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WO2025235682A1 - Peptides targeting tfrc for blood-brain barrier crossing - Google Patents

Peptides targeting tfrc for blood-brain barrier crossing

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Publication number
WO2025235682A1
WO2025235682A1 PCT/US2025/028247 US2025028247W WO2025235682A1 WO 2025235682 A1 WO2025235682 A1 WO 2025235682A1 US 2025028247 W US2025028247 W US 2025028247W WO 2025235682 A1 WO2025235682 A1 WO 2025235682A1
Authority
WO
WIPO (PCT)
Prior art keywords
aav
protein
cell
aav capsid
tfrc
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
PCT/US2025/028247
Other languages
French (fr)
Inventor
Matthew R. TIFFANY
David S. OJALA
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Sangamo Therapeutics Inc
Original Assignee
Sangamo Therapeutics Inc
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Filing date
Publication date
Application filed by Sangamo Therapeutics Inc filed Critical Sangamo Therapeutics Inc
Publication of WO2025235682A1 publication Critical patent/WO2025235682A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • 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
    • A61K48/0008—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • This application relates to peptides and engineered AAV (adeno-associated virus) capsids that mediate blood-brain barrier crossing by interacting with TFRC (Transferrin receptor, also known as transferrin receptor protein 1, TFR1, and CD71).
  • AAV adeno-associated virus
  • AAV capsids with improved properties e.g., improved tropism to a target cell or tissue upon systemic administration
  • AAV capsid modifications influence targeting of cellular receptors.
  • a target cell or tissue e.g., a CNS cell or tissue
  • BBB blood-brain barrier
  • One approach to achieving this goal is to engineer capsids that interact with receptors that are highly expressed on the human bloodbrain barrier.
  • targeting molecules i.e., targeting peptides
  • a CNS-targeting molecule interacts with (e.g., binds to) TFRC (transferrin receptor), thereby enabling the CNS- targeting molecule to cross the BBB.
  • the CNS-targeting molecule comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1-7.
  • the amino acid sequence is fused or conjugated to a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.
  • a method for identifying CNS-targeting molecule that crosses the BBB comprising selecting for variant AAV capsids that interact with TFRC.
  • an adeno-associated virus (AAV) capsid protein that interacts with (e.g., binds to) TFRC (transferrin receptor), thereby enabling the AAV capsid protein to cross the BBB.
  • the AAV capsid protein comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1-7.
  • the amino acid sequence is inserted into a parent capsid (“Parent Capsid”) at an insertion site (“Peptide Insertion Site”), optionally as shown in a single row in Tables 1 to 7.
  • the amino acid sequence comprises a peptide sequence as indicated in a single row in Tables 1 to 7, and optionally wherein the parent capsid and/or the insertion site is/are as indicated in the same single row as shown in a single row in Tables 1 to 7.
  • the amino acid sequence is inserted into a parental capsid, optionally wherein the parental capsid is selected from any one of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC-BBB (i.e., CNSRCV300 in US Application No.63/606,012).
  • an engineered AAV capsid protein is provided, wherein the engineered AAV capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to or comprises a sequence set forth in SEQ ID NOs: 251-268 designated as CNSRCV420, CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427, CNSRCV428, CNSRCV429, CNSRCV431, CNSRCV432, CNSRCV433, CNSRCV434, CNSRCV436, CNSRCV437, CNSRCV438, or CNSRCV439.
  • SEQ ID NOs: 251-268 designated as CNSRCV420, CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427, CNSRCV428, CNSRCV429, CNSRCV431,
  • nucleic acid molecule encoding an engineered AAV capsid protein described herein is provided.
  • a host cell comprising such a nucleic acid molecule is provided.
  • a composition comprising: 1) an adeno- associated virus (AAV) capsid protein as described herein; and 2) an expression construct comprising a coding sequence for a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and/or therapeutic payload.
  • the payload of interest is a therapeutic payload
  • the therapeutic payload comprises a DNA binding domain, optionally wherein the therapeutic payload comprises a fusion protein.
  • the payload of interest comprises a therapeutic protein, a zinc finger protein, a CRISPR-associated DNA binding protein, a TALE protein, an antibody, an enzyme, a regulatory RNA, a Bxbl serine recombinase, or a DNA recombinase protein.
  • a method of delivering a payload of interest to a cell or a tissue wherein a coding sequence for the therapeutic payload is encapsidated in an AAV capsid protein as described herein, and wherein the AAV capsid protein interacts with TFRC.
  • delivering the payload of interest to the cell or the tissue comprises crossing a BBB.
  • a method of expressing or repressing a therapeutically relevant gene of interest in a cell comprising contacting the cell with the composition as described herein.
  • a method of treating a disease in a subject comprising administering to the subject the composition as described herein.
  • an AAV capsid protein, a nucleic acid construct, or a host cell as described herein is provided for the manufacture of a medicament in a method as described herein.
  • a method for identifying an AAV capsid variant that crosses the BBB or exhibits enhanced delivery to a cell or a tissue comprising selecting for variant AAV capsids that interact with TFRC.
  • FIG. 1 illustrates a schematic of the approach used to pan AAV capsid libraries against an immobilized receptor.
  • the receptor in this case human transferrin receptor, is biotinylated and then immobilized on a streptavidin coated bead.
  • the AAV capsid libraries are then panned in parallel against the immobilized receptor or a streptavidin bead control without a receptor.
  • AAV library genomes are extracted from each sample, amplified via PCR, and subjected to next-generation sequencing to quantify the enrichment of capsid variants. Capsids that specifically interact with the receptor are identified by comparing enrichment in the immobilized receptor condition relative to the bead only control.
  • FIG. 2 illustrates a schematic of the approach used to assess AAV library transduction in cells overexpressing a receptor, in this case human transferrin receptor, relative to AAV library transduction in cells expressing a fluorescent protein transfection control.
  • a receptor in this case human transferrin receptor
  • FIG. 2 illustrates a schematic of the approach used to assess AAV library transduction in cells overexpressing a receptor, in this case human transferrin receptor, relative to AAV library transduction in cells expressing a fluorescent protein transfection control.
  • Cells are transfected with a plasmid encoding a strong ubiquitous promoter that drives the expression of the receptor or a negative control fluorescent protein. Forty-eight hours after transfection, the cells are transduced with the AAV capsid libraries. Cells are incubated for 72 hours to allow for AAV-mediated expression of transgene mRNA.
  • RNA is extracted from cells, reverse transcribed to cDNA, and then PCR is used to amplify the AAV cDNA.
  • FIG. 3 illustrates a schematic of the approach used to assess AAV library binding in cells overexpressing a receptor, in this case human transferrin receptor, relative to AAV library binding in cells expressing a fluorescent protein transfection control.
  • a receptor in this case human transferrin receptor
  • FIG. 3 illustrates a schematic of the approach used to assess AAV library binding in cells overexpressing a receptor, in this case human transferrin receptor, relative to AAV library binding in cells expressing a fluorescent protein transfection control.
  • Cells are transfected with a plasmid encoding a strong ubiquitous promoter that drives the expression of the receptor or a negative control fluorescent protein. Forty-eight hours after transfection, the cells are transduced with the AAV capsid libraries. Cells are incubated for 1 hour to allow for AAV binding to cells. DNA is extracted from cells and then PCR is used to amplify the AAV genome. Next-generation sequencing analysis is used to quantify enrichment of capsid variants. Capsids that specifically interact with the receptor are
  • FIG. 4 The figure shows that AAV library screens can identify capsid variants that specifically target human or cynomolgus macaque transferrin receptor. Three representative examples of capsid library screens that were conducted for transferrin receptor are shown:
  • FIG. 5 The figure shows that AAV library screens can identify capsid variants that specifically target human or cynomolgus macaque transferrin receptor. Two representative examples of capsid library screens that were conducted for transferrin receptor are shown:
  • capsids that exhibited specific enrichment for human or cynomolgus macaque transferrin receptor orthologs are colored in green.
  • the marginal axis histograms represent capsids that were identified in only the receptor condition (y-axis) or the negative control (x-axis).
  • FIG. 6 shows the performance of five exemplary capsid variants that exhibit enrichment for binding to immobilized human transferrin receptor, and transduction of cells overexpressing either human or cynomolgus macaque transferrin receptor in library assessments.
  • FIG. 7(A)-(G) shows Tables 1, 2, 3, 4, 5, 6, and 7.
  • FIG. 8 shows the transgene expression mediated by individual engineered capsids in cells overexpressing human or cynomolgus macaque transferrin receptor, relative to the transgene expression in cells expressing a fluorescent protein transfection control.
  • Representative engineered capsids, designed to target transferrin receptor and described herein, are presented as illustrative examples.
  • Neuro2A cells were transfected with a plasmid encoding a strong ubiquitous promoter that drives the expression of human transferrin receptor, cynomolgus macaque transferrin receptor, or a negative control fluorescent protein.
  • the cells were transduced with the AAV capsid at a multiplicity of infection of 3E2, 1E3, 3E3, or 1E4 vector genomes per cell. Cells were incubated for 72 hours to allow for AAV-mediated expression of transgene mRNA. RNA was extracted from cells and RT-qPCR was conducted to quantify mRNA transgene expression. Data were normalized to expression of the housekeeping gene GAPDH.
  • Another capsid exhibits enhanced transduction only in cells expressing human transferrin receptor, but not in cells expressing cynomolgus macaque transferrin receptor.
  • the parent capsid AAV9 shows no significant increase in transgene expression in cells overexpressing human or cynomolgus macaque transferrin receptor relative to cells overexpressing a fluorescent protein transfection control.
  • FIG. 9 shows the fold change improvement in AAV mediated transgene expression in cells overexpressing human or cynomolgus macaque transferrin receptor relative to cells expressing the transfection control.
  • Representative engineered capsids, designed to target transferrin receptor and described herein, are presented as illustrative examples.
  • a nonlinear regression model was used to interpolate relative transgene expression values for each capsid-receptor condition. These values were then scaled to the transfection control value for each capsid.
  • FIG. 10 shows the fold change improvement in AAV mediated transgene expression in cells overexpressing human or cynomolgus macaque transferrin receptor relative to the parent capsid AAV9.
  • Representative engineered capsids, designed to target transferrin receptor and described herein, are presented as illustrative examples.
  • FIG. 11 shows representative examples of the binding kinetics of capsids described herein that were engineered to target transferrin receptor.
  • Bio-layer interferometry experiments were performed to assess the binding of capsids to human or cynomolgus macaque transferrin receptor.
  • Human or cynomolgus macaque transferrin receptors were immobilized on Octet BLI sensors and binding to each capsid analyte was measured across a range of capsid concentrations.
  • Two examples of capsids that exhibit cross-reactive binding responses to both human and cynomolgus macaque transferrin receptor are shown.
  • FIG. 12 shows a representative example of an engineered capsid described herein that exhibits a human specific binding response.
  • Bio-layer interferometry experiments were performed to assess the binding of capsids to human or cynomolgus macaque transferrin receptor.
  • Human or cynomolgus macaque transferrin receptors were immobilized on Octet BLI sensors and binding to each capsid analyte was measured across a range of capsid concentrations.
  • FIG. 13 shows the binding kinetics of the parent capsid AAV9, an AAV serotype that does not target transferrin receptor.
  • Bio-layer interferometry experiments were performed to assess the binding of AAV9 to human or cynomolgus macaque transferrin receptor.
  • Human or cynomolgus macaque transferrin receptors were immobilized on Octet BLI sensors and binding to the AAV9 capsid analyte was measured across a range of capsid concentrations.
  • AAV9 exhibits no binding response to human or cynomolgus macaque transferrin receptor.
  • targeting molecules i.e., targeting peptides
  • a specific cellular receptor preferably where in the receptor is expressed on the blood brain barrier.
  • the receptor is the transferrin receptor, TFRC.
  • CNS-targeting molecules comprising a targeting peptide sequence indicated in Tables 1-7 are provided.
  • engineered AAV capsid proteins are provided.
  • a targeting peptide is inserted into a parental AAV capsid, for example an AAV3B capsid protein (SEQ ID NO: 246), an AAV5 capsid protein (SEQ ID NO: 247), an AAV6 capsid protein (SEQ ID NO: 248), an AAV8 capsid protein (SEQ ID NO: 249), or an AAV9 capsid protein (SEQ ID NO: 250).
  • the targeting peptide is any of the targeting peptides disclosed in Tables 1-7.
  • the peptide sequence is inserted into the parental capsid at any of the peptide insertion sites disclosed in Tables 1-7.
  • the targeting peptide functions to target the CNS-targeting molecule to a specific target tissue (e.g., CNS tissue).
  • disclosed herein is the development of libraries encoding engineered AAV capsid proteins, wherein members of the library encode engineered AAV capsid proteins having different sequences, and wherein some members of the library encode an AAV capsid protein having a desired characteristic compared to a natural/wild-type AAV serotype.
  • disclosed herein is the development of libraries encoding engineered AAV capsid proteins with a desired characteristic compared to a parent capsid.
  • libraries of AAV capsid proteins with a desired characteristic compared to a parent capsid are described herein.
  • the desired characteristic is enhanced cell or tissue tropism as compared to the parent capsid, for example, enhanced cell or tissue tropism to the central nervous system (CNS) as compared to the parent capsid.
  • the desired characteristic is increased penetrance through the blood brain barrier following administration to a subject.
  • the desired characteristic is wider distribution throughout the multiple brain regions, e.g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and/or hippocampus.
  • the desired characteristic is elevated genetic material expression in multiple brain regions.
  • the desired characteristic is delivery of genetic material of interest to a desired tissue, cell, or organelle.
  • each member of a library comprises one or more of a) a nucleic acid sequence encoding an AAV capsid protein comprising an engineered variant AAV sequence; b) a nucleic acid sequence encoding barcode; c) nucleic acid sequence(s) encoding a promoter(s); d) a nucleic acid sequence encoding a unique molecular identifier (UMI); and combinations thereof.
  • each member of the library also includes genetic material to be delivered to and expressed in a cell or tissue of interest.
  • each member of the library also includes a polyA sequence.
  • each engineered AAV capsid protein was synthesized as an oligo pool.
  • each member of a library comprises one or more (such as 1-10) of a nucleic acid sequence encoding an AAV capsid protein comprising a) nucleic acid sequences encoding one or more (such as 1-10) barcodes: b) nucleic acid sequences encoding one or more (such as 1-10) promoters; c) nucleic acid sequences encoding one or more (such as 1-10,000) unique molecular identifiers (UMIs); or combinations thereof.
  • each member of the library also includes genetic material to be delivered to a cell or tissue of interest.
  • each member of the library also includes a polyA sequence.
  • each of the one or more (such as 1-10) barcodes is linked to the identity of a single engineered AAV capsid protein.
  • each of the barcodes is linked to one or more (such as 1-10,000) UMIs.
  • a nucleic acid comprising a barcode is added to the genome of each AAV capsid in a library.
  • a unique barcode is bioinformatically linked to each different variant sequence that is represented within the library, for example, each different variant AAV sequence.
  • the DNA sequences encoding an AAV variant sequence are synthesized to further comprise a random or specified barcode.
  • the barcode may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more nucleotides.
  • each AAV variant sequence is linked to at least 2 distinct barcodes.
  • each barcode is linked to one or more (such as 1-10,000) UMIs.
  • each member of the library comprises a nucleic acid comprising more than one barcode sequences (such as 1-10). In some embodiments, each member of the library comprises two or more nucleic acids (such as 1-10) each comprising a barcode sequence. In some embodiments, each member of the library comprises a first nucleic acid comprising a first barcode and a second nucleic acid comprising a second barcode. In some embodiments, the first nucleic acid comprising the first barcode and the second nucleic acid comprising the second barcode are different. In some embodiments, each of the first nucleic acid comprising the first barcode and the second nucleic acid comprising the second barcode is independently operatively linked to a promoter.
  • each capsid is linked to at least one unique barcode. In some embodiments, each capsid is linked to at least two unique barcodes using a bioinformatic look-up table. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from the neuron specific promoter. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from the neuron specific human Synapsin 1 promoter. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from the ubiquitous CMV promoter.
