG. 18. NHP animals are first subject to a mobilization regimen as described above. Briefly, AMD3100 is administered for 3 consecutive days wherein VLP is administered roughly 2 hours after each mobilization at a controlled rate dosing. Without wishing to be bound by theory, mobilization of cells from the bone marrow to the peripheral blood is generally at its peak 1.5-3 hours post AMD treatment. Peripheral blood is collected throughout the study period, while bone marrow is collected through aspirates bi-monthly. Additional needle aspirates of the liver are performed to assess off-target editing as a function of safety and biodistribution. Animals are sacrificed at 9 months post vector dose for assessing permanent gene modification of the long-term HSC in the bone marrow and potential editing in off-target tissues.
[1436] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure. Sequences

Claims

Claims What is claimed is:
1. A targeted lipid particle, comprising a lipid bilayer, a fusogen, and a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3, wherein the fusogen and the targeting agent are exposed on the surface of the lipid bilayer.
2. The particle of claim 1, wherein the targeting agent is fused to a transmembrane domain incorporated into the lipid bilayer.
3. The particle of claim 1, wherein the targeting agent is fused to the fusogen.
4. The particle of any of claims 1-3, wherein the particle is a viral particle or virus-like particle.
5. The particle of any of claims 1-4, wherein the lipid bilayer is derived from a membrane of a producer cell used for producing a viral particle or virus-like particle.
6. The particle of claim 4 or claim 5, wherein the viral particle or virus-like particle is a retroviral particle or retrovirus-like particle.
7. The particle of any of claims 4-6, wherein the viral particle or virus-like particle is a lentivirus-like particle.
8. The particle of any of claims 4-6, wherein the viral particle or virus-like particle is a lentiviral particle.
9. A targeted lentiviral particle, comprising a fusogen and a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3, wherein the fusogen and the targeting agent are exposed on the surface of the lentiviral particle.
10. The particle of any of claims 4-9, wherein the particle is pseudotyped with the fusogen.
11. The particle of claim 9 or claim 10, wherein the targeting agent is fused to the fusogen.
12. The particle of any of claims 1 and 3-11, wherein the targeting agent is fused to the fusogen directly.
13. The particle of any of claims 1 and 3-11, wherein the targeting agent is fused to the fusogen via a linker.
14. The particle of any of claims 4-10, wherein the targeting agent is fused to a transmembrane domain incorporated into the viral envelope.
15. The particle of any of claims 1-14, wherein the particle is prepared by a method comprising transducing a producer cell with plasmids encoding the fusogen and targeting agent and a Gag-pol and Rev.
16. The particle of any of claims 5-8 and 10-15, wherein the producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells.
17. The particle of any of claims 5-8 and 10-16, wherein the producer cell is a 293T cell.
18. The particle of any of claims 4-17, wherein the particle is replication defective.
19. The particle of any of claims 1-6 and 8-18, wherein the particle comprises a viral nucleic acid.
20. The particle of claim 19, wherein the viral nucleic acid is a retroviral nucleic acid, optionally wherein the viral nucleic acid is a lentiviral nucleic acid.
21. The particle of any of claims 19-20, wherein the viral nucleic acid comprises one or more, optionally all, of the following nucleic acid sequences: 5’ LTR (e.g., comprising U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT)/central termination sequence (CTS) (e.g., DNA flap), Poly A tail sequence, a posttranscriptional regulatory element (e.g., WPRE), a Rev response element (RRE), and 3’ LTR (e.g., comprising U5 and lacking a functional U3).
22. The particle of any of claims 1-7 and 10-18, wherein the particle is devoid of viral genomic nucleic acids.
23. The particle of any of claims 1-22, wherein the target molecule is ASCT2.
24. The particle of any of claims 1-22, wherein the target molecule is CD117.
25. The particle of any of claims 1-24, wherein the fusogen is a viral fusion protein.
26. The particle of any of claims 1-25, wherein the fusogen is a viral envelope protein.
27. The particle of any of claims 1-26, wherein the fusogen is a VSV-G protein or a functional variant thereof.
28. The particle of any of claims 1-26, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
29. A targeted lipid particle, comprising a lipid bilayer, a fusogen, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof and is exposed on the surface of the lipid bilayer.
30. The particle of claim 28 or 29, wherein the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild-type BaEV inhibitory R peptide.
31. The particle of claim 28, 29 or 30, wherein the fusogen is set forth in any of SEQ ID NO:254-260.
32. The particle of claim 28 or 29, wherein the fusogen is set forth in SEQ ID NO:261 (BaEVRLess).
33. The particle of claim 29, wherein the fusogen is set forth in SEQ ID NO:262 (BaEVTR).
34. The particle of any of claims 1-26, wherein the fusogen is a Cocal virus G protein or a functional variant thereof.
35. The particle of any of claims 1-26, wherein the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
36. The particle of any of claims 1-26, wherein the fusogen is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof.
37. The particle of any of claims 1-26 and 36, wherein the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des-petits- ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
38. The particle of any of claims 1-26 and 36, wherein the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
39. The particle of any of claims 1-26, 36, and 38, wherein the fusogen is a Nipah virus fusion protein or a functional variant thereof.
40. The particle of any of claims 1-26, 36, 38, and 39, wherein the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
41. The particle of claim 40, wherein the targeting agent is fused to the NiV-G protein or biologically active portion thereof.
42. The particle of claim 40 or claim 41, wherein the targeting agent is fused to the C- terminus of the NiV-G protein or biologically active portion thereof.
43. The particle of any of claims 40-42, wherein the NiV-G or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof.
44. The particle of any of claims 40-43, wherein the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4.
45. The particle of any of claims 40-44, wherein the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4.
46. The particle of any of claims 40-45, wherein the NiV-G protein or biologically active portion thereof does not include an initial methionine.
47. The particle of any of claims 40-46, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42.
48. The particle of any of claims 40-47, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 42.
49. The particle of any of claims 1-48, wherein the fusogen comprises one or more modifications to reduce binding to its native receptor.
50. The particle of any of claims 40-46 and 49, wherein the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
51. The particle of any of claims 40-46, 49, and 50, wherein the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
52. The particle of any of claims 40-46 and 49-51, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17.
53. The particle of any of claims 40-46 and 49-52, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17.
54. The particle of any of claims 40-46 and 49-51, wherein the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18.
55. The particle of any of claims 40-46, 49-51, and 54, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
56. The particle of any of claims 40-55, wherein the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.
57. The particle of any of claims 40-56, wherein the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30.
58. The particle of any of claims 40-57, wherein the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30.
59. The particle of any of claims 40-58, wherein the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30.
60. The particle of any of claims 40-59, wherein the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16.
61. The particle of any of claims 40-60, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16.
62. The particle of any of claims 40-59, wherein the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21.
63. The particle of any of claims 40-59 and 62, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
64. The particle of any of claims 40-46, 49-51, 54-59, 62, and 63, wherein the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
65. The particle of any of claims 1-64, wherein the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule.
66. The particle of any of claims 1-65, wherein the targeting agent is a single domain antibody that binds to the target molecule.