  • libraries are created encoding engineered AAV capsid proteins that comprise at least one mutation relative to a parent capsid, for example, the parent capsid AAV3B, AAV5, AAV6, AAV8, or AAV9 (SEQ ID NOs: 246-250).
  • the engineered AAV capsid proteins contain a peptide sequence inserted within a parent capsid protein, for example, the parent capsid AAV3B, AAV5, AAV6, AAV8, or AAV9 (SEQ ID NOs: 246-250).
  • the engineered AAV capsid proteins contain a peptide sequence inserted within a surface exposed loop of a parent capsid protein, for example, the parent capsid AAV3B, AAV5, AAV6, AAV8, or AAV9 (SEQ ID NOs: 246-250).
  • the libraries are packaged in HEK293 cells where the helper functions (e.g. E2A, E4, VA, El A and E1B) are supplied in trans.
  • the AAV rep function comprises rep78, rep 68, rep 52, and rep40 genes.
  • the rep genes are supplied in trans.
  • the start codon of the rep78 and/or the rep68 gene is altered from ACG to ATG to increase replication of the capsid library construct containing inverted terminal repeats (ITRs), thereby improving AAV library manufacturing yield.
  • the cap genes are supplied as genetic material that is packaged into the manufactured AAVs.
  • the capsid gene is controlled by the p40 promoter such that it is only expressed during manufacturing in HEK293 cells in the presence of helper virus functions.
  • a method of identifying an engineered AAV capsid protein with a desired characteristic compared to a natural/wild-type AAV serotype comprising: (i) contacting an immobilized receptor protein, a cell, a cell line, or tissue in vitro or in vivo with any one of the libraries of engineered AAV capsid proteins, (ii) allowing the engineered AAV capsid proteins in said library to transduce the cell, cell line, or tissue; (iii) recovering from the immobilized receptor protein, cell, cell line, or tissue the AAV variant; and (iv) identifying the engineered AAV capsid protein with the desired characteristic.
  • the steps for directed evolution of engineered AAV capsid proteins to identify engineered AAV capsid proteins with a desired characteristic compared to a natural/wild-type AAV serotype comprise (i) modifying the natural/wild-type AAV serotype to create variant capsids; (ii) packaging of the variant AAVs in producer cells wherein adenovirus helper and AAV rep functions are supplied in trans; (iii) purification of viral capsid library pools; (iv) administration of the pools in vitro or in vivo; (v) recovery of engineered AAV capsid proteins from target tissues or cell lines; (vi) next-generation sequencing to determine the identity of the engineered variant capsid sequences; (vii) repeated rounds of in vitro or in vivo selection where
  • the desired characteristic includes enhanced tissue tropism as compared to the natural/wild-type AAV serotype. In some embodiments, the desired characteristic includes enhanced tissue tropism for tissues of the peripheral nervous system as compared to the natural/wild-type AAV serotype. In some embodiments, the desired characteristic includes enhanced tissue tropism of the central nervous system as compared to the natural/wild-type AAV serotype.
  • compositions comprising engineered AAV capsid proteins and methods of making and using the same.
  • the engineered AAV capsid proteins interact with a receptor, e.g. TFRC.
  • the engineered AAV capsid proteins demonstrate binding to a receptor, e.g. TFRC.
  • the engineered AAV capsid proteins may be delivered to one or more of target cells, tissues, organs, or organisms.
  • the engineered AAV capsid protein has enhanced tropism for a cell or tissue, e.g., for the delivery of genetic material to a specific cell or tissue, for example a CNS tissue or a CNS cell, or cells and tissues of a muscle.
  • the engineered AAV capsid proteins may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ.
  • the engineered AAV capsid proteins that are desired to have tropism for CNS cells may have enhanced tropism for neurons, astrocytes, oligodendrocytes, microglia, endothelial cells, Schwann cells, and reduced tropism for liver and dorsal root ganglion.
  • an engineered AAV capsid protein comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all contiguous amino acids of a peptide sequence inserted within a parent capsid protein.
  • the peptide sequence is inserted within or near a surface-exposed loop of the parent capsid protein.
  • the parent capsid is AAV3B, AAV5, AAV6, AAV8, or AAV9 (SEQ ID NOs: 246-250).
  • the peptide sequence is inserted within or near amino acids 588 through 589 corresponding to the sequence of AAV3B (SEQ ID NO: 246).
  • the peptide sequence is inserted within or near amino acids 577 through 578 corresponding to the sequence of AAV5 (SEQ ID NO: 247). In some embodiments the peptide sequence is inserted within or near amino acids 589 through 590 corresponding to the sequence of AAV6 (SEQ ID NO: 248). In some embodiments the peptide sequence is inserted within or near amino acids 590 through 591 corresponding to the sequence of AAV8 (SEQ ID NO: 249). In some embodiments the peptide sequence is inserted within or near amino acids 587 through 590 corresponding to the sequence of AAV9 (SEQ ID NO: 250).
  • an amino acid sequence is inserted into a parent capsid (“Parent Capsid”) at an insertion site (“Peptide Insertion Site”).
  • the inserted amino acid sequence comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1 to 7.
  • an engineered AAV capsid protein is provided comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1 to 7.
  • the amino acid sequence comprises a peptide sequence as indicated in a single row in Tables 1 to 7, and optionally wherein the parent capsid and/or the insertion site is/are as indicated in the same single row.
  • an amino acid sequence is inserted into a parental capsid, optionally wherein the parental capsid is selected from any one of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC- BBB.
  • the inserted amino acid sequence comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1 to 7.
  • the engineered AAV capsid protein is at least 80%, 85%,
  • CNSRCV420 CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427, CNSRCV428, CNSRCV429, CNSRCV431, CNSRCV432, CNSRCV433, CNSRCV434, CNSRCV436, CNSRCV437, CNSRCV438, or CNSRCV439.
  • the engineered AAV capsid proteins have advantages over wild-type AAV capsid proteins. In some embodiments, these advantages including (i) enhanced cell or tissue tropism as compared to the natural/wild-type AAV serotype, for example, enhanced cell or tissue tropism to the central nervous system (CNS) as compared to the natural/wild-type AAV serotype (ii) increased penetrance through the blood brain barrier following administration to a subject, (iii) wider distribution throughout the multiple brain regions, for example, the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and/or hippocampus, (iv) elevated expression of genetic material in multiple brain regions.
  • CNS central nervous system
  • the engineered AAV capsids enhance the delivery of genetic material to multiple regions of the brain including for example, the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and/or hippocampus, (v) is delivery of genetic material of interest to a desired tissue, cell, or organelle.
  • the engineered AAV capsid proteins and genetic material described herein may be delivered to one or more (such as 1-10) target cells, tissues, organs, or organisms.
  • the engineered AAV capsid proteins have enhanced tropism for a specific target cell type, tissue or organ.
  • the engineered AAV capsid protein has enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively).
  • engineered AAV capsid proteins are produced recombinantly and are an adeno-associated virus (AAV) serotype such as AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAV3, AAV4, AAV7, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC-BBB, or a combination thereof.
  • AAV adeno-associated virus
  • engineered AAV capsid proteins are produced recombinantly and are based on any one or more (such as 1-15) AAV serotypes known in the art.
  • AAV Adeno-associated virus
  • AAV are capable of infecting a wide range of cells including quiescent cells and dividing cells.
  • AAV can be modified so that it contains the components necessary for the assembly of a functional recombinant virus or viral particle.
  • the AAV is engineered to interact with a specific receptor, e.g. TFRC.
  • the AAV is engineered to target a specific tissue and/or cell, for example, CNS tissue and/or cell.
  • the AAV is engineered to deliver specific genetic material to a tissue and/or cell.
  • the AAV is engineered to target a blood brain barrier receptor, for example, TFRC.
  • an engineered AAV may be based on any natural or recombinant AAV serotype. Different AAV serotypes have different characteristics such as different packaging, tropism, and transduction profiles.
  • the engineered AAV capsid proteins are based on a wild-type AAV serotype.
  • the AAV serotype comprises AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, or AAV9.
  • the AAV serotype comprises less well-characterized AAV serotypes such as AAV3, AAV4, AAV7, AAV11, AAVrhlO, AAVrh39, or AAVrh74.
  • the AAV serotype is an engineered AAV serotype such as STAC-BBB.
  • the engineered AAV capsid protein is derived from multiple AAV serotypes, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more AAV serotypes.
  • AAV variant capsid proteins derived from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more AAV serotypes are combined to create chimeric capsids.
  • combinatorial libraries are generated by modifying nucleic acids encoding AAV capsid proteins from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more serotypes in the same pool.
  • different AAV serotypes are different in their ability to direct or modulate an AAV particle to a particular cell or tissue.
  • the AAV serotype can be modified to interact with a receptor, e.g. TFRC.
  • the AAV serotype modified to interact with a receptor has an altered tropism.
  • the AAV serotype can be modified to increase the tropism of the AAV particle to cells or tissues of the central nervous system (CNS).
  • the AAV serotype can be modified to increase tropism of the AAV particle to cells or tissues of the peripheral nervous system (PNS).
  • the modified AAV serotype has a desired characteristic compared to a parental AAV serotype.
  • the modified AAV serotype allows for increased penetration of the blood brain barrier following administration to a subject.
  • the modified AAV serotype causes increased biodistribution to a brain region.
  • the brain region comprises the frontal cortex, the sensory cortex, the motor cortex, the cerebellar cortex, the hippocampus, the thalamus, or the putamen.
  • the brain comprises any brain region known in the art.
  • the modified AAV serotype causes increased biodistribution to more than one brain regions, for example, 2 brain regions, 3 brain regions, 4 brain regions, 5 brain regions, 6 brain regions, 7 brain regions, 8 brain regions, 9 brain regions, or 10 brain regions. In some embodiments, the modified AAV serotype causes increased biodistribution to 1- 10 brain regions. In some embodiments, the modified AAV serotype are useful in elevating genetic material expression in multiple brain regions. In some embodiments, the modified AAV serotype are used to deliver genetic material of interest to a desired tissue, cell, or organelle.
  • the modified AAV serotype causes increased biodistribution to regions of the spinal cord.
  • the region of the spinal cord comprises any of the thoracic spinal cord region, the lumbar spinal cord region, and/or the cervical spinal cord region.
  • the region of the spinal cord includes any region of the spinal cord known in the art.
  • the modified AAV serotype comprises an inserted sequence having at least 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of a Peptide Sequence shown in Tables 1-7.
  • the inserted sequence is inserted into a parent capsid serotype as shown in Tables 1-7.
  • the inserted sequence is inserted into a parent capsid sequence at or near the Peptide Insertion Site as shown in Tables 1-7.
  • the modified AAV serotype comprises an inserted peptide sequence described herein (e.g., a Peptide Sequence shown in Tables 1-7).
  • the modified AAV serotype comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences corresponding to SEQ ID NOs: 251-268 designated as CNSRCV420, CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427, CNSRCV428, CNSRCV429, CNSRCV431, CNSRCV432, CNSRCV433, CNSRCV434, CNSRCV436, CNSRCV437, CNSRCV438, or CNSRCV439.
  • SEQ ID NOs: 251-268 designated as CNSRCV420, CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427
  • the modified AAV sequence comprises any one of the sequences corresponding to SEQ ID NOs: 251-268 designated as CNSRCV420, CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427, CNSRCV428, CNSRCV429, CNSRCV431, CNSRCV432, CNSRCV433, CNSRCV434, CNSRCV436, CNSRCV437, CNSRCV438, or CNSRCV439.
  • the genome of the AAV comprises a single-strand DNA (ssDNA) molecule that is approximately between about 2.5 kb and about 5.0 kb in length. In some embodiments, the genome of the AAV comprises a self-complementary DNA (scDNA) molecule that is approximately between about 0.5 kb and about 2.5 kb in length. In some embodiments, the AAV genome contains inverted terminal repeats (ITRs) that flank the 5’ and 3’ ends of the AAV molecule. In some embodiments, the ITRs contain origins of replication for the viral genome. In some embodiments, the length of the ITRs is about 145 bp in length, for example, between about 130 bp in length and 160 bp in length.
  • the AAV genome comprises the rep and cap genes.
  • the AAV genome nucleotide includes nucleotide sequences that encode four non- structural Rep proteins (Rep 78, Rep68, Rep52, Rep40, encoded by Rep genes).
  • the AAV viral genome includes nucleotide sequences that encode the three capsid, or structural, proteins (i.e., VP1, VP2, VP3, encoded by the cap gene).
  • the rep proteins are used for replication and packaging.
  • the capsid proteins are assembled to create the protein shell of the AAV.
  • the engineered AAV capsid proteins assemble to form AAV particles.
  • the engineered AAV capsid proteins interact with a receptor expressed at the blood brain barrier, for example, TFRC.
  • the AAV particles that have enhanced tropism for a target tissue e.g., CNS and PNS
  • the AAV particles include engineered AAV variant sequences that alter tropism to a particular cell-type, tissue, organ or organism, in vivo, ex vivo or in vitro.
  • the AAV particles are capable of penetrating the blood brain barrier.
  • the AAV particles may be delivered to one or more target cells, tissues, organs, or organisms.
  • the AAV particles demonstrate enhanced tropism for a target cell type, tissue or organ.
  • the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively), or cells and tissues of a muscle.
  • the AAV particles may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ.
  • the AAV particles can be used to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating. In some embodiments, the AAV particles are used to deliver any cargoes of interest, or example, therapeutic cargoes.
  • the AAV particles are used to deliver a viral genome (i.e., a genetic payload) to a tissue or cells such as CNS or PNS cell or tissue.
  • a viral genome i.e., a genetic payload
  • the delivered viral genome may include genetic material of interest, such as, for example, genetic material that encodes an engineered DNA recombinase protein, a fusion protein comprising a DNA-binding domain (e.g., a zinc finger or a TALE protein) fused to a functional domain (e.g., to modulate DNA function or to cleave DNA), an antibody, an enzyme, regulatory RNA, a CRISPR protein, or a cDNA, amongst others.
  • the viral genome includes 2 ITR sequences.
  • the ITR sequences flank the genetic material of interest.
  • the ITR sequences are complementary to each other. In some embodiments, the ITR sequences are not complementary to each other.
  • one ITR sequence is a self-complementary ITR.
  • the ITR regions are derived from the same serotype as the capsid protein.
  • the ITR regions are derived from AAV2 serotype.
  • the ITR regions are derived from a serotype known to the art. ITR regions may be between 100 and 150 nucleotides in length.
  • targeting peptide sequences are disclosed herein.
  • the sequences enhance or enable interaction with transferrin receptor (TFRC).
  • the sequences can function to target a cell or a tissue, for example, as a general CNS-targeting molecule or sequence.
  • the targeting sequences function as a general CNS-targeting molecule sequence.
  • the CNS-targeting sequence is fused or conjugated to a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.
  • any one of the targeting sequences described herein may be fused or conjugated to a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.
  • CNS-targeting sequences may be utilized to enable a small molecule, an antibody, zinc finger protein, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein to cross the blood brain barrier.
  • the targeting sequences are part of an engineered AAV capsid protein.
  • the engineered AAV capsid protein is any engineered AAV capsid protein disclosed herein.
  • the targeting sequences enable the binding of an AAV capsid protein to a specific receptor, e.g. TFRC. In some embodiments, the targeting sequences enable the binding of an AAV capsid protein to a specific receptor expressed at the blood brain barrier, e.g. TFRC. In some embodiments, the targeting sequences modulate the binding affinity of an AAV capsid protein to a specific receptor, e.g. TFRC.
  • the sequences may increase tropism of an AAV capsid protein to a cell or tissue of the CNS.
  • the cell of the CNS is a neuron (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), a glial cells (e.g., microglia, astrocytes, oligodendrocytes) and/or a supporting cells of the brain such as immune cells (e.g., T cells).
  • a neuron e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.
  • a glial cells e.g., microglia, astrocytes, oligodendrocytes
  • T cells e.g., T cells
  • the CNS tissue is the cortex (e.g., frontal, parietal, occipital, temporal), thalamus, hypothalamus, striatum, caudate nucleus, hippocampus, putamen, basal ganglia, entorhinal cortex, cerebellum, or spinal cord.