67. The particle of any of claims 1-66, wherein the targeting agent is a VHH that binds to the target molecule.
68. The particle of any of claims 1-66, wherein the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
69. The particle of any of claims 1-68, wherein the target molecule is expressed on a target cell.
70. The particle of any of claims 1-69, wherein the particle comprises an exogenous agent for delivery to a target cell.
71. The particle of any one of claims 1-69, wherein the particle comprises a fusion protein between a viral structural protein and an exogenous agent.
72. The particle of claim 71, wherein the fusion protein is a cleavable fusion protein comprising a cleavable linker positioned between the viral structural protein and the exogenous agent.
73. The particle of claim 71 or 72, wherein the fusion protein comprises one or more nuclear localization sequences.
74. The particle of claim 71 or 72, wherein the fusion protein comprises one or more nuclear export sequences.
75. The particle of claim 70 or claim 71, wherein the target molecule is expressed on the target cell.
76. The particle of any of claims 70-75, wherein the exogenous agent is present in the lumen.
77. The particle of any of claims 70-76, wherein the exogenous agent is a protein or a nucleic acid.
78. The particle of any of claims 70-77, wherein the exogenous agent is or encodes a therapeutic agent or a diagnostic agent.
79. The particle of any of claims 70-78, wherein the exogenous agent is or encodes a factor associated with gene editing.
80. The particle of any of claims 70-79, wherein the exogenous agent is or encodes a genome-modifying protein for gene editing a target gene encoding an endogenous protein.
81. The particle of claim 80, wherein the genome-modifying protein is associated with gene editing by a sequence-specific nuclease, a CRISPR-associated transposase (CAST), prime editing, or Programmable Addition via Site-specific Targeting Elements (PASTE).
82. The particle of claim 80 or claim 81, wherein the genome-modifying protein is a sequence-specific nuclease.
83. The particle of claim 81 or claim 82, wherein the sequence-specific nuclease is selected from the group consisting of an RNA-guided nuclease, a meganuclease, a transcription activator-like effector nuclease (TALEN), and a zinc-finger nuclease (ZFN).
84. The particle of any of claims 81-83, wherein the sequence-specific nuclease is an RNA- guided nuclease.
85. The particle of claim 83 or claim 84, wherein the RNA-guided nuclease is TnpB.
86. The particle of claim 83 or claim 84, wherein the RNA-guided nuclease comprises a Cas nuclease and a guide RNA (CRISPR-Cas combination).
87. The particle of claim 86, wherein the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease.
88. The particle of claim 86 or claim 87, wherein the Cas nuclease is a Type II or Type V Cas protein.
89. The particle of any of claims 80-84 and 86-88, wherein the genome-modifying protein is selected from the group consisting of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxl l, Csyl, Csy2, Csy3, Mad7.
90. The particle of any of claims 86-89, wherein the Cas is a Cas9 or a Casl2.
91. The particle of any of claims 70-81, wherein the exogenous agent is or encodes a factor associated with base editing or prime editing (e.g., target-primed reverse transcription (TPRT)).
92. The particle of any of claims 70-81 and 91, wherein the exogenous agent is or encodes a transposase, integrase, or recombinase.
93. The particle of any of claims 70-81 and 91, wherein the exogenous agent is or encodes a DNA polymerase, RNA polymerase, or reverse-transcriptase.
94. The particle of any of claims 70-93, wherein the exogenous agent is for use in gene therapy to correct a genetic deficiency in the target cell.
95. The particle of claim 94, wherein the exogenous agent is a nucleic acid comprising a payload gene for correcting the genetic deficiency.
96. The particle of any of claims 70-78, wherein the exogenous agent is or encodes a membrane protein.
97. The particle of claim 96, wherein the membrane protein is an antigen receptor.
98. The particle of claim 97, wherein the antigen receptor binds to an antigen expressed on cells associated with a disease or condition.
99. The particle of claim 98, wherein the disease or condition is a cancer.
100. The particle of any of claims 97-99, wherein the antigen receptor binds to an antigen expressed on tumor cells.
101. The particle of any of claims 97-100, wherein the antigen receptor is a chimeric antigen receptor (CAR).
102. The particle of any of claims 97-100, wherein the antigen receptor is an engineered T cell receptor (TCR).
103. The particle of any of claims 69-102, wherein the target cell is a hematopoietic cell.
104. The particle of any of claims 69-103, wherein the target cell is CD34+.
105. The particle of any of claims 69-104, wherein the target cell is a CD34+ progenitor cell.
106. The particle of any of claims 69-105, wherein the target cell is a hematopoietic stem cell.
107. The particle of any of claims 70-106, wherein the delivery to target cells expressing the target molecule is increased by or by greater than 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5- fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, or more compared to delivery to nontarget cells not expressing the target molecule.
108. A polynucleotide comprising a nucleic acid sequence encoding a fusogen and a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3.
109. The polynucleotide of claim 108, wherein the polynucleotide encodes a transmembrane domain that is fused to the targeting agent.
110. The polynucleotide of claim 108, wherein the targeting agent is fused to the fusogen.
111. The polynucleotide of claim 108 or claim 110, wherein the targeting agent is fused to the fusogen directly.
112. The polynucleotide of any of claims 108, 110, and 111, wherein the targeting agent is fused to the fusogen via a linker.
113. The polynucleotide of any of claims 108-112, wherein the target molecule is ASCT2.
114. The polynucleotide of any of claims 108-112, wherein the target molecule is CD117.
115. The polynucleotide of any of claims 108-114, wherein the fusogen is a viral fusion protein.
116. The polynucleotide of any of claims 108-115, wherein the fusogen is a viral envelope protein.
117. The polynucleotide of any of claims 108-116, wherein the fusogen is a VSV-G protein or a functional variant thereof.
118. The polynucleotide of any of claims 108-116, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
119. A polynucleotide comprising a nucleic acid sequence encoding a fusogen, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
120. The polynucleotide of claim 118 or 119, wherein the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild- type BaEV inhibitory R peptide.