  • cortex e.g., frontal, parietal, occipital, temporal
  • thalamus e.g., hypothalamus, striatum, caudate nucleus, hippocampus, putamen, basal ganglia, entorhinal cortex, cerebellum, or spinal cord.
  • the sequences increase tropism of an AAV capsid protein to a cell, region, or tissue of the PNS.
  • the cell or tissue of the PNS is dorsal root ganglion (DRG).
  • the sequences decrease tropism of an AAV capsid protein to a cell, region, or tissue of the PNS.
  • the cell or tissue of the PNS is dorsal root ganglion (DRG).
  • the targeting sequence comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1 to 7.
  • the sequence comprises the amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1 to 7.
  • the targeting peptide sequence is inserted within a parent AAV capsid sequence.
  • the inserted sequence is inserted into a parent capsid serotype as shown in Tables 1-7.
  • the inserted sequence is inserted into a parent capsid sequence at or near the Peptide Insertion Site as shown in Tables 1-7.
  • the targeting peptide sequence is inserted within or near a surface-exposed loop of a parent AAV capsid sequence.
  • the parent capsid sequence is any of the serotypes AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, or STAC-BBB.
  • the peptide sequence is inserted within or near amino acids 588 through 589 corresponding to the sequence of AAV3B (SEQ ID NO: 246). In some embodiments the peptide sequence is inserted within or near amino acids 577 through 578 corresponding to the sequence of AAV5 (SEQ ID NO: 247). In some embodiments the peptide sequence is inserted within or near amino acids 589 through 590 corresponding to the sequence of AAV6 (SEQ ID NO: 248). In some embodiments the peptide sequence is inserted within or near amino acids 590 through 591 corresponding to the sequence of AAV8 (SEQ ID NO: 249). In some embodiments the peptide sequence is inserted within or near amino acids 587 through 590 corresponding to the sequence of AAV9 (SEQ ID NO: 250). Administering Engineered AAV Capsid Proteins to Subjects
  • AAV capsid proteins containing the targeting peptides described herein mediate enhanced delivery to cells and tissues relative to AAV capsid proteins that lack the targeting peptides.
  • the AAV capsid protein administered to subjects comprises an engineered AAV sequence described herein.
  • the engineered AAV capsid proteins described herein encapsidate genetic material of interest to be delivered to a cell of interest.
  • the genetic material of interest may be a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and/or therapeutic payload.
  • the engineered AAV capsid proteins described herein enable delivery of genetic material to a cell of interest.
  • the genetic material may encode a research, diagnostic, and/or therapeutic payload.
  • the genetic material encodes a zinc finger protein, a TALE protein, a recombinase protein, and/or a CRISPR protein, or fragments thereof.
  • the genetic material encodes one or more antibodies or an antibody fragments.
  • the genetic material encodes one or more regulatory RNA, such as RNAi agents or microRNAs.
  • the genetic material can include sequences that are coding sequences. In some embodiments, the genetic material can include sequences that are non-coding sequences. In some embodiments, the genetic material can include sequences that are both coding sequences and non-coding sequences. In some embodiments, the expression of the genetic material is capable of being regulated. In some embodiments, the genetic material comprises elements that are regulatable.
  • mRNA is encoded in the genetic material. In some embodiments, the mRNA is codon optimized.
  • the genetic material encodes a gene therapy product.
  • a gene therapy product can include a peptide, a polypeptide, or an RNA molecule that when expressed carries out a desired therapeutic effect.
  • the therapeutic effect is treating any one or more diseases or disorders described herein.
  • a promoter is operably linked to the genetic material to be delivered to the cell.
  • the promoter comprises a tissue and/or cell specific promoter.
  • the one more promoters comprise a ubiquitous promoter.
  • ubiquitous promoters include cytomegalovirus (CMV), chicken P-actin (CBA), ubiquitin C (UBC), and elongation factor la-subunit (EFl -a), amongst others.
  • CMV cytomegalovirus
  • CBA chicken P-actin
  • UBC ubiquitin C
  • EFl -a elongation factor la-subunit
  • the promoter comprises a cell type and/or tissue specific type promoter.
  • Exemplary cell type and/or tissue specific promoters include the human synapsin promoter (hSynl), only expressed in neurons, or the transthyretin promoter (TTR), expressed in hepatocytes.
  • tissue specific promoters for use in the methods and compositions of the invention include cytokeratin 18 and 19 (epithelial cell specific, Other cell-specific promoters include GFAP promoter (astrocytes), TBG promoter (liver), MHCK promoter (skeletal muscle), MYH6 promoter (cardiomyocytes).
  • tissue specific or cell specific promoters can restrict expression to tissues or cells of the CNS or PNS.
  • tissue specific or cell specific promoters can be used to restrict expression to neurons of the sympathetic system, the parasympathetic system, astrocytes, microglia, oligodendrocytes, and/or Schwann cells.
  • the promoters are naturally occurring promoters. In some embodiments, the promoter is synthetic. In some embodiments, the promoter is derived from mammals, humans, viruses, or plants. In some embodiments, the promoters are truncated. In some embodiments, the promoter is mutated.
  • the genetic material of interest comprises a gene editing system or portions of a gene editing system.
  • the gene editing system is capable of inducing single or double-stranded breaks into nucleic acid sequences at one or more site of interest.
  • the gene editing system is capable of inserting, substituting, or deleting a base or a sequence of bases into nucleic acid sequences at one or more site of interest.
  • the gene editing system includes a CRISPR-Cas system.
  • the gene editing system includes a TALEN.
  • the gene editing system includes a zinc finger nuclease.
  • the gene editing system includes a modified recombinase protein.
  • the genetic material of interest comprises an epigenetic regulation system or components of an epigenetic regulation system for general or targeted gene regulation.
  • the epigenetic regulation system is capable of modifying chromatin structure or altering epigenetic marks on nucleic acid sequences.
  • the epigenetic regulation system is capable of promoting or repressing gene expression without altering the underlying DNA sequence.
  • the epigenetic regulation system includes a CRISPR-dCas system fused to epigenetic effector domains.
  • the epigenetic regulation system includes a transcription activator or repressor domain tethered to a programmable DNA-binding protein.
  • the epigenetic regulation system includes hi stone-modifying enzymes or DNA methyltransferases targeted to a specific genomic locus.
  • the epigenetic regulation system includes a TALEN.
  • the epigenetic regulation system includes a zinc finger protein fused to an epigenetic effector domain, for example, a zinc finger repressor or a zinc finger activator.
  • engineered AAV sequences described herein are fused or coupled to an active agent.
  • a sequence is fused or coupled to an active agent through conjugation.
  • the active agent comprises a therapeutic agent.
  • the therapeutic agent comprises an antibody or a portion of an antibody (e.g., Fc region).
  • the sequence is fused to a Fc region of an antibody.
  • the sequence is fused to the C-terminus of the Fc region.
  • the sequence is fused to the N-terminus of the Fc region.
  • the therapeutic agent comprises an RNAi agent (e.g., siRNA, shRNA, IncRNA, piRNA, snoRNA, or miRNA).
  • RNAi agent e.g., siRNA, shRNA, IncRNA, piRNA, snoRNA, or miRNA.
  • the sequence is fused or coupled directly to at least on strand of the RNAi.
  • the sequence is fused or coupled to at least one strand of RNAi using a linker.
  • the sequence is fused or coupled to the sense strand of RNAi.
  • the sequence is fused or coupled to the antisense strand of RNAi.
  • the active agent comprises a diagnostic agent.
  • the diagnostic agent comprises a detectable moiety such as a fluorophore.
  • the active agent is a small molecule.
  • compositions herein can be included in pharmaceutical compositions.
  • the pharmaceutical compositions can include one or more excipients or diluents to (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release of the genetic material; (4) alter the biodistribution (e.g., target the composition to specific tissues or cell types); (5) increase the translation of encoded protein; (6) alter the release profile of encoded protein and/or (7) allow for regulatable expression of the genetic material.
  • compositions described herein can be administered periodically, such as once or twice a day, or any other suitable time period.
  • pharmaceutical compositions may be administered to a subject in need once a week, once every other week, once every three weeks, once a month, every other month, every three months, every six months, every nine months, once a year, every eighteen months, every two years, every thirty months, or every three years.
  • compositions described herein can be formulated in a wide variety of dosage forms, including but not limited to nasal, pulmonary, oral, topical, or parenteral dosage forms for clinical.
  • Each of the dosage forms can comprise various solubilizing agents, disintegrating agents, surfactants, fillers, thickeners, binders, diluents such as wetting agents or other pharmaceutically acceptable excipients.
  • the compositions described herein can also be formulated for injection, insufflation, infusion, or intradermal exposure.
  • an injectable formulation may comprise the disclosed compositions in an aqueous or non-aqueous solution at a suitable pH and tonicity.
  • the compositions can be included liquid dosage form for oral administration, such as suspensions, emulsions, or syrups.
  • the pharmaceutical compositions described herein function to increase the stability, increase transduction or transfection efficiency, impact biodistribution, increase expression of the protein, and/or alter the release profile.
  • compositions described herein e.g., engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins
  • methods for introducing the compositions described herein e.g., engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins
  • the methods comprise introducing into cells and/or tissues any of the compositions described herein in an amount sufficient to modulate, e.g., increase, the production of a target mRNA and/or protein in the cells and/or tissues.
  • the compositions described herein are delivered via a localized delivery route.
  • the localized delivery route includes any one or more of intramuscular administration, intraparenchymal administration, and intracerebral administration, amongst others.
  • the compositions described herein are administered via a localized delivery route through a bolus infusion.
  • compositions described herein are administered through systemic administration.
  • systemic administration includes intravenous administration.
  • compositions described herein are administered to the central nervous system of via intraventricular administration and/or intrathecal administration. In some embodiments, the compositions described herein are administered to the central nervous system via systemic administration. In some embodiments, the systemic administration is intravenous (IV) injection. In some embodiments, the compositions described herein are administered to the central nervous system via administration into the cerebrospinal fluid.
  • IV intravenous
  • the compositions described herein are administered to the central nervous system via administration into the cerebrospinal fluid.
  • compositions can be delivered to target cells or target tissue including, but not limited to, the CNS, heart, lung, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testes, prostate, eye, blood, lymph, or oral mucosa.
  • target cells or target tissue including, but not limited to, the CNS, heart, lung, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testes, prostate, eye, blood, lymph, or oral mucosa.
  • the target cell or tissue includes, but is not limited to CNS, heart, lung, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testes, prostate, eye, blood, lymph, or oral mucosa.
  • the target cell or target tissue is a CNS cell or tissue.
  • the target cell or tissue is liver cell or tissue.
  • the target cell includes, but is not limited to, neurons, glial cells, astrocytes, oligodendroglia, microglia, Schwann cells, ependymal cells, hepatocytes, stellate fat storing cells, Kupffer cells, liver endothelial cells, epithelial cells, cardiomyocytes, smooth muscle cells, T-cells, B cells, hematopoietic stem cells, and embryonic stem cells.
  • compositions described herein are delivered to the central nervous system through the cerebral spinal fluid pathway. In some embodiments, compositions described herein are administered to the central nervous system via intraparenchymal delivery. In some embodiments, the compositions described herein are administered to the central nervous system via intracranial delivery. In some embodiments, the compositions described herein are delivered to the central nervous system via intraocular delivery. In some embodiments, the compositions described herein are administered to the brain. In some embodiments, the compositions described herein are administered to the brain via injection into the brain. In some embodiments, the compositions described herein are administered to the brain via intrahippocampal injection.
  • compositions described herein are administered as part of a composition that allows for extended release.
  • the compositions comprises a formulation that includes a depot.
  • compositions described herein engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins.
  • the disclosed compositions can be used to treat any one or more of muscular or neuromuscular disorders, neurooncological disorders, neurological diseases/disorders, and neurodegenerative disorders, amongst others.
  • the disclosed compositions can be used to treat any one or more of Alzheimer’s disease, Huntington’s disease; autism; Parkinson’s disease; Spinal muscular atrophy, Friedreich’s ataxia.
  • the disclosed compositions are used in treatments through any of the methods of delivery described herein.
  • compositions described herein e.g., engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins
  • delivering the compositions described herein into targeted cells inhibiting or activating the gene expression and protein production, and ameliorating symptoms of the disease or condition in the subject.
  • the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.
  • An adeno-associated virus (AAV) capsid protein comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 1-245 as shown in a single row in Tables 1 to 7.
  • AAV capsid protein of any one of embodiments 1-3 wherein the amino acid sequence is inserted into a parental capsid, optionally wherein the parental capsid is selected from any one of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC-BBB.
  • An AAV capsid protein wherein the AAV capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to or comprises a sequence set forth in SEQ ID NOs: 251-268 designated as CNSRCV420, CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427, CNSRCV428, CNSRCV429, CNSRCV431, CNSRCV432, CNSRCV433, CNSRCV434, CNSRCV436, CNSRCV437, CNSRCV438, or CNSRCV439.
  • SEQ ID NOs: 251-268 designated as CNSRCV420, CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427, CNSRCV428, CNSRCV429, CNSRCV431, CNSRCV432,
  • AAV capsid protein of any one of embodiments 1-5 wherein the AAV capsid protein interacts with transferrin receptor (TFRC), thereby enabling delivery of the AAV capsid protein to a cell or a tissue, optionally wherein interaction with TFRC enables the AAV capsid protein to cross a blood-brain barrier (BBB).
  • TFRC transferrin receptor
  • BBB blood-brain barrier
  • AAV capsid protein of any one of embodiments 6-8 wherein the amino acid sequence is inserted into a parental capsid, optionally wherein the parental capsid is selected from any one of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC-BBB.
  • AAV adeno-associated virus
  • a host cell comprising the nucleic acid molecule of embodiment 11.
  • a composition comprising: 1) an AAV capsid protein comprising of any one of embodiments 1-10; and 2) an expression construct comprising a coding sequence for a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and/or therapeutic payload.
  • composition of embodiment 13, wherein the payload of interest is a therapeutic payload, and wherein the therapeutic payload comprises a DNA binding domain, optionally wherein the therapeutic payload comprises a fusion protein.
  • the payload of interest comprises a therapeutic protein, a zinc finger protein, a CRISPR-associated DNA binding protein, a TALE protein, an antibody, an enzyme, a regulatory RNA, a Bxbl serine recombinase, or a DNA recombinase protein.
  • a method of activating, expressing, repressing, or modulating the expression of a therapeutically relevant gene of interest in a cell comprising contacting the cell with a composition of any one of embodiments 13-15.
  • a method of treating a disease in a subject comprising administering to the subject a composition of any one of embodiments 13-15.
  • a method for identifying an AAV capsid variant that crosses the BBB or exhibits enhanced delivery to a cell or a tissue comprising selecting for AAV capsids that interact with TFRC, optionally comprising selecting for AAV capsids that interact with both human and cynomolgus macaque TFRC.
  • a method for identifying an AAV capsid variant that crosses the BBB or exhibits enhanced delivery to a cell or a tissue comprising selecting for AAV capsids that comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 1-245 as shown in a single row in Tables 1 to 7.
  • a targeting molecule that interacts with TFRC thereby enabling delivery of the targeting molecule to a cell or a tissue, optionally wherein the interaction with TFRC enables the targeting molecule to cross the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • AAV AAV
  • small molecule an antibody
  • zinc finger protein Cas protein
  • exosome exosome
  • scFV scFV
  • ASO antisense oligonucleotide
  • siRNA siRNA
  • lipid lipid nanoparticle
  • polymer virus-like particle (VLP)
  • VLP virus-like particle
  • bocavirus dendrimer
  • aptamer or recombinant protein.
  • a composition comprising: 1) a targeting molecule of any one of embodiments 23-26; and 2) a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and/or therapeutic payload.
  • the payload of interest encodes or comprises a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.
  • a method of delivering a therapeutically relevant gene or protein of interest to a cell comprising contacting the cell with the composition of any one of embodiments 27-28.
  • a method of modulating the expression or activity of a therapeutically relevant gene of interest or protein in a cell comprising contacting the cell with the composition of any one of embodiments 27-28.
  • a method of treating a disease in a subject comprising administering to the subject the composition of any one of embodiments 27-28.