121. The particle of any of claims 118-120, wherein the fusogen is set forth in any of SEQ ID NO:254-260.
122. The particle of claim 118 or 119, wherein the fusogen is set forth in SEQ ID NO:261 (BaEVRLess).
123. The particle of claim 118 or 119, wherein the fusogen is set forth in SEQ ID NO:262 (BaEVTR).
124. The polynucleotide of any of claims 108-116, wherein the fusogen is a Cocal virus G protein or a functional variant thereof.
125. The polynucleotide of any of claims 108-116, wherein the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
126. The polynucleotide of any of claims 108-116, wherein the fusogen is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof.
127. The polynucleotide of any of claims 108-116 and 126, wherein the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
128. The polynucleotide of any of claims 108-116 and 126, wherein the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
129. The polynucleotide of any of claims 108-116, 126, and 128, wherein the fusogen is a Nipah virus fusion protein or a functional variant thereof.
130. The polynucleotide of any of claims 108-116, 126, 128, and 129, wherein the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
131. The polynucleotide of claim 130, wherein the targeting agent is fused to the NiV-G protein or biologically active portion thereof.
132. The polynucleotide of claim 130 or claim 131, wherein the targeting agent is fused to the C-terminus of the NiV-G protein or biologically active portion thereof.
133. The polynucleotide of any of claims 130-132, wherein the NiV-G or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof.
134. The polynucleotide of any of claims 130-133, wherein the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4.
135. The polynucleotide of any of claims 130-134, wherein the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4.
136. The polynucleotide of any of claims 130-135, wherein the NiV-G protein or biologically active portion thereof does not include an initial methionine.
137. The polynucleotide of any of claims 130-136, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42.
138. The polynucleotide of any of claims 130-137, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
139. The polynucleotide of any of claims 108-138, wherein the fusogen comprises one or more modifications to reduce binding to its native receptor.
140. The polynucleotide of any of claims 130-136 and 139, wherein the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
141. The polynucleotide of any of claims 130-136, 139, and 140, wherein the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
142. The polynucleotide of any of claims 130-136 and 139-141, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17.
143. The polynucleotide of any of claims 130-136 and 139-142, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17.
144. The polynucleotide of any of claims 130-136 and 139-141, wherein the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18.
145. The polynucleotide of any of claims 130-136, 139-141, and 144, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
146. The polynucleotide of any of claims 130-145, wherein the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.
147. The polynucleotide of any of claims 130-146, wherein the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30.
148. The polynucleotide of any of claims 130-147, wherein the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30.
149. The polynucleotide of any of claims 130-148, wherein the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30.
150. The polynucleotide of any of claims 130-149, wherein the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16.
151. The polynucleotide of any of claims 130-150, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16.
152. The polynucleotide of any of claims 130-149, wherein the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21.
153. The polynucleotide of any of claims 130-149 and 152, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
154. The polynucleotide of any of claims 130-136, 139-141, 144-149, 152, and 153, wherein the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
155. The polynucleotide of any of claims 108-154, wherein the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule.
156. The polynucleotide of any of claims 108-155, wherein the targeting agent is a single domain antibody that binds to the target molecule.
157. The polynucleotide of any of claims 108-156, wherein the targeting agent is a VHH that binds to the target molecule.
158. The polynucleotide of any of claims 108-156, wherein the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
159. The polynucleotide of any of claims 108-158, wherein the polynucleotide is codon optimized.
160. The polynucleotide of any of claims 108-159, wherein the polynucleotide comprises at least one promoter that is operatively linked to control expression of the targeting agent and fusogen.
161. The polynucleotide of claim 160, wherein the promoter is a constitutive promoter.
162. The polynucleotide of claim 160, wherein the promoter is an inducible promoter.
163. A plasmid, comprising the polynucleotide of any of claims 108-162 or 420.
164. The plasmid of claim 163, wherein the plasmid comprises one or more nucleic acids encoding proteins for lentivirus production.
165. A vector, comprising the polynucleotide of any of claims 108-162 or 420.
166. A cell comprising the polynucleotide of any of claims 108-162 or 420, the plasmid of claim 163 or claim 164, or the vector of claim 165.
167. A cell comprising (i) a first polynucleotide comprising a nucleic acid sequence encoding a fusogen and (ii) a second polynucleotide comprising a nucleic acid sequence encoding a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3.
168. The cell of claim 167, wherein the second polynucleotide encodes a transmembrane domain that is fused to the targeting agent.
169. A cell comprising (i) a first polynucleotide encoding a nucleic acid sequence encoding a fusogen, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
170. The cell of any of claims 167-169, wherein the first and/or second polynucleotide is a plasmid.
171. The cell of claim 170, wherein the plasmid of the first and/or second polynucleotide comprises one or more nucleic acids encoding proteins for lentivirus production.
172. The cell of any of claims 167-169, wherein the first and/or second polynucleotide is a vector.
173. The cell of any of claims 167-172, wherein the first and/or second polynucleotide is codon optimized.
174. The cell of any of claims 167-173, wherein: the first polynucleotide comprises at least one promoter that is operatively linked to control expression of the fusogen; and/or the second polynucleotide comprises at least one promoter that is operatively linked to control expression of the targeting agent.
175. The cell of claim 174, wherein the promoter of the first and/or second polynucleotide is a constitutive promoter.
176. The cell of claim 174, wherein the promoter of the first and/or second polynucleotide is an inducible promoter.
177. The cell of any of claims 166-176, wherein the cell is a producer cell used for producing a viral particle or virus-like particle.
178. The cell of claim 177, wherein the viral particle or virus-like particle is a retroviral particle or retrovirus-like particle.
179. The cell of claim 177 or claim 178, wherein the viral particle or virus-like particle is a lentiviral particle or lentivirus-like particle.
180. The cell of any of claims 166-179, wherein the cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells.
181. The cell of any of claims 166-180, wherein the cell is a 293T cell.
182. The cell of any of claims 166-181, wherein the cell comprises a viral nucleic acid.
183. The cell of claim 182, wherein the viral nucleic acid is a retroviral nucleic acid.
184. The cell of claim 182 or claim 183, wherein the viral nucleic acid is a lentiviral nucleic acid.
185. The cell of any of claims 182-184, wherein the viral nucleic acid lacks one or more genes involved in viral replication.
186. The cell of any of claims 182-185, wherein the viral nucleic acid comprises a nucleic acid sequence encoding a viral packaging protein selected from one or more of Gag, Pol, and Rev.
187. The cell of any of claims 182-186, wherein the viral nucleic acid comprises one or more, optionally all, of the following nucleic acid sequences: 5’ LTR (e.g., comprising U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT)/central termination sequence (CTS) (e.g., DNA flap), Poly A tail sequence, a posttranscriptional regulatory element (e.g., WPRE), a Rev response element (RRE), and 3’ LTR (e.g., comprising U5 and lacking a functional U3).
188. The cell of any of claims 166-187, wherein the target molecule is ASCT2.
189. The cell of any of claims 166-187, wherein the target molecule is CD117.
190. The cell of any of claims 166-189, wherein the fusogen is a viral fusion protein.
191. The cell of any of claims 166-190, wherein the fusogen is a viral envelope protein.
192. The cell of any of claims 166-191, wherein the fusogen is a VSV-G protein or a functional variant thereof.
193. The cell of any of claims 166-191, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
194. The cell of any of claims 166-191 and 193, wherein the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild- type BaEV inhibitory R peptide.
195. The cell of claim 168 or 169, wherein the fusogen is set forth in any of SEQ ID NO:254- 260.
196. The cell of claim 168, 169, or 195, wherein the fusogen is set forth in SEQ ID NO:261 (BaEVRLess).
197. The cell of claim 168, 169 or 195, wherein the fusogen is set forth in SEQ ID NO:262 (BaEVTR).
198. The cell of any of claims 166-191, wherein the fusogen is a Cocal virus G protein or a functional variant thereof.
199. The cell of any of claims 166-191, wherein the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
200. The cell of any of claims 166-191, wherein the fusogen is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof.
201. The cell of any of claims 166-191 and 200, wherein the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des-petits- ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
202. The cell of any of claims 166-191 and 200, wherein the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
203. The cell of any of claims 166-191, 200, and 202, wherein the fusogen is a Nipah virus fusion protein or a functional variant thereof.
204. The cell of any of claims 166-191, 200, 202, and 203, wherein the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
205. The cell of claim 204, wherein the NiV-G or biologically active portion thereof is a wildtype NiV-G protein or a functionally active variant or biologically active portion thereof.
206. The cell of claim 204 or claim 205, wherein the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N- terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4.
207. The cell of any of claims 204-206, wherein the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4.
208. The cell of any of claims 204-207, wherein the NiV-G protein or biologically active portion thereof does not include an initial methionine.
209. The cell of any of claims 204-208, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42.
210. The cell of any of claims 204-209, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
211. The cell of any of claims 166-210, wherein the fusogen comprises one or more modifications to reduce binding to its native receptor.
212. The cell of any of claims 204-208 and 211, wherein the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
213. The cell of any of claims 204-208, 211, and 212, wherein the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
214. The cell of any of claims 204-208 and 211-213, wherein the NiV-G protein or biologically active portion thereof comprises the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17.
215. The cell of any of claims 204-208 and 211-214, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17.
216. The cell of any of claims 204-208 and 211-213, wherein the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18.
217. The cell of any of claims 204-208, 211-213, and 216, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
218. The cell of any of claims 204-217, wherein the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.
219. The cell of any of claims 204-218, wherein the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30.
220. The cell of any of claims 204-219, wherein the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30.
221. The cell of any of claims 204-220, wherein the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:30.
222. The cell of any of claims 204-221, wherein the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16.
223. The cell of any of claims 204-222, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16.
224. The cell of any of claims 204-221, wherein the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21.
225. The cell of any of claims 204-221 and 224, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
226. The cell of any of claims 204-208, 211-213, 216-221, 224, and 225, wherein the NiV- G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
227. The cell of any of claims 166-226, wherein the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule.
228. The cell of any of claims 166-227, wherein the targeting agent is a single domain antibody that binds to the target molecule.
229. The cell of any of claims 166-228, wherein the targeting agent is a VHH that binds to the target molecule.
230. The cell of any of claims 166-228, wherein the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
231. A method of making a targeted lipid particle, comprising:
(a) introducing into a source cell the polynucleotide of any of claims 108-162 or 420, the plasmid of claim 163 or claim 164, or the vector of claim 165;
(b) culturing the source cell under conditions for producing a lipid particle comprising a lipid bilayer and the targeting agent and fusogen exposed on the surface of the lipid bilayer; and
(c) separating, enriching, or purifying the lipid particle from the source cell, thereby making the lipid particle.
232. A method of making a targeted lipid particle, comprising:
(a) introducing into a source cell (i) a first polynucleotide comprising a nucleic acid sequence encoding a fusogen and (ii) a second polynucleotide comprising a nucleic acid sequence encoding a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3;
(b) culturing the source cell under conditions for producing a lipid particle comprising a lipid bilayer and the targeting agent and fusogen exposed on the surface of the lipid bilayer; and
(c) separating, enriching, or purifying the lipid particle from the source cell, thereby making the lipid particle.
233. The method of claim 232, wherein the second polynucleotide encodes a transmembrane domain that is fused to the targeting agent.
234. The method of claim 232 or claim 233, wherein the first and/or second polynucleotide is a plasmid.
235. The method of claim 234, wherein the plasmid of the first and/or second polynucleotide comprises one or more nucleic acids encoding proteins for lentivirus production.
236. The method of claim 232 or claim 233, wherein the first and/or second polynucleotide is a vector.
237. The method of any of claims 226-230, wherein the first and/or second polynucleotide is codon optimized.
238. The method of any of claims 226-231, wherein: the first polynucleotide comprises at least one promoter that is operatively linked to control expression of the fusogen; and/or the second polynucleotide comprises at least one promoter that is operatively linked to control expression of the targeting agent.
239. The method of claim 232, wherein the promoter of the first and/or second polynucleotide is a constitutive promoter.
240. The method of claim 232, wherein the promoter of the first and/or second polynucleotide is an inducible promoter.
241. The method of any of claims 225-234, wherein the source cell is a mammalian cell.
242. The method of any of claims 225-235, wherein the source cell is a producer cell used for producing a viral particle or virus-like particle.
243. The method of claim 236, wherein the particle is a viral particle or virus-like particle.
244. The method of claim 236 or claim 237, wherein the viral particle or virus-like particle is a retroviral particle or retrovirus-like particle.
245. The method of any of claims 236-238, wherein the viral particle or virus-like particle is a lentivirus-like particle.
246. The method of any of claims 236-238, wherein the viral particle or virus-like particle is a lentiviral particle.
247. The method of any of claims 225-240, wherein the particle is pseudotyped with the fusogen.
248. The method of any of claims 236-241, wherein the producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells, optionally wherein the producer cell is a 293T cell.
249. The method of any of claims 231-248, wherein the method comprises transducing the source cell with packaging plasmids encoding a Gag-pol and Rev.