  • 34. A method for identifying a targeting molecule that targets a cell or a tissue, optionally wherein the targeting molecule crosses a BBB, comprising selecting for targeting molecules that interact with TFRC, optionally comprising selecting for targeting molecules that interact with both human and cynomolgus macaque TFRC.
  • a method for identifying a targeting molecule that crosses the BBB or exhibits enhanced delivery to a cell or a tissue comprising selecting for targeting molecules that comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 1-245 as shown in a single row in Tables 1 to 7.
  • Capsid libraries used in round 1 screening were constructed by insertion of peptides into the exposed loops of the parent capsid proteins. Gibson assembly was used to generate the capsid libraries where the introduced peptides were encoded by the primers used to amplify the assembly fragments. Two PCR products from the parent capsid gene sequence were amplified (left and right fragments), these included an overlap region to facilitate assembly using the Gibson assembly procedure into a plasmid backbone (see e.g. Gibson et al (2009) Nat Meth 6(5):343-345).
  • AAV9 capsid library used in round 1 screening was constructed by inserting trimer-19 synthesized codon block oligos using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs catalog number E2621) between amino acids 587 and 590 with replacement of amino acids 588 and 589.
  • capsid variants for library screening were synthesized as an oligo pool.
  • Each capsid peptide was synthesized with unique nucleotide sequences encoding the peptide, and each peptide was linked to at least three distinct barcodes.
  • the oligo pool was cloned into a linearized intermediate plasmid, followed by cloning of a constant donor sequence to separate the barcode and peptide region and generate the full AAV vector construct. Expression of barcodes was driven by a ubiquitous CMV promoter.
  • the peptide sequences listed in Table 1 were inserted into AAV3B (SEQ ID NO: 246) between amino acids 588 and 589.
  • the peptide sequences listed in Table 2 were inserted into AAV5 (SEQ ID NO: 247) between amino acids 577 and 578.
  • the peptide sequences listed in Table 3 were inserted into AAV6 (SEQ ID NO: 248) between amino acids 589 and 590.
  • the peptide sequences listed in Tables 4 and 6 were inserted into AAV8 (SEQ ID NO: 249) between amino acids 590 and 591.
  • the peptide sequences listed in Tables 5 and 7 were inserted into AAV9 (SEQ ID NO: 250) between amino acids 454 and 455 or between 588 and 589 or between 587 and 590 with replacement of 588 or 589, as indicated.
  • AAV capsid libraries were manufactured in HEK293 cells. Briefly, AAV libraries were produced by triple transient transfection of the capsid library plasmid, pXX6 helper (encodes essential adenovirus genes E4, E2A, and VA), and with supplementation of Rep in trans. Capsids were purified by cesium density centrifugation and buffer exchanged into PBS plus 0.001% PF-68 by dialysis. DNase-resistant viral genomic titers were measured by quantitative real time PCR.
  • Recombinant human transferrin receptor was diluted to 2 mg/mL in PBS (without calcium and magnesium).
  • EZ-Link NHS-LC-Biotin (Thermo Fisher Catalog #21336) was added to a final concentration of 10 mM and the mixture was incubated on ice for 2 hours. After incubation, the biotinylation reaction was quenched by adding 500 mM glycine to a final concentration of 50 mM and incubating on ice for 30 minutes. The same procedure was applied to biotinylate bovine serum albumin for use as a blocking agent.
  • the pre-cleared AAV capsid library was added to the receptor-coated beads and rotated for an hour at room temperature.
  • the unbound capsids were separated from the bead bound capsids using a tube magnet.
  • the beads were washed three times with SuperBlock (PBS) Blocking Buffer (ThermoFisher Catalog #37515).
  • the receptor bound AAV capsids were then eluted from the beads with Pierce IgG Elution Buffer pH 2.0 (ThermoFisher Catalog #21028).
  • Vector genomes were extracted using the Maxwell RSC Viral Total Nucleic Acid Purification Kit (Promega Catalog # AS 1330) and samples were prepared for NGS using Kapa HiFi Hotstart ReadyMix (Roche Catalog #KK2602).
  • Amplification of library specific amplicons was performed with the following cycling conditions: 95oC for 3:00 min; 25 cycles at 98oC for 20 sec; 58oC for 15 sec; 72oC for 30 sec followed by 72oC for 1 minute. Amplification was qualitatively confirmed by agarose gel electrophoresis and relative apparent amplification was used to determine the dilution of amplicons needed for indexing. Illumina plate level i5 and well level i7 indices were added to the amplicons with 10 cycles of amplification: 95oC for 3:00 min; 10 cycles at 98oC for 20 sec; 60oC for 15 sec; 72oC for 30 sec followed by 72oC for 1 minute.
  • samples were pooled and purified using Qiagen GeneRead Size Selection Kit following the manufacturer’s protocol. Samples were sequenced on Illumina MiSeq platform using MiSeq Reagent Kit v2. Following NGS of library amplicons the reads were demultiplexed and features were extracted using a custom bioinformatic pipeline.
  • variants interacting with recombinant human TFRC were recovered from the bead bound fraction and re-cloned in a subsequent capsid library, produced and re-screened for binding to recombinant human TFRC. Binding variants went through a total of three selections before being included in the pooled round 2 library for further confirmation both against recombinant human TFRC protein and in cells overexpressing human or cynomolgus macaque TFRC.
  • Neuro2A cells were seeded in 10 cm dishes coated with poly-D-lysine (PDL) at a density of 3E6 cells per dish. 24 hours later cells were transfected with 1 microgram of plasmid encoding human or cynomolgus macaque TFRC under the control of the ubiquitous cytomegalovirus (CMV) promoter. In parallel a transfection control plate was transfected with 1 microgram of plasmid encoding a fluorescent protein (GFP or mRuby) under the control of the ubiquitous cytomegalovirus (CMV) promoter. Lipofectamine 3000 was used for transfection of the plasmid DNA. Cell culture media was changed 24 hours post-transfection to remove transfection reagents.
  • PDL poly-D-lysine
  • Illumina plate level i5 and well level i7 indices were added to the amplicons with 10 cycles of amplification: 95oC for 3:00 min; 10 cycles at 98oC for 20 sec; 60oC for 15 sec; 72oC for 15 sec followed by 72oC for 1 minute. Finally, samples were pooled and purified using Qiagen GeneRead Size Selection Kit following the manufacturer’s protocol. Samples were sequenced on the Illumina MiSeq platform using a MiSeq Reagent Kit v2. Following next-generation sequencing of library amplicons the reads were demultiplexed and features (the barcode or peptide sequence) were extracted using a custom bioinformatic pipeline.
  • the extracted barcode was used to query a pre-determined lookup table and return the identity of the corresponding capsid variant.
  • log2FC log2 fold change
  • Capsid variants that were enriched for binding to recombinant human TFRC protein were synthesized as a pooled round 2 library.
  • capsids were evaluated in cells overexpressing the human or cynomolgus macaque ortholog of TFRC. Both a transduction and a binding assay were completed to assay cell entry and binding, respectively. The fold change enrichment of each capsid was determined by next-generation sequencing and normalized to capsid abundance in the administered library.
  • the average log2FC for each assay is shown in Tables 1, 2, 3, 4, 5, 6 and 7 for peptides inserted into the parent capsid AAV3B (Table 1), AAV5 (Table 2), AAV6 (Table 3), AAV8 (Tables 4 and 6), or AAV9 (Tables 5 and 7). Empty cells indicate that the capsid was not detected in that assay, potentially due to limited sample recovery or sequencing depth.
  • Table 1 Round 2 library performance for peptides inserted into the parent capsid AAV3B [0110] Table 1 is shown in Figure 7.
  • Table 4 and Table 6 are shown in Figure 7.
  • Table 4 shows peptides that exhibit targeting specificity for human transferrin receptor.
  • Table 6 shows peptides that target both human and cynomolgus macaque transferrin receptor.
  • Table 5 and Table 7 are shown in Figure 7.
  • Table 5 shows peptides that exhibit targeting specificity for human transferrin receptor.
  • Table 7 shows peptides that target both human and cynomolgus macaque transferrin receptor.
  • Neuro2A cells were seeded in 96-well tissue culture plates at a density of 3.5E4 cells/well and transfected with 100 ng/well of a TFRC overexpression construct or a fluorescent protein transfection control using Lipofectamine 3000. The media was changed 24 hours post transfection. 48 hours post transfection, the cells were transduced with AAV in reduced serum EMEM media (2% FBS) at a multiplicity of infection of 3E2, 1E3, 3E3, or 1E4 vector genomes per cell.
  • AAV capsids contained an expression cassette encoding a GFP fluorescent protein under the control of the ubiquitous CAG promoter.
  • Capsids engineered to target transferrin receptor exhibit enhanced transduction in cells overexpressing transferrin receptor relative to a transfection control.
  • Representative engineered capsids, designed to target transferrin receptor and described herein, are presented as illustrative examples. Some capsids engineered to target transferrin receptor exhibit higher transduction in cells overexpressing human, or cynomolgus macaque transferrin receptor relative to cells overexpressing a fluorescent protein transfection control.
  • Another capsid exhibits enhanced transduction only in cells expressing human transferrin receptor, but not in cells expressing cynomolgus macaque transferrin receptor.
  • the parental capsid AAV9 shows no significant increase in transgene expression in cells overexpressing human or cynomolgus macaque transferrin receptor relative to cells overexpressing a fluorescent protein transfection control.
  • Binding of AAV capsids to transferrin receptor was measured by bio-layer interferometry on an Octet Red96e instrument. All steps were performed at 30°C shaking at 1,000 rpm. Purified human or cynomolgus macaque transferrin receptors containing an N- terminal Fc domain were diluted to 5 pg/mL in PBS with 0.05% Tween20 and 0.1% BSA and immobilized on Octet BLI anti-human Fc capture biosensors (Sartorius Catalog # 18-5060) to produce a shift of approximately 1.3 nm.
  • Capsids were diluted in PBS with 0.05% Tween20 and 0.1% BSA across a 2-fold capsid dilution series ranging in concentration from 3.125E10 to 1E12 vg/mL. Capsid association to each sensor was performed for 150 seconds followed by a 350 second dissociation step.
  • amino acid sequences of the parent capsids are defined as follows:
  • amino acid sequences of individual capsids are defined as follows (inserted
  • CNSRCV420 capsid amino acid sequence SEQ ID NO: 251:
  • CNSRCV421 capsid amino acid sequence SEQ ID NO: 252:
  • CNSRCV422 capsid amino acid sequence SEQ ID NO: 253:
  • CNSRCV423 capsid amino acid sequence SEQ ID NO: 254:
  • CNSRCV424 capsid amino acid sequence SEQ ID NO: 255:
  • CNSRCV425 capsid animo acid sequence SEQ ID NO: 256:
  • CNSRCV426 capsid animo acid sequence SEQ ID NO: 257:
  • CNSRCV427 capsid animo acid sequence SEQ ID NO: 258:
  • CNSRCV428 capsid animo acid sequence SEQ ID NO: 259:
  • CNSRCV429 capsid animo acid sequence SEQ ID NO: 260:
  • CNSRCV43 1 capsid animo acid sequence SEQ ID NO: 261 :
  • CNSRCV432 capsid animo acid sequence SEQ ID NO: 262:
  • CNSRCV433 capsid animo acid sequence SEQ ID NO: 263:
  • EGVYSEPRPIGTRYLTRNL [0157] CNSRCV434 capsid animo acid sequence SEQ ID NO: 264:
  • CNSRCV436 capsid animo acid sequence SEQ ID NO: 265:
  • CNSRCV437 capsid animo acid sequence SEQ ID NO: 266:
  • CNSRCV438 capsid animo acid sequence SEQ ID NO: 267:
  • CNSRCV439 capsid animo acid sequence SEQ ID NO: 268:

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Abstract

This application relates to targeting peptides and engineering AAV capsids. In some embodiments, the targeting peptides and engineered AAV capsids are capable of interacting with transferrin receptor (TFRC) to mediate delivery to a cell or tissue.

Description

PEPTIDES TARGETING TFRC FOR BLOOD-BRAIN BARRIER CROSSING
FIELD
[0001] This application relates to peptides and engineered AAV (adeno-associated virus) capsids that mediate blood-brain barrier crossing by interacting with TFRC (Transferrin receptor, also known as transferrin receptor protein 1, TFR1, and CD71).
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63/643,687, filed on May 7, 2024 and entitled “PEPTIDES TARGETING TFRC FOR BLOOD-BRAIN BARRIER CROSSING,” the entire contents of which are incorporated by reference herein.
BACKGROUND
[0003] The clinical translation of genomic medicines has been limited by inefficient gene delivery through viral vectors like AAV capsids.
[0004] Attempts at engineering AAV capsids with improved properties, e.g., improved tropism to a target cell or tissue upon systemic administration, have met with limited success due to the unpredictable nature of how AAV capsid modifications influence targeting of cellular receptors. As such, there is a need for improved methods of engineering AAV capsids for delivery of genetic material of interest to a target cell or tissue, e.g., a CNS cell or tissue, including delivery across the blood-brain barrier (BBB). One approach to achieving this goal is to engineer capsids that interact with receptors that are highly expressed on the human bloodbrain barrier.
There remains a need to engineer AAV capsids having enhanced tropism to a target cell or tissue through engineering novel receptor interactions.
SUMMARY
[0005] In an aspect, targeting molecules, i.e., targeting peptides, are provided to enable targeting of a cell or a tissue, for example, the CNS. In some embodiments, a CNS-targeting molecule interacts with (e.g., binds to) TFRC (transferrin receptor), thereby enabling the CNS- targeting molecule to cross the BBB. In some embodiments, the CNS-targeting molecule comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1-7. In some embodiments, the amino acid sequence is fused or conjugated to a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein. In some embodiments, a method for identifying CNS-targeting molecule that crosses the BBB is provided, comprising selecting for variant AAV capsids that interact with TFRC.
[0006] In some embodiments, an adeno-associated virus (AAV) capsid protein is provided that interacts with (e.g., binds to) TFRC (transferrin receptor), thereby enabling the AAV capsid protein to cross the BBB. In some embodiments, the AAV capsid protein comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1-7. In some embodiments, the amino acid sequence is inserted into a parent capsid (“Parent Capsid”) at an insertion site (“Peptide Insertion Site”), optionally as shown in a single row in Tables 1 to 7. In some embodiments, the amino acid sequence comprises a peptide sequence as indicated in a single row in Tables 1 to 7, and optionally wherein the parent capsid and/or the insertion site is/are as indicated in the same single row as shown in a single row in Tables 1 to 7. In some embodiments, the amino acid sequence is inserted into a parental capsid, optionally wherein the parental capsid is selected from any one of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC-BBB (i.e., CNSRCV300 in US Application No.63/606,012).
[0007] In some embodiments, an engineered AAV capsid protein is provided, wherein the engineered AAV capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to or comprises a sequence set forth in SEQ ID NOs: 251-268 designated as CNSRCV420, CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427, CNSRCV428, CNSRCV429, CNSRCV431, CNSRCV432, CNSRCV433, CNSRCV434, CNSRCV436, CNSRCV437, CNSRCV438, or CNSRCV439.
[0008] In some embodiments, a nucleic acid molecule encoding an engineered AAV capsid protein described herein is provided. In some embodiment, a host cell comprising such a nucleic acid molecule is provided.
[0009] In some embodiments, a composition is provided comprising: 1) an adeno- associated virus (AAV) capsid protein as described herein; and 2) an expression construct comprising a coding sequence for a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and/or therapeutic payload. In some embodiments, the payload of interest is a therapeutic payload, and the therapeutic payload comprises a DNA binding domain, optionally wherein the therapeutic payload comprises a fusion protein. In some embodiments, the payload of interest comprises a therapeutic protein, a zinc finger protein, a CRISPR-associated DNA binding protein, a TALE protein, an antibody, an enzyme, a regulatory RNA, a Bxbl serine recombinase, or a DNA recombinase protein.
[0010] In some embodiments, a method of delivering a payload of interest to a cell or a tissue is provided, wherein a coding sequence for the therapeutic payload is encapsidated in an AAV capsid protein as described herein, and wherein the AAV capsid protein interacts with TFRC. In some embodiments, delivering the payload of interest to the cell or the tissue comprises crossing a BBB.