250. The method of any of claims 231-249, wherein the target molecule is ASCT2.
251. The method of any of claims 231-249, wherein the target molecule is CD117.
252. The method of any of claims 231-251, wherein the fusogen is a viral fusion protein.
253. The method of any of claims 231-252, wherein the fusogen is a viral envelope protein.
254. The method of any of claims 231-253, wherein the fusogen is a VSV-G protein or a functional variant thereof.
255. The method of any of claims 231-253, wherein the fusogen is a Cocal virus G protein or a functional variant thereof.
256. The method of any of claims 231-253, wherein the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
257. The method of any of claims 231-253, wherein the fusogen is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof.
258. The method of any of claims 231-253 and 257, wherein the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des- petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
259. The method of any of claims 231-253 and 257, wherein the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
260. The method of any of claims 231-253, 257, and 259, wherein the fusogen is a Nipah virus fusion protein or a functional variant thereof.
261. The method of any of claims 231-253, 257, 259, and 260, wherein the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
262. The method of claim 261, wherein the NiV-G or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof.
263. The method of claim 261 or claim 262, wherein the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4.
264. The method of any of claims 261-263, wherein the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4.
265. The method of any of claims 261-264, wherein the NiV-G protein or biologically active portion thereof does not include an initial methionine.
266. The method of any of claims 261-265, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42.
267. The method of any of claims 261-266, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 42.
268. The method of any of claims 231-267, wherein the fusogen comprises one or more modifications to reduce binding to its native receptor.
269. The method of any of claims 261-265 and 268, wherein the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
270. The method of any of claims 261-265, 268, and 269, wherein the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
271. The method of any of claims 261-265 and 268-270, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17.
272. The method of any of claims 261-265 and 268-271, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17.
273. The method of any of claims 261-265 and 268-270, wherein the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18.
274. The method of any of claims 261-265, 268-270, and 273, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
275. The method of any of claims 261-274, wherein the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.
276. The method of any of claims 261-275, wherein the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30.
277. The method of any of claims 261-276, wherein the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30.
278. The method of any of claims 261-277, wherein the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525- 546 of SEQ ID NO:30.
279. The method of any of claims 261-278, wherein the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16.
280. The method of any of claims 261-279, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16.
281. The method of any of claims 261-278, wherein the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21.
282. The method of any of claims 261-278 and 281, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
283. The method of any of claims 261-265, 268-270, 273-278, 281, and 282, wherein the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
284. The method of any of claims 231-283, wherein the targeting agent is an antibody or antibody fragment, a Design ankyrin repeat protein (DARPin), or an antigen-binding fibronectin type III (Fn3) scaffold that binds to the target molecule.
285. The method of any of claims 231-284, wherein the targeting agent is a single domain antibody that binds to the target molecule.
286. The method of any of claims 231-285, wherein the targeting agent is a VHH that binds to the target molecule.
287. The method of any of claims 231-285, wherein the targeting agent is a single chain variable fragment (scFv) that binds to the target molecule.
288. A targeted lipid particle produced by the method of any of claims 231-287 and 418.
289. A targeted lentiviral particle produced by the method of any of claims 231, 241-287 and 418.
290. A composition comprising a plurality of the lipid particle of any of claims 1-8, 10-107,
288 and 419.
291. A composition comprising a plurality of the lentiviral particle of any of claims 8-107,
289 and 419.
292. The composition of claim 290 or claim 291, wherein the composition further comprises a pharmaceutically acceptable excipient.
293. A method of transducing a target cell, the method comprising contacting a target cell with the lentiviral particle of any of claims 8-107, 289, and 419, or the composition of claim 291 or claim 292.
294. The method of claim 293, wherein the particle comprises an exogenous agent, and the transduction introduces the exogenous agent into the target cell.
295. A method of delivering an exogenous agent to a target cell, the method comprising contacting a target cell with the particle of any of claims 70-107, 288, 289, and 419, or the composition of any of claims 290-292, the particle or plurality of particles comprising an exogenous agent for delivery to the target cell.
296. The method of any of claims 293-295, wherein the contacting is in vitro or ex vivo.
297. The method of any of claims 293-295, wherein the contacting is in vivo in a subject.
298. A method of delivering an exogenous agent to a target cell in a subject, comprising administering to a subject the particle of any of claims 70-107, 288, 289, and 419, or the composition of any of claims 290-292, the particle or plurality of particles comprising an exogenous agent for delivery to a target cell in the subject.
299. The method of any of claims 294-298, wherein the exogenous agent is or encodes a therapeutic agent or a diagnostic agent.
300. The method of any of claims 297-299, wherein the exogenous agent is for treating a disease or condition in the subject.
301. A method of treating a disease or condition in a subject, comprising administering to a subject the particle of any of claims 70-107, 288, 289, or 419, or the composition of any of claims 290- 292, the particle or plurality of particles comprising an exogenous agent for treating a disease or condition in the subject.
302. The method of any of claims 297-301, wherein the method comprises administering to the subject one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood.
303. The method of any of claims 297-301, wherein the subject has previously been administered one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood.
304. A method of treating a disease or condition in a subject, comprising administering to a subject:
(a) one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood; and
(b) a lipid particle comprising a lipid bilayer, a fusogen exposed on the surface of the lipid bilayer, and an exogenous agent for treating a disease or condition in the subject.
305. The method of claim 302 or claim 304, wherein the administration of at least one of the one or more agents that stimulate mobilization is initiated prior to the administration of the particle.
306. A method of treating a disease or condition in a subject, comprising administering to a subject a lipid particle comprising a lipid bilayer, a fusogen exposed on the surface of the lipid bilayer, and an exogenous agent for treating a disease or condition in the subject, wherein the subject has previously been administered one or more agents that stimulate mobilization of bone marrow cells from the bone marrow to the peripheral blood.
307. The method of any of claims 301-306, wherein the exogenous agent is for delivery to a target cell for treating the disease or condition in the subject.
308. The method of any of claims 295-307, wherein the particle is a viral particle or virus-like particle.
309. The method of any of claims 293-308, wherein the lipid bilayer is derived from a membrane of a producer cell used for producing a viral particle or virus-like particle.
310. The method of any of claims 293-309, wherein the particle is prepared by a method comprising transducing a producer cell with plasmids encoding the fusogen and a Gag-pol and Rev.
311. The method of any of claims 308-310, wherein the viral particle or virus-like particle is a retroviral particle or retrovirus-like particle.
312. The method of any of claims 308-311, wherein the viral particle or virus-like particle is a lentivirus-like particle.
313. The method of any of claims 308-311, wherein the viral particle or virus-like particle is a lentiviral particle.
314. The method of any of claims 293-303, 305, and 307-313, wherein the particle is pseudotyped with the fusogen.
315. The method of any of claims 309-314, wherein the producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells.
316. The method of any of claims 309-315, wherein the producer cell is a 293T cell.
317. The method of any of claims 293-303, 305, and 307-316, wherein the particle is replication defective.
318. The method of any of claims 293-311 and 313-317, wherein the particle comprises a viral nucleic acid.
319. The method of claim 318, wherein the viral nucleic acid is a retroviral nucleic acid.
320. The method of claim 318 or claim 319, wherein the viral nucleic acid is a lentiviral nucleic acid.
321. The method of any of claims 318-320, wherein the viral nucleic acid comprises one or more, optionally all, of the following nucleic acid sequences: 5’ LTR (e.g., comprising U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT)/central termination sequence (CTS) (e.g., DNA flap), Poly A tail sequence, a posttranscriptional regulatory element (e.g., WPRE), a Rev response element (RRE), and 3’ LTR (e.g., comprising U5 and lacking a functional U3).
322. The method of any of claims 295-312 and 314-317, wherein the particle is devoid of viral genomic nucleic acids.
323. The method of any of claims 293-322, wherein the fusogen is a viral fusion protein.
324. The method of any of claims 293-323, wherein the fusogen is a viral envelope protein.
325. The method of any of claims 293-324, wherein the fusogen is a VSV-G protein or a functional variant thereof.
326. The method of any of claims 293-324, wherein the fusogen is a baboon endogenous virus (BaEV) envelope glycoprotein or a functional variant thereof.
327. The method of any of claims 293-324 and 326, wherein the fusogen is a truncated BaEV envelope glycoprotein that comprises a cytoplasmic tail with a partial inhibitory R peptide that is less than the full length wild- type BaEV inhibitory R peptide.
328. The method of any of claims 293-324, 326, and 326, wherein the fusogen is set forth in any of SEQ ID NO:254-260.
329. The method of any of claims 293-324 and 326, wherein the fusogen is set forth in SEQ ID NO:261 (BaEVRLess).
330. The method of any of claims 293-324 and 326, wherein the fusogen is set forth in SEQ ID NO:262 (BaEVTR).
331. The method of any of claims 293-324, wherein the fusogen is a Cocal virus G protein or a functional variant thereof.
332. The method of any of claims 293-324, wherein the fusogen is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
333. The method of any of claims 293-324, wherein the fusogen is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof.
334. The method of any of claims 293-324 and 333, wherein the fusogen is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des- petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
335. The method of any of claims 293-324 and 333, wherein the fusogen is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
336. The method of any of claims 293-324, 333, and 335, wherein the fusogen is a Nipah virus fusion protein or a functional variant thereof.
337. The method of any of claims 293-324, 333, 335, and 336, wherein the fusogen comprises a Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
338. The method claim 337, wherein the NiV-G or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof.
339. The method of claim 337 or claim 338, wherein the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4.
340. The method of any of claims 337-339, wherein the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4.
341. The method of any of claims 337-340, wherein the NiV-G protein or biologically active portion thereof does not include an initial methionine.
342. The method of any of claims 337-341, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42.
343. The method of any of claims 337-342, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 42.
344. The method of any of claims 293-343, wherein the fusogen comprises one or more modifications to reduce binding to its native receptor.
345. The method of any of claims 337-341 and 344, wherein the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
346. The method of any of claims 337-341, 344, and 345, wherein the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
347. The method of any of claims 337-341 and 344-346, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17.
348. The method of any of claims 337-341 and 344-347, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17.
349. The method of any of claims 337-341 and 344-346, wherein the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18.
350. The method of any of claims 337-341, 344-346, and 349, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
351. The method of any of claims 337-350, wherein the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.
352. The method of any of claims 337-351, wherein the NiV-F protein or biologically active portion thereof is a truncated NiV-F that is truncated by or by at least 22 amino acids or by or by at least 20 amino acids at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:30.
353. The method of any of claims 337-352, wherein the NiV-F protein or biologically active portion thereof has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein set forth in SEQ ID NO:30.
354. The method of any of claims 337-353, wherein the NiV-F protein or biologically active portion thereof comprises a deletion in its cytoplasmic tail and lacks amino acid residues 525- 546 of SEQ ID NO:30.
355. The method of any of claims 337-354, wherein the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16.
356. The method of any of claims 337-355, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO: 16.
357. The method of any of claims 337-354, wherein the NiV-F protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:21 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:21.
358. The method of any of claims 337-354 and 357, wherein the NiV-F protein or biologically active portion thereof is set forth in SEQ ID NO:21.
359. The method of any of claims 337-341, 344-346, 349-354, 357, and 358, wherein the NiV-G protein or biologically active portion thereof has the sequence set forth in SEQ ID NO: 18, and the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:21.
360. The method of any of claims 302-359, wherein the bone marrow cells are CD34+.
361. The method of any of claims 302-360, wherein the bone marrow cells are CD34+ progenitor cells.
362. The method of any of claims 302-361, wherein the bone marrow cells are hematopoietic stem cells.
363. The method of any of claims 302-362, wherein the one or more agents that stimulate mobilization are selected from the group consisting of stem cell factor (SCF), small molecule VLA-4 inhibitor BI05192, BOP (N-(henzenesulfonyl)-L-prolyl-L-0-(l-pyrrolidinylcarbonyl)tyrosine), heparin, granulocyte colony-stimulating factor (G-CSF), MGTA-145, and plerixafor (AMD3100).
364. The method of any of claims 302-362, wherein the one or more agents that stimulate mobilization comprise G-CSF.
365. The method of claim 364, wherein the G-CSF is administered to the subject daily on the two days, three days, four days, or five days prior to the administration of the first dose of the particle.
366. The method of claim 364 or claim 365, wherein the G-CSF is administered to the subject on the day of the administration of the first dose of the particle.
367. The method of any of claims 364-366, wherein the G-CSF is administered to the subject on the day of administration of a second or later dose of the particle.
368. The method of any of claims 302-367, wherein the one or more agents that stimulate mobilization comprise plerixafor.
369. The method of claim 368, wherein the plerixafor is administered to the subject on the day of the administration of the first dose of the particle.
370. The method of claim 368 or claim 369, wherein the plerixafor is administered to the subject on the day of the administration of a second or later dose of the particle.
371. The method of any of claims 302-370, wherein the one or more agents that stimulate mobilization are G-CSF and plerixafor.
372. The method of claim 371, wherein: the G-CSF is administered to the subject daily on the four days prior to the administration of the first dose of the particle; and the plerixafor is administered to the subject on the day of the administration of the first dose of the particle.