[0011] In some embodiments, a method of expressing or repressing a therapeutically relevant gene of interest in a cell, comprising contacting the cell with the composition as described herein.
[0012] In some embodiments, a method of treating a disease in a subject is provided, comprising administering to the subject the composition as described herein.
[0013] In some embodiments, use of an AAV capsid protein, a nucleic acid construct, or a host cell as described herein is provided for the manufacture of a medicament in a method as described herein.
[0014] In some embodiments, a method for identifying an AAV capsid variant that crosses the BBB or exhibits enhanced delivery to a cell or a tissue is provided, comprising selecting for variant AAV capsids that interact with TFRC.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 illustrates a schematic of the approach used to pan AAV capsid libraries against an immobilized receptor. The receptor, in this case human transferrin receptor, is biotinylated and then immobilized on a streptavidin coated bead. The AAV capsid libraries are then panned in parallel against the immobilized receptor or a streptavidin bead control without a receptor. AAV library genomes are extracted from each sample, amplified via PCR, and subjected to next-generation sequencing to quantify the enrichment of capsid variants. Capsids that specifically interact with the receptor are identified by comparing enrichment in the immobilized receptor condition relative to the bead only control. [0016] FIG. 2 illustrates a schematic of the approach used to assess AAV library transduction in cells overexpressing a receptor, in this case human transferrin receptor, relative to AAV library transduction in cells expressing a fluorescent protein transfection control. Cells are transfected with a plasmid encoding a strong ubiquitous promoter that drives the expression of the receptor or a negative control fluorescent protein. Forty-eight hours after transfection, the cells are transduced with the AAV capsid libraries. Cells are incubated for 72 hours to allow for AAV-mediated expression of transgene mRNA. RNA is extracted from cells, reverse transcribed to cDNA, and then PCR is used to amplify the AAV cDNA. Next-generation sequencing analysis is used to quantify enrichment of capsid variants. Capsids that specifically interact with the receptor are identified by comparing enrichment in the receptor expression condition relative to the fluorescent protein transfection control.
[0017] FIG. 3 illustrates a schematic of the approach used to assess AAV library binding in cells overexpressing a receptor, in this case human transferrin receptor, relative to AAV library binding in cells expressing a fluorescent protein transfection control. Cells are transfected with a plasmid encoding a strong ubiquitous promoter that drives the expression of the receptor or a negative control fluorescent protein. Forty-eight hours after transfection, the cells are transduced with the AAV capsid libraries. Cells are incubated for 1 hour to allow for AAV binding to cells. DNA is extracted from cells and then PCR is used to amplify the AAV genome. Next-generation sequencing analysis is used to quantify enrichment of capsid variants. Capsids that specifically interact with the receptor are identified by comparing enrichment in the receptor expression condition relative to the fluorescent protein transfection control.
[0018] FIG. 4 The figure shows that AAV library screens can identify capsid variants that specifically target human or cynomolgus macaque transferrin receptor. Three representative examples of capsid library screens that were conducted for transferrin receptor are shown:
1) Immobilized human transferrin receptor versus a bead only control. Data shown are for the round 2 library.
2) Binding to cells overexpressing human transferrin receptor versus a transfection control. Data shown are for the round 2 capsid library.
3) Binding to cells overexpressing cynomolgus macaque transferrin receptor versus a transfection control. Data shown are for the round 2 capsid library. [0019] A subset of capsids that exhibited specific enrichment for human or cynomolgus macaque transferrin receptor orthologs are colored in green. The marginal axis histograms represent capsids that were identified in only the receptor condition (y-axis) or the negative control (x-axis).
[0020] FIG. 5 The figure shows that AAV library screens can identify capsid variants that specifically target human or cynomolgus macaque transferrin receptor. Two representative examples of capsid library screens that were conducted for transferrin receptor are shown:
1) Transduction of cells overexpressing human transferrin receptor versus a transfection control. Data shown are for the round 2 capsid library.
2) Transduction of cells overexpressing cynomolgus macaque transferrin receptor versus a transfection control. Data shown are for the round 2 capsid library.
[0021] A subset of capsids that exhibited specific enrichment for human or cynomolgus macaque transferrin receptor orthologs are colored in green. The marginal axis histograms represent capsids that were identified in only the receptor condition (y-axis) or the negative control (x-axis).
[0022] FIG. 6 shows the performance of five exemplary capsid variants that exhibit enrichment for binding to immobilized human transferrin receptor, and transduction of cells overexpressing either human or cynomolgus macaque transferrin receptor in library assessments.
[0023] FIG. 7(A)-(G) shows Tables 1, 2, 3, 4, 5, 6, and 7.
[0024] FIG. 8 shows the transgene expression mediated by individual engineered capsids in cells overexpressing human or cynomolgus macaque transferrin receptor, relative to the transgene expression in cells expressing a fluorescent protein transfection control. Representative engineered capsids, designed to target transferrin receptor and described herein, are presented as illustrative examples. Neuro2A cells were transfected with a plasmid encoding a strong ubiquitous promoter that drives the expression of human transferrin receptor, cynomolgus macaque transferrin receptor, or a negative control fluorescent protein. Forty-eight hours after transfection, the cells were transduced with the AAV capsid at a multiplicity of infection of 3E2, 1E3, 3E3, or 1E4 vector genomes per cell. Cells were incubated for 72 hours to allow for AAV-mediated expression of transgene mRNA. RNA was extracted from cells and RT-qPCR was conducted to quantify mRNA transgene expression. Data were normalized to expression of the housekeeping gene GAPDH. Some capsids engineered to target transferrin receptor exhibit higher transduction in cells overexpressing human, or cynomolgus macaque transferrin receptor relative to cells overexpressing a fluorescent protein transfection control. Another capsid exhibits enhanced transduction only in cells expressing human transferrin receptor, but not in cells expressing cynomolgus macaque transferrin receptor. In contrast, the parent capsid AAV9 shows no significant increase in transgene expression in cells overexpressing human or cynomolgus macaque transferrin receptor relative to cells overexpressing a fluorescent protein transfection control.
[0025] FIG. 9 shows the fold change improvement in AAV mediated transgene expression in cells overexpressing human or cynomolgus macaque transferrin receptor relative to cells expressing the transfection control. Representative engineered capsids, designed to target transferrin receptor and described herein, are presented as illustrative examples. A nonlinear regression model was used to interpolate relative transgene expression values for each capsid-receptor condition. These values were then scaled to the transfection control value for each capsid.
[0026] FIG. 10 shows the fold change improvement in AAV mediated transgene expression in cells overexpressing human or cynomolgus macaque transferrin receptor relative to the parent capsid AAV9. Representative engineered capsids, designed to target transferrin receptor and described herein, are presented as illustrative examples.
[0027] FIG. 11 shows representative examples of the binding kinetics of capsids described herein that were engineered to target transferrin receptor. Bio-layer interferometry experiments were performed to assess the binding of capsids to human or cynomolgus macaque transferrin receptor. Human or cynomolgus macaque transferrin receptors were immobilized on Octet BLI sensors and binding to each capsid analyte was measured across a range of capsid concentrations. Two examples of capsids that exhibit cross-reactive binding responses to both human and cynomolgus macaque transferrin receptor are shown.
[0028] FIG. 12 shows a representative example of an engineered capsid described herein that exhibits a human specific binding response. Bio-layer interferometry experiments were performed to assess the binding of capsids to human or cynomolgus macaque transferrin receptor. Human or cynomolgus macaque transferrin receptors were immobilized on Octet BLI sensors and binding to each capsid analyte was measured across a range of capsid concentrations.
[0029] FIG. 13 shows the binding kinetics of the parent capsid AAV9, an AAV serotype that does not target transferrin receptor. Bio-layer interferometry experiments were performed to assess the binding of AAV9 to human or cynomolgus macaque transferrin receptor. Human or cynomolgus macaque transferrin receptors were immobilized on Octet BLI sensors and binding to the AAV9 capsid analyte was measured across a range of capsid concentrations. AAV9 exhibits no binding response to human or cynomolgus macaque transferrin receptor.
DETAILED DESCRIPTION
[0030] In an aspect, targeting molecules, i.e., targeting peptides, are provided for interacting with a specific cellular receptor, preferably where in the receptor is expressed on the blood brain barrier. In embodiments, the receptor is the transferrin receptor, TFRC. In an aspect, delivery to and/or targeting of a cell or a tissue, preferably wherein the molecules are CNS (central nervous system)-targeting. In embodiments, CNS-targeting molecules comprising a targeting peptide sequence indicated in Tables 1-7 are provided. In another aspect, engineered AAV capsid proteins are provided. In embodiments, a targeting peptide is inserted into a parental AAV capsid, for example an AAV3B capsid protein (SEQ ID NO: 246), an AAV5 capsid protein (SEQ ID NO: 247), an AAV6 capsid protein (SEQ ID NO: 248), an AAV8 capsid protein (SEQ ID NO: 249), or an AAV9 capsid protein (SEQ ID NO: 250). In embodiments, the targeting peptide is any of the targeting peptides disclosed in Tables 1-7. In embodiments, the peptide sequence is inserted into the parental capsid at any of the peptide insertion sites disclosed in Tables 1-7. In some embodiments, the targeting peptide functions to target the CNS-targeting molecule to a specific target tissue (e.g., CNS tissue).
Generation of an Engineered AAV Capsid Library
[0031] In one embodiment, disclosed herein is the development of libraries encoding engineered AAV capsid proteins, wherein members of the library encode engineered AAV capsid proteins having different sequences, and wherein some members of the library encode an AAV capsid protein having a desired characteristic compared to a natural/wild-type AAV serotype. In one embodiment, disclosed herein is the development of libraries encoding engineered AAV capsid proteins with a desired characteristic compared to a parent capsid. Thus, described herein are libraries of AAV capsid proteins with a desired characteristic compared to a parent capsid. In some embodiments, the desired characteristic is enhanced cell or tissue tropism as compared to the parent capsid, for example, enhanced cell or tissue tropism to the central nervous system (CNS) as compared to the parent capsid. In some embodiments, the desired characteristic is increased penetrance through the blood brain barrier following administration to a subject. In some embodiments, the desired characteristic is wider distribution throughout the multiple brain regions, e.g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and/or hippocampus. In some embodiments, the desired characteristic is elevated genetic material expression in multiple brain regions. In some embodiments, the desired characteristic is delivery of genetic material of interest to a desired tissue, cell, or organelle.
[0032] In some embodiments, each member of a library comprises one or more of a) a nucleic acid sequence encoding an AAV capsid protein comprising an engineered variant AAV sequence; b) a nucleic acid sequence encoding barcode; c) nucleic acid sequence(s) encoding a promoter(s); d) a nucleic acid sequence encoding a unique molecular identifier (UMI); and combinations thereof. In some embodiments, each member of the library also includes genetic material to be delivered to and expressed in a cell or tissue of interest. In some embodiments, each member of the library also includes a polyA sequence.
[0033] In some embodiments, each engineered AAV capsid protein was synthesized as an oligo pool. In some embodiments, each member of a library comprises one or more (such as 1-10) of a nucleic acid sequence encoding an AAV capsid protein comprising a) nucleic acid sequences encoding one or more (such as 1-10) barcodes: b) nucleic acid sequences encoding one or more (such as 1-10) promoters; c) nucleic acid sequences encoding one or more (such as 1-10,000) unique molecular identifiers (UMIs); or combinations thereof. In some embodiments, each member of the library also includes genetic material to be delivered to a cell or tissue of interest. In some embodiments, each member of the library also includes a polyA sequence. In some embodiments, each of the one or more (such as 1-10) barcodes is linked to the identity of a single engineered AAV capsid protein. In some embodiments, each of the barcodes is linked to one or more (such as 1-10,000) UMIs.
[0034] In some embodiments, a nucleic acid comprising a barcode is added to the genome of each AAV capsid in a library. In some embodiments, a unique barcode is bioinformatically linked to each different variant sequence that is represented within the library, for example, each different variant AAV sequence. In some embodiments, the DNA sequences encoding an AAV variant sequence are synthesized to further comprise a random or specified barcode. The barcode may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more nucleotides. In some embodiments, each AAV variant sequence is linked to at least 2 distinct barcodes. In some embodiments, each barcode is linked to one or more (such as 1-10,000) UMIs.
[0035] In some embodiments, each member of the library comprises a nucleic acid comprising more than one barcode sequences (such as 1-10). In some embodiments, each member of the library comprises two or more nucleic acids (such as 1-10) each comprising a barcode sequence. In some embodiments, each member of the library comprises a first nucleic acid comprising a first barcode and a second nucleic acid comprising a second barcode. In some embodiments, the first nucleic acid comprising the first barcode and the second nucleic acid comprising the second barcode are different. In some embodiments, each of the first nucleic acid comprising the first barcode and the second nucleic acid comprising the second barcode is independently operatively linked to a promoter. In some embodiments, each capsid is linked to at least one unique barcode. In some embodiments, each capsid is linked to at least two unique barcodes using a bioinformatic look-up table. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from the neuron specific promoter. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from the neuron specific human Synapsin 1 promoter. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from the ubiquitous CMV promoter.
[0036] In some embodiments, libraries are created encoding engineered AAV capsid proteins that comprise at least one mutation relative to a parent capsid, for example, the parent capsid AAV3B, AAV5, AAV6, AAV8, or AAV9 (SEQ ID NOs: 246-250). In some embodiments, the engineered AAV capsid proteins contain a peptide sequence inserted within a parent capsid protein, for example, the parent capsid AAV3B, AAV5, AAV6, AAV8, or AAV9 (SEQ ID NOs: 246-250). In some embodiments, the engineered AAV capsid proteins contain a peptide sequence inserted within a surface exposed loop of a parent capsid protein, for example, the parent capsid AAV3B, AAV5, AAV6, AAV8, or AAV9 (SEQ ID NOs: 246-250).
[0037] In some embodiments, the libraries are packaged in HEK293 cells where the helper functions (e.g. E2A, E4, VA, El A and E1B) are supplied in trans. In some embodiments, the AAV rep function comprises rep78, rep 68, rep 52, and rep40 genes. In some embodiments, the rep genes are supplied in trans. In some embodiments, the start codon of the rep78 and/or the rep68 gene is altered from ACG to ATG to increase replication of the capsid library construct containing inverted terminal repeats (ITRs), thereby improving AAV library manufacturing yield. In some embodiments, the cap genes are supplied as genetic material that is packaged into the manufactured AAVs. In some embodiments, the capsid gene is controlled by the p40 promoter such that it is only expressed during manufacturing in HEK293 cells in the presence of helper virus functions.
Methods of Screening Libraries of Engineered AAV Capsid Proteins
[0038] In some embodiments, a method of identifying an engineered AAV capsid protein with a desired characteristic compared to a natural/wild-type AAV serotype is provided comprising: (i) contacting an immobilized receptor protein, a cell, a cell line, or tissue in vitro or in vivo with any one of the libraries of engineered AAV capsid proteins, (ii) allowing the engineered AAV capsid proteins in said library to transduce the cell, cell line, or tissue; (iii) recovering from the immobilized receptor protein, cell, cell line, or tissue the AAV variant; and (iv) identifying the engineered AAV capsid protein with the desired characteristic.