373. The method of claim 371, wherein: the G-CSF is administered to the subject (i) daily on the two days prior to the administration of the particle; (ii) on the day of the administration of the first dose of the particle; and (iii) on the day of administration of a second or later dose of the particle; and the plerixafor is administered to the subject on the day of administration of a second or later dose of the particle.
374. The method of any of claims 297-373, wherein the method comprises administering to the subject a transduction adjuvant.
375. The method of claim 374, wherein the transduction adjuvant is administered to the subject on the day of the administration of at least one dose of the particle.
376. The method of claim 374 or claim 375, wherein the transduction adjuvant is administered to the subject on the days of the administration of at least two doses of the particle.
377. The method of any of claims 374-376, wherein the transduction adjuvant is a cationic peptide.
378. The method of any of claims 374-377, wherein the transduction adjuvant is a histidine- rich cationic peptide.
379. The method of any of claims 374-378, wherein the transduction adjuvant is a cationic amphipathic peptide.
380. The method of any of claims 374-379, wherein the transduction adjuvant is derived from the LAH4 peptide family.
381. The method of any of claims 374-380, wherein the transduction adjuvant is Vectofusin- 1.
382. The method of any of claims 293-300, 302, 303, 305, and 307-381, wherein the target molecule is expressed on the target cell.
383. The method of any of claims 293-300, 302, 303, 305, and 307-382, wherein the target cell is a hematopoietic cell.
384. The method of any of claims 293-300, 302, 303, 305, and 307-383, wherein the target cell is CD34+.
385. The method of any of claims 293-300, 302, 303, 305, and 307-384, wherein the target cell is a CD34+ progenitor cell.
386. The method of any of claims 293-300, 302, 303, 305, and 307-385, wherein the hematopoietic cell is a hematopoietic stem cell.
387. The particle of any of claims 294-386, wherein the exogenous agent is present in the lumen.
388. The method of any of claims 294-387, wherein the exogenous agent is a protein or a nucleic acid.
389. The method of any of claims 294-388, wherein the exogenous agent is or encodes a factor associated with gene editing.
390. The method of any of claims 294-389, wherein the exogenous agent is or encodes a genome-modifying protein for gene editing a target gene encoding an endogenous protein.
391. The method of any one of claims 294-390, wherein the exogenous agent is a fusion protein between a viral structural protein and an exogenous agent.
392. The method of claim 391, wherein the fusion protein is a cleavable fusion protein comprising a cleavable linker positioned between the viral structural protein and the exogenous agent.
393. The method of claim 391 or 392, wherein the fusion protein comprises one or more nuclear localization sequences.
394. The particle of claim 391 or 392, wherein the fusion protein comprises one or more nuclear export sequences.
395. The method of any of claims 390-394, wherein the genome-modifying protein is associated with gene editing by a sequence-specific nuclease, a CRISPR-associated transposase (CAST), prime editing, or Programmable Addition via Site-specific Targeting Elements (PASTE).
396. The method of any of claims 390-395, wherein the genome-modifying protein is a sequence-specific nuclease.
397. The method of any of claims 390-396, wherein the sequence-specific nuclease is selected from the group consisting of an RNA-guided nuclease, a meganuclease, a transcription activatorlike effector nuclease (TALEN), and a zinc-finger nuclease (ZFN).
398. The method of any of claims 395-397, wherein the sequence-specific nuclease is an RNA-guided nuclease.
399. The method of claim 397 or claim 398, wherein the RNA-guided nuclease is TnpB.
400. The method of claim 397 or claim 398, wherein the RNA-guided nuclease comprises a Cas nuclease and a guide RNA (CRISPR-Cas combination).
401. The method of claim 400, wherein the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease.
402. The method of claim 400 or claim 401, wherein the Cas nuclease is a Type II or Type V Cas protein.
403. The method of any of claims 390-398 and 400-402, wherein the genome-modifying protein is selected from the group consisting of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxl l, Csyl, Csy2, Csy3, Mad7.
404. The method of any of claims 400-403, wherein the Cas is a Cas9 or a Casl2.
405. The method of any of claims 294-395, wherein the exogenous agent is or encodes a factor associated with base editing or prime editing (e.g., target-primed reverse transcription (TPRT)).
406. The method of any of claims 294-395 and 405, wherein the exogenous agent is or encodes a transposase, integrase, or recombinase.
407. The method of any of claims 294-395 and 405, wherein the exogenous agent is or encodes a DNA polymerase, RNA polymerase, or reverse-transcriptase.
408. The method of any of claims 294-407, wherein the exogenous agent is for use in gene therapy to correct a genetic deficiency in the target cell.
409. The method of claim 408, wherein the exogenous agent is a nucleic acid comprising a payload gene for correcting the genetic deficiency.
410. The method of any of claims 294-388, wherein the exogenous agent is or encodes a membrane protein.
411. The method of claim 410, wherein the membrane protein is an antigen receptor.
412. The method of claim 411, wherein the antigen receptor binds to an antigen expressed on cells associated with a disease or condition in the subject.
413. The method of claim 412, wherein the disease or condition is a cancer.
414. The method of any of claims 411-413, wherein the antigen receptor binds to an antigen expressed on tumor cells in the subject.
415. The method of any of claims 411-414, wherein the antigen receptor is a chimeric antigen receptor (CAR).
416. The method of any of claims 411-414, wherein the antigen receptor is an engineered T cell receptor (TCR).
417. The method of any of claims 293-300, 302, 303, 305, and 307-416, wherein the delivery to target cells expressing the target molecule is increased by or by greater than 1.1 -fold, 1.2-fold, 1.3- fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5- fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, or more compared to delivery to non-target cells not expressing the target molecule.
418. The method of any of claims 297-417, wherein the subject is a human subject.
419. The particle of any of claims 1-22 and 25-107, wherein the target molecule is CD 133.
420. The polynucleotide of any of claims 108-112 and 115-162, wherein the target molecule is CD133.
421. The cell of any of claims 167-187 and 190-230, wherein the target molecule is CD133.
422. The method of making a targeted lipid particle of any of claims 232-249 and 252-287, wherein the target molecule is CD 133.
423. The method of any of claims 306-418, wherein the fusogen is fused to a targeting agent that binds to a target molecule selected from the group consisting of ASCT1, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3.
424. The method of claim 423, wherein the target molecule is ASCT2.
425. The method of claim 423, wherein the target molecule is CD117.
426. The method of claim 423, wherein the target molecule is CD133.
427. The particle of claim 419, the polynucleotide of claim 420, the cell of claim 421, the method of claims 422, 423 or claim 426, wherein the targeting agent is a CD 133 binding agent that is an antibody or antigen-binding fragment comprising a variable heavy (VH) chain and a variable light (VL) chain selected from a) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 271, 272, 273, respectively, and a VL chain comprising a CDR- Ll, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 280, 281, 282, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 284, 285, and 286, respectively; (c) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 298, 299, and 300, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively; (d) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 307, 308, and 309, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 311, 312, and 313, respectively; or (e) a VH chain comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 298, 290, and 291, respectively, and a VL chain comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 293, 294, and 295, respectively.