[0039] In another embodiment, disclosed herein are methods for directed evolution of engineered AAV capsid proteins and identification of an engineered AAV capsid protein with a desired characteristic compared to a natural/wild-type AAV serotype. In some embodiments, the steps for directed evolution of engineered AAV capsid proteins to identify engineered AAV capsid proteins with a desired characteristic compared to a natural/wild-type AAV serotype comprise (i) modifying the natural/wild-type AAV serotype to create variant capsids; (ii) packaging of the variant AAVs in producer cells wherein adenovirus helper and AAV rep functions are supplied in trans; (iii) purification of viral capsid library pools; (iv) administration of the pools in vitro or in vivo; (v) recovery of engineered AAV capsid proteins from target tissues or cell lines; (vi) next-generation sequencing to determine the identity of the engineered variant capsid sequences; (vii) repeated rounds of in vitro or in vivo selection where variants are isolated from a target tissue or cell line; and (viii) full evaluation of enriched variants. In some embodiments, the desired characteristic includes enhanced tissue tropism as compared to the natural/wild-type AAV serotype. In some embodiments, the desired characteristic includes enhanced tissue tropism for tissues of the peripheral nervous system as compared to the natural/wild-type AAV serotype. In some embodiments, the desired characteristic includes enhanced tissue tropism of the central nervous system as compared to the natural/wild-type AAV serotype. Engineered AAV Capsid Proteins
[0040] In one embodiment, described herein are compositions comprising engineered AAV capsid proteins and methods of making and using the same. In some embodiments, the engineered AAV capsid proteins interact with a receptor, e.g. TFRC. In some embodiments, the engineered AAV capsid proteins demonstrate binding to a receptor, e.g. TFRC. The engineered AAV capsid proteins may be delivered to one or more of target cells, tissues, organs, or organisms. In some embodiments, the engineered AAV capsid protein has enhanced tropism for a cell or tissue, e.g., for the delivery of genetic material to a specific cell or tissue, for example a CNS tissue or a CNS cell, or cells and tissues of a muscle. The engineered AAV capsid proteins may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ. As a non-limiting example, the engineered AAV capsid proteins that are desired to have tropism for CNS cells may have enhanced tropism for neurons, astrocytes, oligodendrocytes, microglia, endothelial cells, Schwann cells, and reduced tropism for liver and dorsal root ganglion.
[0041] In some embodiments, an engineered AAV capsid protein is provided comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all contiguous amino acids of a peptide sequence inserted within a parent capsid protein. In some embodiments, the peptide sequence is inserted within or near a surface-exposed loop of the parent capsid protein. In a preferred embodiment, the parent capsid is AAV3B, AAV5, AAV6, AAV8, or AAV9 (SEQ ID NOs: 246-250). In some embodiments, the peptide sequence is inserted within or near amino acids 588 through 589 corresponding to the sequence of AAV3B (SEQ ID NO: 246). In some embodiments the peptide sequence is inserted within or near amino acids 577 through 578 corresponding to the sequence of AAV5 (SEQ ID NO: 247). In some embodiments the peptide sequence is inserted within or near amino acids 589 through 590 corresponding to the sequence of AAV6 (SEQ ID NO: 248). In some embodiments the peptide sequence is inserted within or near amino acids 590 through 591 corresponding to the sequence of AAV8 (SEQ ID NO: 249). In some embodiments the peptide sequence is inserted within or near amino acids 587 through 590 corresponding to the sequence of AAV9 (SEQ ID NO: 250).
[0042] In some embodiments, an amino acid sequence is inserted into a parent capsid (“Parent Capsid”) at an insertion site (“Peptide Insertion Site”). In some embodiments, the inserted amino acid sequence comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1 to 7. In some embodiments, an engineered AAV capsid protein is provided comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1 to 7. In some embodiments, the amino acid sequence comprises a peptide sequence as indicated in a single row in Tables 1 to 7, and optionally wherein the parent capsid and/or the insertion site is/are as indicated in the same single row.
[0043] In some embodiments, an amino acid sequence is inserted into a parental capsid, optionally wherein the parental capsid is selected from any one of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC- BBB. In some embodiments, the inserted amino acid sequence comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1 to 7.
[0044] In some embodiments, the engineered AAV capsid protein is at least 80%, 85%,
90%, 95%, or 99% identical to or comprises any one of the sequences corresponding to SEQ ID NOs: 251-268 designated as CNSRCV420, CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427, CNSRCV428, CNSRCV429, CNSRCV431, CNSRCV432, CNSRCV433, CNSRCV434, CNSRCV436, CNSRCV437, CNSRCV438, or CNSRCV439.
[0045] In some embodiments, the engineered AAV capsid proteins have advantages over wild-type AAV capsid proteins. In some embodiments, these advantages including (i) enhanced cell or tissue tropism as compared to the natural/wild-type AAV serotype, for example, enhanced cell or tissue tropism to the central nervous system (CNS) as compared to the natural/wild-type AAV serotype (ii) increased penetrance through the blood brain barrier following administration to a subject, (iii) wider distribution throughout the multiple brain regions, for example, the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and/or hippocampus, (iv) elevated expression of genetic material in multiple brain regions. In some embodiments, the engineered AAV capsids enhance the delivery of genetic material to multiple regions of the brain including for example, the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and/or hippocampus, (v) is delivery of genetic material of interest to a desired tissue, cell, or organelle.
[0046] In embodiments, the engineered AAV capsid proteins and genetic material described herein may be delivered to one or more (such as 1-10) target cells, tissues, organs, or organisms. In some embodiments, the engineered AAV capsid proteins have enhanced tropism for a specific target cell type, tissue or organ. As a non-limiting example, the engineered AAV capsid protein has enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively). In some embodiments, engineered AAV capsid proteins are produced recombinantly and are an adeno-associated virus (AAV) serotype such as AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAV3, AAV4, AAV7, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC-BBB, or a combination thereof. In some embodiments, engineered AAV capsid proteins are produced recombinantly and are based on any one or more (such as 1-15) AAV serotypes known in the art.
Adeno-associated virus (AAV)
[0047] AAV are capable of infecting a wide range of cells including quiescent cells and dividing cells. In some embodiments, AAV can be modified so that it contains the components necessary for the assembly of a functional recombinant virus or viral particle. In some embodiments, the AAV is engineered to interact with a specific receptor, e.g. TFRC. In some embodiments, the AAV is engineered to target a specific tissue and/or cell, for example, CNS tissue and/or cell. In some embodiments, the AAV is engineered to deliver specific genetic material to a tissue and/or cell. In some embodiments, the AAV is engineered to target a blood brain barrier receptor, for example, TFRC.
Modified AAV Serotypes
[0048] In some embodiments, an engineered AAV may be based on any natural or recombinant AAV serotype. Different AAV serotypes have different characteristics such as different packaging, tropism, and transduction profiles. In some embodiments, the engineered AAV capsid proteins are based on a wild-type AAV serotype. In some embodiments, the AAV serotype comprises AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, or AAV9. In some embodiments, the AAV serotype comprises less well-characterized AAV serotypes such as AAV3, AAV4, AAV7, AAV11, AAVrhlO, AAVrh39, or AAVrh74. In some embodiments, the AAV serotype is an engineered AAV serotype such as STAC-BBB. In some embodiments, the engineered AAV capsid protein is derived from multiple AAV serotypes, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more AAV serotypes. In some embodiments, AAV variant capsid proteins derived from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more AAV serotypes are combined to create chimeric capsids. In some embodiments, combinatorial libraries are generated by modifying nucleic acids encoding AAV capsid proteins from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more serotypes in the same pool.
[0049] In some embodiments, different AAV serotypes are different in their ability to direct or modulate an AAV particle to a particular cell or tissue. In some embodiments, the AAV serotype can be modified to interact with a receptor, e.g. TFRC. In some embodiments, the AAV serotype modified to interact with a receptor has an altered tropism. In some embodiments, the AAV serotype can be modified to increase the tropism of the AAV particle to cells or tissues of the central nervous system (CNS). In some embodiments, the AAV serotype can be modified to increase tropism of the AAV particle to cells or tissues of the peripheral nervous system (PNS).
[0050] In some embodiments, the modified AAV serotype has a desired characteristic compared to a parental AAV serotype. In some embodiments, the modified AAV serotype allows for increased penetration of the blood brain barrier following administration to a subject. In some embodiments, the modified AAV serotype causes increased biodistribution to a brain region. In some embodiments, the brain region comprises the frontal cortex, the sensory cortex, the motor cortex, the cerebellar cortex, the hippocampus, the thalamus, or the putamen. In some embodiments, the brain comprises any brain region known in the art. In some embodiments, the modified AAV serotype causes increased biodistribution to more than one brain regions, for example, 2 brain regions, 3 brain regions, 4 brain regions, 5 brain regions, 6 brain regions, 7 brain regions, 8 brain regions, 9 brain regions, or 10 brain regions. In some embodiments, the modified AAV serotype causes increased biodistribution to 1- 10 brain regions. In some embodiments, the modified AAV serotype are useful in elevating genetic material expression in multiple brain regions. In some embodiments, the modified AAV serotype are used to deliver genetic material of interest to a desired tissue, cell, or organelle.
[0051] In some embodiments, the modified AAV serotype causes increased biodistribution to regions of the spinal cord. In some embodiments, the region of the spinal cord comprises any of the thoracic spinal cord region, the lumbar spinal cord region, and/or the cervical spinal cord region. In some embodiments, the region of the spinal cord includes any region of the spinal cord known in the art.
[0052] In some embodiments, the modified AAV serotype comprises an inserted sequence having at least 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of a Peptide Sequence shown in Tables 1-7. In some embodiments, the inserted sequence is inserted into a parent capsid serotype as shown in Tables 1-7. In some embodiments, the inserted sequence is inserted into a parent capsid sequence at or near the Peptide Insertion Site as shown in Tables 1-7. In some embodiments, the modified AAV serotype comprises an inserted peptide sequence described herein (e.g., a Peptide Sequence shown in Tables 1-7). In some embodiments, the modified AAV serotype comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences corresponding to SEQ ID NOs: 251-268 designated as CNSRCV420, CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427, CNSRCV428, CNSRCV429, CNSRCV431, CNSRCV432, CNSRCV433, CNSRCV434, CNSRCV436, CNSRCV437, CNSRCV438, or CNSRCV439. In some embodiments, the modified AAV sequence comprises any one of the sequences corresponding to SEQ ID NOs: 251-268 designated as CNSRCV420, CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427, CNSRCV428, CNSRCV429, CNSRCV431, CNSRCV432, CNSRCV433, CNSRCV434, CNSRCV436, CNSRCV437, CNSRCV438, or CNSRCV439.
Structure of AAV
[0053] In some embodiments, the genome of the AAV comprises a single-strand DNA (ssDNA) molecule that is approximately between about 2.5 kb and about 5.0 kb in length. In some embodiments, the genome of the AAV comprises a self-complementary DNA (scDNA) molecule that is approximately between about 0.5 kb and about 2.5 kb in length. In some embodiments, the AAV genome contains inverted terminal repeats (ITRs) that flank the 5’ and 3’ ends of the AAV molecule. In some embodiments, the ITRs contain origins of replication for the viral genome. In some embodiments, the length of the ITRs is about 145 bp in length, for example, between about 130 bp in length and 160 bp in length.
[0054] In some embodiments, the AAV genome comprises the rep and cap genes. In some embodiments, the AAV genome nucleotide includes nucleotide sequences that encode four non- structural Rep proteins (Rep 78, Rep68, Rep52, Rep40, encoded by Rep genes). In some embodiments, the AAV viral genome includes nucleotide sequences that encode the three capsid, or structural, proteins (i.e., VP1, VP2, VP3, encoded by the cap gene). In some embodiments, the rep proteins are used for replication and packaging. In some embodiments, the capsid proteins are assembled to create the protein shell of the AAV.
AAV particles [0055] In some embodiments, the engineered AAV capsid proteins assemble to form AAV particles. In some embodiments, the engineered AAV capsid proteins interact with a receptor expressed at the blood brain barrier, for example, TFRC. In some embodiments, the AAV particles that have enhanced tropism for a target tissue (e.g., CNS and PNS) are provided. In some embodiments, the AAV particles include engineered AAV variant sequences that alter tropism to a particular cell-type, tissue, organ or organism, in vivo, ex vivo or in vitro. In some embodiments, the AAV particles are capable of penetrating the blood brain barrier.
Delivery of AAV particles
[0056] The AAV particles may be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, the AAV particles demonstrate enhanced tropism for a target cell type, tissue or organ. As a non-limiting example, the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively), or cells and tissues of a muscle. The AAV particles may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ.
[0057] In some embodiments, the AAV particles can be used to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating. In some embodiments, the AAV particles are used to deliver any cargoes of interest, or example, therapeutic cargoes.
AAV viral genomes
[0058] In some embodiments, the AAV particles are used to deliver a viral genome (i.e., a genetic payload) to a tissue or cells such as CNS or PNS cell or tissue.
[0059] The delivered viral genome may include genetic material of interest, such as, for example, genetic material that encodes an engineered DNA recombinase protein, a fusion protein comprising a DNA-binding domain (e.g., a zinc finger or a TALE protein) fused to a functional domain (e.g., to modulate DNA function or to cleave DNA), an antibody, an enzyme, regulatory RNA, a CRISPR protein, or a cDNA, amongst others. In some embodiments, the viral genome includes 2 ITR sequences. In some embodiments, the ITR sequences flank the genetic material of interest. In some embodiments, the ITR sequences are complementary to each other. In some embodiments, the ITR sequences are not complementary to each other. In some embodiments, one ITR sequence is a self-complementary ITR. In some embodiments, the ITR regions are derived from the same serotype as the capsid protein. In some embodiments, the ITR regions are derived from AAV2 serotype. In some embodiments, the ITR regions are derived from a serotype known to the art. ITR regions may be between 100 and 150 nucleotides in length.
Targeting Peptide Sequences
[0060] In embodiments, targeting peptide sequences (e.g., targeting molecules or sequences) are disclosed herein. In some embodiments, the sequences enhance or enable interaction with transferrin receptor (TFRC). In some embodiments, the sequences can function to target a cell or a tissue, for example, as a general CNS-targeting molecule or sequence.
In embodiments, the targeting sequences function as a general CNS-targeting molecule sequence. In some embodiments, the CNS-targeting sequence is fused or conjugated to a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein. In some embodiments, any one of the targeting sequences described herein may be fused or conjugated to a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein. In some embodiments, CNS-targeting sequences may be utilized to enable a small molecule, an antibody, zinc finger protein, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein to cross the blood brain barrier.
[0061] In some embodiments, the targeting sequences are part of an engineered AAV capsid protein. In some embodiments, the engineered AAV capsid protein is any engineered AAV capsid protein disclosed herein.
[0062] In some embodiments, the targeting sequences enable the binding of an AAV capsid protein to a specific receptor, e.g. TFRC. In some embodiments, the targeting sequences enable the binding of an AAV capsid protein to a specific receptor expressed at the blood brain barrier, e.g. TFRC. In some embodiments, the targeting sequences modulate the binding affinity of an AAV capsid protein to a specific receptor, e.g. TFRC.
[0063] In some embodiments, the sequences may increase tropism of an AAV capsid protein to a cell or tissue of the CNS. In some embodiments, the cell of the CNS is a neuron (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), a glial cells (e.g., microglia, astrocytes, oligodendrocytes) and/or a supporting cells of the brain such as immune cells (e.g., T cells). In some embodiments, the CNS tissue is the cortex (e.g., frontal, parietal, occipital, temporal), thalamus, hypothalamus, striatum, caudate nucleus, hippocampus, putamen, basal ganglia, entorhinal cortex, cerebellum, or spinal cord.
[0064] In some embodiments, the sequences increase tropism of an AAV capsid protein to a cell, region, or tissue of the PNS. In some embodiments, the cell or tissue of the PNS is dorsal root ganglion (DRG).
[0065] In some embodiments, the sequences decrease tropism of an AAV capsid protein to a cell, region, or tissue of the PNS. In some embodiments, the cell or tissue of the PNS is dorsal root ganglion (DRG).
[0066] In some embodiments, the targeting sequence comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1 to 7. In some embodiments, the sequence comprises the amino acid sequence (“Peptide Sequence”) as shown in a single row in the first column of Tables 1 to 7.
[0067] In some embodiments, the targeting peptide sequence is inserted within a parent AAV capsid sequence. In some embodiments, the inserted sequence is inserted into a parent capsid serotype as shown in Tables 1-7. In some embodiments, the inserted sequence is inserted into a parent capsid sequence at or near the Peptide Insertion Site as shown in Tables 1-7. In some embodiments, the targeting peptide sequence is inserted within or near a surface-exposed loop of a parent AAV capsid sequence. In some embodiments, the parent capsid sequence is any of the serotypes AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, or STAC-BBB.