428. The particle of claim 419, the polynucleotide of claim 420, the cell of claim 421, the method of claims 422, 423 or claim 426, wherein the targeting agent is a CD 133 binding agent that is an antibody or antigen-binding fragment comprising a variable heavy (VH) chain and a variable light (VL) chain selected from a) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 314, 315, and 273, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 316, 317, and 282, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 284, 285, and 286, respectively; (c) a VH comprising a a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 318, 319, and 300, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR- L3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively; (d) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 320, 321, and 309, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 311, 312, and 313, respectively; and (e) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 322, 323, and 291, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 293, 294, and 295, respectively.
429. The particle, the polynucleotide, the cell, or the method of claim 427 or claim 428, wherein the CD 133 binding agent is selected from a) a CD 133 binding agent comprising a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 270, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 274, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; b) a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 279, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 283, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; c) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 297, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 301, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; d) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 306, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 310, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; e) a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 288, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 292, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
430. The particle, the polynucleotide, the cell, or the method of any of claims 427-429, wherein the CD 133 binding agent is a scFv.
431. The particle, the polynucleotide, the cell, or the method of any of claims 427-430, wherein the CD 133 binding agent comprises the amino acid sequence set forth in any one of SEQ ID NOs: 269, 278, 287, 296 or 305, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
432. The particle of claim 24, the polynucleotide of claim 114, the cell of claim 189, the method of claims 251 or claim 425, wherein the targeting agent is a CD117 binding agent that is a VHH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
433. The particle, the polynucleotide, the cell or the method of claim 432, wherein the CD117 binding agent comprises the amino acid sequence set froth in any one of SEQ ID NOS: 324-374.
434. An anti-CDl 17 binding agent comprising a VHH single domain antibody comprising a CDR-H1, a CDR-H2, and a CDR-H3 contained within an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374.
435. An anti-CDl 17 binding agent comprising a VHH single domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 324-374, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
436. The anti-CDl 17 binding agent of claim 434 or claim 435, wherein the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOS: 324-374.
437. A viral fusion protein comprising a viral envelope protein and the anti-CDl 17 binding agent of any one of claims 434-436.
438. A viral fusion protein comprising a viral envelope protein and an anti-CDl 33 binding agent, wherein the anti-CDl 33 binding agent is an antibody or antigen-binding fragment comprising a variable heavy (VH) chain and a variable light (VL) chain selected from a) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 314, 315, and 273, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 275, 276, 277, respectively; (b) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 316, 317, and 282, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 284, 285, and 286, respectively; (c) a VH comprising a a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 318, 319, and 300, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively; (d) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 320, 321, and 309, respectively, and a VL comprising a CDR- Ll, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 311, 312, and 313, respectively; and (e) a VH comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 322, 323, and 291, respectively, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 293, 294, and 295, respectively.
439. The viral fusion protein of claim 438, wherein the CD133 binding agent is selected from a) a CD 133 binding agent comprising a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 270, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 274, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; b) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 279, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 283, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; c) a CD 133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 297, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 301, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; d) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 306, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 310, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; e) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 288, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO: 292, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
440. The viral fusion protein of claim 438 or claim 439, wherein the CD133 binding agent is a scFv.
441. The viral fusion protein of any of claims 438-440, wherein the CD133 binding agent comprises the amino acid sequence set forth in any one of SEQ ID NOs: 269, 278, 287, 296 or 305, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
442. The viral fusion protein of any of claims 437-441, wherein the viral envelope protein is a VSV-G protein or a functional variant thereof.
443. The viral fusion protein of any of claims 437-441, wherein the viral envelope protein is a Cocal virus G protein or a functional variant thereof.
444. The viral fusion protein of any of claims 437-441, wherein the viral envelope protein is an Alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof.
445. The viral fusion protein of any of claims 437-441, wherein the viral envelope protein is a Paramyxovirus fusion protein (e.g., a Morbillivirus or Henipavirus) or a functional variant thereof.
446. The viral fusion protein of any of claims 437-441 and 445, wherein the viral envelope protein is a Morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des-petits-ruminants virus, Phocine distemper virus, or Rinderpest virus) or a functional variant thereof.
447. The viral fusion protein of any of claims 437-441 and 445, wherein the viral envelope protein is a Henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus, or Langya virus) or a functional variant thereof.
448. The viral fusion protein of any of claims 437-441, 445 and 447, wherein the viral envelope protein is a Nipah virus fusion protein or a functional variant thereof.
449. The viral fusion protein of any of claims 437-441, 445, 447 and 448, wherein the viral envelope protein comprises a Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof.
450. The viral fusion protein of claim 449, wherein the binding agent is fused to the C- terminus of the NiV-G protein or biologically active portion thereof.
451. The viral fusion protein of claim 449 or claim 450, wherein the NiV-G or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof.
452. The viral fusion protein of any of claims 449-451, wherein the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein set forth in SEQ ID NO:4.
453. The viral fusion protein of any of claims 449-452, wherein the NiV-G protein or biologically active portion thereof is a biologically active portion that is a truncated NiV-G that has a deletion of amino acids 2-34 at or near the N-terminus of wild-type NiV-G set forth in SEQ ID NO:4.
454. The viral fusion protein of any of claims 449-453, wherein the NiV-G protein or biologically active portion thereof does not include an initial methionine.
455. The viral fusion protein of any of claims 449-454, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 42 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42.
456. The viral fusion protein of any of claims 449-455, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO:42.
457. The viral fusion protein of any of claims 437-456, wherein the viral envelope protein comprises one or more modifications to reduce binding to its native receptor.
458. The viral fusion protein of any of claims 449-457, wherein the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein or biologically active portion thereof that exhibits reduced binding to Ephrin B2 or Ephrin B3.
459. The viral fusion protein of any of claims 449-458, wherein the NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to numbering set forth in SEQ ID NO:4.
460. The viral fusion protein of any of claims 449-459, wherein the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 17.
461. The viral fusion protein of any of claims 449-460, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 17.
462. The viral fusion protein of any of claims 449-461, wherein the NiV-G protein or the biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least at or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18.
463. The viral fusion protein of any of claims 449-462, wherein the NiV-G protein or biologically active portion thereof is set forth in SEQ ID NO: 18.
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