[0068] In some embodiments the peptide sequence is inserted within or near amino acids 588 through 589 corresponding to the sequence of AAV3B (SEQ ID NO: 246). In some embodiments the peptide sequence is inserted within or near amino acids 577 through 578 corresponding to the sequence of AAV5 (SEQ ID NO: 247). In some embodiments the peptide sequence is inserted within or near amino acids 589 through 590 corresponding to the sequence of AAV6 (SEQ ID NO: 248). In some embodiments the peptide sequence is inserted within or near amino acids 590 through 591 corresponding to the sequence of AAV8 (SEQ ID NO: 249). In some embodiments the peptide sequence is inserted within or near amino acids 587 through 590 corresponding to the sequence of AAV9 (SEQ ID NO: 250). Administering Engineered AAV Capsid Proteins to Subjects
[0069] In some embodiments, when administered to subjects, AAV capsid proteins containing the targeting peptides described herein mediate enhanced delivery to cells and tissues relative to AAV capsid proteins that lack the targeting peptides. In some embodiments, the AAV capsid protein administered to subjects comprises an engineered AAV sequence described herein.
Genetic Material
[0070] In some embodiments, the engineered AAV capsid proteins described herein encapsidate genetic material of interest to be delivered to a cell of interest. In some embodiment, the genetic material of interest may be a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and/or therapeutic payload. As such, in embodiments the engineered AAV capsid proteins described herein enable delivery of genetic material to a cell of interest. In embodiments, the genetic material may encode a research, diagnostic, and/or therapeutic payload. In embodiments, the genetic material encodes a zinc finger protein, a TALE protein, a recombinase protein, and/or a CRISPR protein, or fragments thereof. In embodiments, the genetic material encodes one or more antibodies or an antibody fragments. In some embodiments the genetic material encodes one or more regulatory RNA, such as RNAi agents or microRNAs.
[0071] In some embodiments, the genetic material can include sequences that are coding sequences. In some embodiments, the genetic material can include sequences that are non-coding sequences. In some embodiments, the genetic material can include sequences that are both coding sequences and non-coding sequences. In some embodiments, the expression of the genetic material is capable of being regulated. In some embodiments, the genetic material comprises elements that are regulatable.
[0072] In some embodiments, mRNA is encoded in the genetic material. In some embodiments, the mRNA is codon optimized.
[0073] In some embodiments, the genetic material encodes a gene therapy product. A gene therapy product can include a peptide, a polypeptide, or an RNA molecule that when expressed carries out a desired therapeutic effect. In some embodiments, the therapeutic effect is treating any one or more diseases or disorders described herein. [0074] In some embodiments, a promoter is operably linked to the genetic material to be delivered to the cell. In some embodiments, the promoter comprises a tissue and/or cell specific promoter. In some embodiments, the one more promoters comprise a ubiquitous promoter. Examples of ubiquitous promoters include cytomegalovirus (CMV), chicken P-actin (CBA), ubiquitin C (UBC), and elongation factor la-subunit (EFl -a), amongst others. In some embodiments, the promoter comprises a cell type and/or tissue specific type promoter. Exemplary cell type and/or tissue specific promoters include the human synapsin promoter (hSynl), only expressed in neurons, or the transthyretin promoter (TTR), expressed in hepatocytes. Other non-limiting cell type and/or tissue specific promoters for use in the methods and compositions of the invention include cytokeratin 18 and 19 (epithelial cell specific, Other cell-specific promoters include GFAP promoter (astrocytes), TBG promoter (liver), MHCK promoter (skeletal muscle), MYH6 promoter (cardiomyocytes). In embodiments, tissue specific or cell specific promoters can restrict expression to tissues or cells of the CNS or PNS. In embodiments, tissue specific or cell specific promoters can be used to restrict expression to neurons of the sympathetic system, the parasympathetic system, astrocytes, microglia, oligodendrocytes, and/or Schwann cells.
[0075] In some embodiments, the promoters are naturally occurring promoters. In some embodiments, the promoter is synthetic. In some embodiments, the promoter is derived from mammals, humans, viruses, or plants. In some embodiments, the promoters are truncated. In some embodiments, the promoter is mutated.
Gene Editing System
[0076] In some embodiments, the genetic material of interest comprises a gene editing system or portions of a gene editing system. In some embodiments, the gene editing system is capable of inducing single or double-stranded breaks into nucleic acid sequences at one or more site of interest. In some embodiments, the gene editing system is capable of inserting, substituting, or deleting a base or a sequence of bases into nucleic acid sequences at one or more site of interest. In some embodiments, the gene editing system includes a CRISPR-Cas system. In some embodiments, the gene editing system includes a TALEN. In some embodiments, the gene editing system includes a zinc finger nuclease. In some embodiments, the gene editing system includes a modified recombinase protein. Epigenetic Regulation System
[0077] In some embodiments, the genetic material of interest comprises an epigenetic regulation system or components of an epigenetic regulation system for general or targeted gene regulation. In some embodiments, the epigenetic regulation system is capable of modifying chromatin structure or altering epigenetic marks on nucleic acid sequences. In some embodiments, the epigenetic regulation system is capable of promoting or repressing gene expression without altering the underlying DNA sequence. In some embodiments, the epigenetic regulation system includes a CRISPR-dCas system fused to epigenetic effector domains. In some embodiments, the epigenetic regulation system includes a transcription activator or repressor domain tethered to a programmable DNA-binding protein. In some embodiments, the epigenetic regulation system includes hi stone-modifying enzymes or DNA methyltransferases targeted to a specific genomic locus. In some embodiments, the epigenetic regulation system includes a TALEN. In some embodiments, the epigenetic regulation system includes a zinc finger protein fused to an epigenetic effector domain, for example, a zinc finger repressor or a zinc finger activator.
Active Agents
[0078] In some embodiments, engineered AAV sequences described herein are fused or coupled to an active agent. In some embodiments, a sequence is fused or coupled to an active agent through conjugation. In some embodiments, the active agent comprises a therapeutic agent. In some embodiments, the therapeutic agent comprises an antibody or a portion of an antibody (e.g., Fc region). In some embodiments, the sequence is fused to a Fc region of an antibody. In some embodiments, the sequence is fused to the C-terminus of the Fc region. In some embodiments, the sequence is fused to the N-terminus of the Fc region. In some embodiments, the therapeutic agent comprises an RNAi agent (e.g., siRNA, shRNA, IncRNA, piRNA, snoRNA, or miRNA). In some embodiments, the sequence is fused or coupled directly to at least on strand of the RNAi. In some embodiments, the sequence is fused or coupled to at least one strand of RNAi using a linker. In some embodiments, the sequence is fused or coupled to the sense strand of RNAi. In some embodiments, the sequence is fused or coupled to the antisense strand of RNAi.
[0079] In some embodiments the active agent comprises a diagnostic agent. In some embodiments, the diagnostic agent comprises a detectable moiety such as a fluorophore. In some embodiments, the active agent is a small molecule. Pharmaceutical Compositions and Dosage Forms
[0080] Compositions herein (e.g., engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins) can be included in pharmaceutical compositions. In some embodiments, the pharmaceutical compositions can include one or more excipients or diluents to (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release of the genetic material; (4) alter the biodistribution (e.g., target the composition to specific tissues or cell types); (5) increase the translation of encoded protein; (6) alter the release profile of encoded protein and/or (7) allow for regulatable expression of the genetic material.
[0081] The pharmaceutical compositions described herein can be administered periodically, such as once or twice a day, or any other suitable time period. For example, pharmaceutical compositions may be administered to a subject in need once a week, once every other week, once every three weeks, once a month, every other month, every three months, every six months, every nine months, once a year, every eighteen months, every two years, every thirty months, or every three years.
[0082] In some embodiments, the compositions described herein (e.g., engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins) can be formulated in a wide variety of dosage forms, including but not limited to nasal, pulmonary, oral, topical, or parenteral dosage forms for clinical. Each of the dosage forms can comprise various solubilizing agents, disintegrating agents, surfactants, fillers, thickeners, binders, diluents such as wetting agents or other pharmaceutically acceptable excipients. The compositions described herein can also be formulated for injection, insufflation, infusion, or intradermal exposure. For instance, an injectable formulation may comprise the disclosed compositions in an aqueous or non-aqueous solution at a suitable pH and tonicity. The compositions can be included liquid dosage form for oral administration, such as suspensions, emulsions, or syrups.
[0083] In some embodiments, the pharmaceutical compositions described herein function to increase the stability, increase transduction or transfection efficiency, impact biodistribution, increase expression of the protein, and/or alter the release profile.
Methods of Delivery and Treatment
[0084] In some embodiments, methods for introducing the compositions described herein (e.g., engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins) into cells and/or tissues are provided. In some embodiments, the methods comprise introducing into cells and/or tissues any of the compositions described herein in an amount sufficient to modulate, e.g., increase, the production of a target mRNA and/or protein in the cells and/or tissues.
[0085] In some embodiments, the compositions described herein are delivered via a localized delivery route. In some embodiments, the localized delivery route includes any one or more of intramuscular administration, intraparenchymal administration, and intracerebral administration, amongst others. In some embodiments, the compositions described herein are administered via a localized delivery route through a bolus infusion.
[0086] In some embodiments, the compositions described herein are administered through systemic administration. In some embodiments, systemic administration includes intravenous administration.
[0087] In some embodiments, the compositions described herein are administered to the central nervous system of via intraventricular administration and/or intrathecal administration. In some embodiments, the compositions described herein are administered to the central nervous system via systemic administration. In some embodiments, the systemic administration is intravenous (IV) injection. In some embodiments, the compositions described herein are administered to the central nervous system via administration into the cerebrospinal fluid.
[0088] In some embodiments the compositions can be delivered to target cells or target tissue including, but not limited to, the CNS, heart, lung, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testes, prostate, eye, blood, lymph, or oral mucosa. In some embodiments, the target cell or tissue includes, but is not limited to CNS, heart, lung, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testes, prostate, eye, blood, lymph, or oral mucosa. In some embodiments, the target cell or target tissue is a CNS cell or tissue. In some embodiments, the target cell or tissue is liver cell or tissue.
[0089] In some embodiments, the target cell includes, but is not limited to, neurons, glial cells, astrocytes, oligodendroglia, microglia, Schwann cells, ependymal cells, hepatocytes, stellate fat storing cells, Kupffer cells, liver endothelial cells, epithelial cells, cardiomyocytes, smooth muscle cells, T-cells, B cells, hematopoietic stem cells, and embryonic stem cells.
[0090] In some embodiments, the compositions described herein are delivered to the central nervous system through the cerebral spinal fluid pathway. In some embodiments, compositions described herein are administered to the central nervous system via intraparenchymal delivery. In some embodiments, the compositions described herein are administered to the central nervous system via intracranial delivery. In some embodiments, the compositions described herein are delivered to the central nervous system via intraocular delivery. In some embodiments, the compositions described herein are administered to the brain. In some embodiments, the compositions described herein are administered to the brain via injection into the brain. In some embodiments, the compositions described herein are administered to the brain via intrahippocampal injection.
[0091] In some embodiments, the compositions described herein are administered as part of a composition that allows for extended release. In some embodiments, the compositions comprises a formulation that includes a depot.
[0092] Disclosed herein are methods of treatment using any of the compositions described herein (engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins). In embodiments, the disclosed compositions can be used to treat any one or more of muscular or neuromuscular disorders, neurooncological disorders, neurological diseases/disorders, and neurodegenerative disorders, amongst others. In embodiments, the disclosed compositions can be used to treat any one or more of Alzheimer’s disease, Huntington’s disease; autism; Parkinson’s disease; Spinal muscular atrophy, Friedreich’s ataxia. In embodiments, the disclosed compositions are used in treatments through any of the methods of delivery described herein.
[0093] In some embodiments, disclosed are methods for treating, or ameliorating a disease or condition associated with abnormal gene and/or protein in a subject in need of treatment, the methods comprising administering to the subject any effective amount of at least one of the compositions described herein (e.g., engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins), delivering the compositions described herein into targeted cells, inhibiting or activating the gene expression and protein production, and ameliorating symptoms of the disease or condition in the subject. Equivalents and Scope
[0094] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of neurology, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.
[0095] The disclosure includes many equivalents to the specific embodiments described herein. A person of skill in the art will be able to ascertain equivalents to the specific embodiments, through routine experimentation.
[0096] It is assumed that words of this disclosure are for the purpose of description and not limitation. Changes to words in the claims can be made, while still retaining the scope of the disclosure in its broad embodiment. Specific embodiments of the disclosure have been described herein. However, these embodiments are not intended to be limiting of the broad scope of this disclosure. [0097] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.
Exemplary Embodiments
[0098] Non-limiting exemplary embodiments of the present disclosure are described below.
1. An adeno-associated virus (AAV) capsid protein comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 1-245 as shown in a single row in Tables 1 to 7.
2. The AAV capsid protein of embodiment 1, wherein the amino acid sequence is inserted into a parent capsid (“Parent Capsid”) at an insertion site (“Peptide Insertion Site”) as shown in a single row in Tables 1 to 7.
3. The AAV capsid protein of embodiment 1, wherein the amino acid sequence comprises a peptide sequence of any one of SEQ ID NOs: 1-245 as shown in a single row in Tables 1 to 7, and optionally wherein the parent capsid and/or the insertion site is/are as indicated in the same single row as shown in Tables 1 to 7.
4. The AAV capsid protein of any one of embodiments 1-3, wherein the amino acid sequence is inserted into a parental capsid, optionally wherein the parental capsid is selected from any one of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC-BBB.
5. An AAV capsid protein, wherein the AAV capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to or comprises a sequence set forth in SEQ ID NOs: 251-268 designated as CNSRCV420, CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427, CNSRCV428, CNSRCV429, CNSRCV431, CNSRCV432, CNSRCV433, CNSRCV434, CNSRCV436, CNSRCV437, CNSRCV438, or CNSRCV439.
6. The AAV capsid protein of any one of embodiments 1-5, wherein the AAV capsid protein interacts with transferrin receptor (TFRC), thereby enabling delivery of the AAV capsid protein to a cell or a tissue, optionally wherein interaction with TFRC enables the AAV capsid protein to cross a blood-brain barrier (BBB). 7. The AAV capsid protein of embodiment 6, wherein the AAV capsid protein interacts with both human and cynomolgus macaque TFRC, thereby enabling the AAV capsid protein to cross the BBB in both species.
8. The AAV capsid protein of embodiment 6 or embodiment 7, wherein the AAV capsid protein comprises a peptide sequence as shown in a single row in Tables 6 to 7.
9. The AAV capsid protein of any one of embodiments 6-8, wherein the amino acid sequence is inserted into a parental capsid, optionally wherein the parental capsid is selected from any one of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC-BBB.
10. An adeno-associated virus (AAV) capsid protein that interacts with both human and cynomolgus macaque TFRC, thereby enabling delivery of the AAV capsid protein to a cell or a tissue, optionally wherein interaction with TFRC enables the AAV capsid protein to cross a BBB.
11. A nucleic acid molecule encoding an AAV capsid protein of any one of embodiments 1-10.
12. A host cell comprising the nucleic acid molecule of embodiment 11.
13. A composition comprising: 1) an AAV capsid protein comprising of any one of embodiments 1-10; and 2) an expression construct comprising a coding sequence for a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and/or therapeutic payload.
14. The composition of embodiment 13, wherein the payload of interest is a therapeutic payload, and wherein the therapeutic payload comprises a DNA binding domain, optionally wherein the therapeutic payload comprises a fusion protein.
15. The composition of embodiment 13 or embodiment 14, wherein the payload of interest comprises a therapeutic protein, a zinc finger protein, a CRISPR-associated DNA binding protein, a TALE protein, an antibody, an enzyme, a regulatory RNA, a Bxbl serine recombinase, or a DNA recombinase protein. 16. A method of delivering a payload of interest to a cell or a tissue, wherein a coding sequence for the payload of interest is encapsidated in an AAV capsid protein according to any one of embodiments 1-10, and wherein the AAV capsid protein interacts with TFRC.
17. The method of embodiment 16, wherein delivering the payload of interest to the cell or the tissue comprises crossing a BBB.
18. A method of activating, expressing, repressing, or modulating the expression of a therapeutically relevant gene of interest in a cell, comprising contacting the cell with a composition of any one of embodiments 13-15.
19. A method of treating a disease in a subject, comprising administering to the subject a composition of any one of embodiments 13-15.
20. Use of an AAV capsid protein of any one of embodiments 1-10, a nucleic acid construct of embodiment 11, or a host cell of embodiment 12 for the manufacture of a medicament in the method of any one of embodiments 16-19.
21. A method for identifying an AAV capsid variant that crosses the BBB or exhibits enhanced delivery to a cell or a tissue, comprising selecting for AAV capsids that interact with TFRC, optionally comprising selecting for AAV capsids that interact with both human and cynomolgus macaque TFRC.
22. A method for identifying an AAV capsid variant that crosses the BBB or exhibits enhanced delivery to a cell or a tissue, comprising selecting for AAV capsids that comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 1-245 as shown in a single row in Tables 1 to 7.
23. A targeting molecule that interacts with TFRC, thereby enabling delivery of the targeting molecule to a cell or a tissue, optionally wherein the interaction with TFRC enables the targeting molecule to cross the blood-brain barrier (BBB).
24. The targeting molecule of embodiment 23, wherein the targeting molecule interacts with both human and cynomolgus macaque TFRC. 25. The targeting molecule of embodiment 23 or embodiment 24, comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 1-245 as shown in a single row in Tables 1 to 7.
26. The targeting molecule of any one of embodiments 23-25, wherein the targeting molecule is fused or conjugated to an AAV, a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.
27. A composition comprising: 1) a targeting molecule of any one of embodiments 23-26; and 2) a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and/or therapeutic payload.
28. The composition of embodiment 27, wherein the payload of interest encodes or comprises a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.
29. A method of delivering a payload of interest to a cell or a tissue, wherein a coding sequence for the payload of interest is associated with a targeting molecule according to any one of embodiments 23-26, and wherein the targeting molecule interacts with TFRC.
30. The method of embodiments 29, wherein delivering the payload of interest to the cell or the tissue comprises crossing a BBB.
31. A method of delivering a therapeutically relevant gene or protein of interest to a cell, comprising contacting the cell with the composition of any one of embodiments 27-28.
32. A method of modulating the expression or activity of a therapeutically relevant gene of interest or protein in a cell, comprising contacting the cell with the composition of any one of embodiments 27-28.
33. A method of treating a disease in a subject, comprising administering to the subject the composition of any one of embodiments 27-28. 34. A method for identifying a targeting molecule that targets a cell or a tissue, optionally wherein the targeting molecule crosses a BBB, comprising selecting for targeting molecules that interact with TFRC, optionally comprising selecting for targeting molecules that interact with both human and cynomolgus macaque TFRC.
35. A method for identifying a targeting molecule that crosses the BBB or exhibits enhanced delivery to a cell or a tissue, comprising selecting for targeting molecules that comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 1-245 as shown in a single row in Tables 1 to 7.
EXAMPLES
Example 1. Methods
1.1. AAV capsid library generation
[0099] Capsid libraries used in round 1 screening were constructed by insertion of peptides into the exposed loops of the parent capsid proteins. Gibson assembly was used to generate the capsid libraries where the introduced peptides were encoded by the primers used to amplify the assembly fragments. Two PCR products from the parent capsid gene sequence were amplified (left and right fragments), these included an overlap region to facilitate assembly using the Gibson assembly procedure into a plasmid backbone (see e.g. Gibson et al (2009) Nat Meth 6(5):343-345).
[0100] An additional AAV9 capsid library used in round 1 screening was constructed by inserting trimer-19 synthesized codon block oligos using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs catalog number E2621) between amino acids 587 and 590 with replacement of amino acids 588 and 589.
[0101] For round 2 selection, capsid variants for library screening were synthesized as an oligo pool. Each capsid peptide was synthesized with unique nucleotide sequences encoding the peptide, and each peptide was linked to at least three distinct barcodes. The oligo pool was cloned into a linearized intermediate plasmid, followed by cloning of a constant donor sequence to separate the barcode and peptide region and generate the full AAV vector construct. Expression of barcodes was driven by a ubiquitous CMV promoter. The peptide sequences listed in Table 1 were inserted into AAV3B (SEQ ID NO: 246) between amino acids 588 and 589. The peptide sequences listed in Table 2 were inserted into AAV5 (SEQ ID NO: 247) between amino acids 577 and 578. The peptide sequences listed in Table 3 were inserted into AAV6 (SEQ ID NO: 248) between amino acids 589 and 590. The peptide sequences listed in Tables 4 and 6 were inserted into AAV8 (SEQ ID NO: 249) between amino acids 590 and 591. The peptide sequences listed in Tables 5 and 7 were inserted into AAV9 (SEQ ID NO: 250) between amino acids 454 and 455 or between 588 and 589 or between 587 and 590 with replacement of 588 or 589, as indicated.
[0102] AAV capsid libraries were manufactured in HEK293 cells. Briefly, AAV libraries were produced by triple transient transfection of the capsid library plasmid, pXX6 helper (encodes essential adenovirus genes E4, E2A, and VA), and with supplementation of Rep in trans. Capsids were purified by cesium density centrifugation and buffer exchanged into PBS plus 0.001% PF-68 by dialysis. DNase-resistant viral genomic titers were measured by quantitative real time PCR.
1.2. Biotinylation of recombinant human TFRC
[0103] Recombinant human transferrin receptor was diluted to 2 mg/mL in PBS (without calcium and magnesium). EZ-Link NHS-LC-Biotin (Thermo Fisher Catalog #21336) was added to a final concentration of 10 mM and the mixture was incubated on ice for 2 hours. After incubation, the biotinylation reaction was quenched by adding 500 mM glycine to a final concentration of 50 mM and incubating on ice for 30 minutes. The same procedure was applied to biotinylate bovine serum albumin for use as a blocking agent.
1.3. In vitro panning with recombinant human TFRC
[0104] To block non-specific interactions 1 mg of DynaBeads M-280 Streptavidin beads were rotated for an hour at room temperature with 4el3 molecules of biotinylated bovine serum albumin. The beads were then rotated for an hour at room temperature with 4el3 vg of AAV capsid library. After the one-hour rotation, the pre-cleared AAV capsid library that did not bind to the streptavidin beads or biotinylated bovine serum albumin was separated using a tube magnet. One mg of Dynabeads M-280 Streptavidin beads were then rotated at room temperature with 4el3 molecules of biotinylated human TFRC. The pre-cleared AAV capsid library was added to the receptor-coated beads and rotated for an hour at room temperature. The unbound capsids were separated from the bead bound capsids using a tube magnet. The beads were washed three times with SuperBlock (PBS) Blocking Buffer (ThermoFisher Catalog #37515). The receptor bound AAV capsids were then eluted from the beads with Pierce IgG Elution Buffer pH 2.0 (ThermoFisher Catalog #21028). Vector genomes were extracted using the Maxwell RSC Viral Total Nucleic Acid Purification Kit (Promega Catalog # AS 1330) and samples were prepared for NGS using Kapa HiFi Hotstart ReadyMix (Roche Catalog #KK2602). Amplification of library specific amplicons was performed with the following cycling conditions: 95oC for 3:00 min; 25 cycles at 98oC for 20 sec; 58oC for 15 sec; 72oC for 30 sec followed by 72oC for 1 minute. Amplification was qualitatively confirmed by agarose gel electrophoresis and relative apparent amplification was used to determine the dilution of amplicons needed for indexing. Illumina plate level i5 and well level i7 indices were added to the amplicons with 10 cycles of amplification: 95oC for 3:00 min; 10 cycles at 98oC for 20 sec; 60oC for 15 sec; 72oC for 30 sec followed by 72oC for 1 minute. Finally, samples were pooled and purified using Qiagen GeneRead Size Selection Kit following the manufacturer’s protocol. Samples were sequenced on Illumina MiSeq platform using MiSeq Reagent Kit v2. Following NGS of library amplicons the reads were demultiplexed and features were extracted using a custom bioinformatic pipeline.
[0105] In parallel to sequencing, variants interacting with recombinant human TFRC were recovered from the bead bound fraction and re-cloned in a subsequent capsid library, produced and re-screened for binding to recombinant human TFRC. Binding variants went through a total of three selections before being included in the pooled round 2 library for further confirmation both against recombinant human TFRC protein and in cells overexpressing human or cynomolgus macaque TFRC.
1.4. Capsid library screening in cells overexpressing TFRC
[0106] Neuro2A cells were seeded in 10 cm dishes coated with poly-D-lysine (PDL) at a density of 3E6 cells per dish. 24 hours later cells were transfected with 1 microgram of plasmid encoding human or cynomolgus macaque TFRC under the control of the ubiquitous cytomegalovirus (CMV) promoter. In parallel a transfection control plate was transfected with 1 microgram of plasmid encoding a fluorescent protein (GFP or mRuby) under the control of the ubiquitous cytomegalovirus (CMV) promoter. Lipofectamine 3000 was used for transfection of the plasmid DNA. Cell culture media was changed 24 hours post-transfection to remove transfection reagents. 48 hours after transfection of plasmid DNA, the cells were transduced with the AAV capsid library at a multiplicity of infection of 3E4 vector genomes (vg) per cell in reduced serum media (0.5% FBS). Capsid delivery was assessed with transduction and binding assays. For cell culture transduction assays the media was changed 24 hours post-transduction to fully supplemented media (10% FBS). 72 hours post-transduction, the plates were washed one time with phosphate buffered saline (with calcium and magnesium), and RNA was extracted using the Qiagen RNeasy kit following the manufacturers protocol and quantified by NanoDrop 8000 spectrophotometer. RNA was reverse transcribed to cDNA using the NEB Induro Reverse Transcriptase kit.
[0107] For cell culture binding assays the cells were incubated with the AAV capsid library for 1 hour at 37°C and washed three times with phosphate buffered saline (with calcium and magnesium). DNA was extracted using the Qiagen DNeasy kit following the manufacturers protocol and quantified by NanoDrop 8000 spectrophotometer.
[0108] Samples from transduction and binding assays were prepared for nextgeneration sequencing (NGS) using Kapa HiFi Hotstart ReadyMix (Roche Catalog #KK2602). PCR amplification of library specific amplicons was performed with the following cycling conditions: 95oC for 3:00 min; 25 cycles at 98oC for 20 sec; 58oC for 15 sec; 72oC for 10 sec followed by 72oC for 1 minute. Amplification was qualitatively confirmed by agarose gel electrophoresis and relative apparent amplification was used to determine the dilution of amplicons needed for indexing. Illumina plate level i5 and well level i7 indices were added to the amplicons with 10 cycles of amplification: 95oC for 3:00 min; 10 cycles at 98oC for 20 sec; 60oC for 15 sec; 72oC for 15 sec followed by 72oC for 1 minute. Finally, samples were pooled and purified using Qiagen GeneRead Size Selection Kit following the manufacturer’s protocol. Samples were sequenced on the Illumina MiSeq platform using a MiSeq Reagent Kit v2. Following next-generation sequencing of library amplicons the reads were demultiplexed and features (the barcode or peptide sequence) were extracted using a custom bioinformatic pipeline. For barcoded libraries the extracted barcode was used to query a pre-determined lookup table and return the identity of the corresponding capsid variant. Finally, the log2 fold change (log2FC) enrichment of each capsid variant was normalized to its relative abundance in the administered AAV library.
Example 2. Results from round 2 capsid library screening
[0109] Capsid variants that were enriched for binding to recombinant human TFRC protein were synthesized as a pooled round 2 library. For the round 2 evaluation capsids were evaluated in cells overexpressing the human or cynomolgus macaque ortholog of TFRC. Both a transduction and a binding assay were completed to assay cell entry and binding, respectively. The fold change enrichment of each capsid was determined by next-generation sequencing and normalized to capsid abundance in the administered library. The average log2FC for each assay is shown in Tables 1, 2, 3, 4, 5, 6 and 7 for peptides inserted into the parent capsid AAV3B (Table 1), AAV5 (Table 2), AAV6 (Table 3), AAV8 (Tables 4 and 6), or AAV9 (Tables 5 and 7). Empty cells indicate that the capsid was not detected in that assay, potentially due to limited sample recovery or sequencing depth.
Table 1. Round 2 library performance for peptides inserted into the parent capsid AAV3B [0110] Table 1 is shown in Figure 7.
Table 2. Round 2 library performance for peptides inserted into the parent capsid AAV5
[OHl] Table 2 is shown in Figure 7.
Table 3. Round 2 library performance for peptides inserted into the parent capsid AAV6
[0112] Table 3 is shown in Figure 7.
Table 4 and Table 6. Round 2 library performance for peptides inserted into the parent capsid AAV8
[0113] Table 4 and Table 6 are shown in Figure 7. Table 4 shows peptides that exhibit targeting specificity for human transferrin receptor. Table 6 shows peptides that target both human and cynomolgus macaque transferrin receptor.
Table 5 and Table 7. Round 2 library performance for peptides inserted into the parent capsid AAV9
[0114] Table 5 and Table 7 are shown in Figure 7. Table 5 shows peptides that exhibit targeting specificity for human transferrin receptor. Table 7 shows peptides that target both human and cynomolgus macaque transferrin receptor.
Example 3. Evaluation of capsids engineered to target TFRC
3.1. Methods for individual evaluation of receptor-targeted capsids in cells overexpressing human or cynomolgus macaque TFRC
[0115] Neuro2A cells were seeded in 96-well tissue culture plates at a density of 3.5E4 cells/well and transfected with 100 ng/well of a TFRC overexpression construct or a fluorescent protein transfection control using Lipofectamine 3000. The media was changed 24 hours post transfection. 48 hours post transfection, the cells were transduced with AAV in reduced serum EMEM media (2% FBS) at a multiplicity of infection of 3E2, 1E3, 3E3, or 1E4 vector genomes per cell. AAV capsids contained an expression cassette encoding a GFP fluorescent protein under the control of the ubiquitous CAG promoter. 72 hours post transduction, the plates were washed one time with phosphate buffered saline (with calcium and magnesium) and cDNA was generated using a Cells-to-CT kit (Thermo Fisher Catalog #21336). Transgene expression was quantified by RT-qPCR using a Qiagen QuantiNova Probe PCR kit. A mRNA-specific primer probe was used to assess transgene expression and normalized to expression of the GAPDH housekeeping gene. Data normalization and analysis was performed using the Bio-Rad CFX Maestro software. For fold change calculations a nonlinear regression model was used to interpolate relative transgene expression values for each capsid-receptor condition. These values were then scaled to the transfection control value for each capsid.
3.2. Results of individual capsid evaluation
[0116] Capsids engineered to target transferrin receptor exhibit enhanced transduction in cells overexpressing transferrin receptor relative to a transfection control. Representative engineered capsids, designed to target transferrin receptor and described herein, are presented as illustrative examples. Some capsids engineered to target transferrin receptor exhibit higher transduction in cells overexpressing human, or cynomolgus macaque transferrin receptor relative to cells overexpressing a fluorescent protein transfection control. Another capsid exhibits enhanced transduction only in cells expressing human transferrin receptor, but not in cells expressing cynomolgus macaque transferrin receptor. In contrast, the parental capsid AAV9 shows no significant increase in transgene expression in cells overexpressing human or cynomolgus macaque transferrin receptor relative to cells overexpressing a fluorescent protein transfection control.
Example 4. Binding kinetics of engineered capsids targeting transferrin receptor
4.1. Methods for bio-layer interferometry (BLI) measurement of binding kinetics for TFRC-targeted capsids
[0117] Binding of AAV capsids to transferrin receptor was measured by bio-layer interferometry on an Octet Red96e instrument. All steps were performed at 30°C shaking at 1,000 rpm. Purified human or cynomolgus macaque transferrin receptors containing an N- terminal Fc domain were diluted to 5 pg/mL in PBS with 0.05% Tween20 and 0.1% BSA and immobilized on Octet BLI anti-human Fc capture biosensors (Sartorius Catalog # 18-5060) to produce a shift of approximately 1.3 nm. Capsids were diluted in PBS with 0.05% Tween20 and 0.1% BSA across a 2-fold capsid dilution series ranging in concentration from 3.125E10 to 1E12 vg/mL. Capsid association to each sensor was performed for 150 seconds followed by a 350 second dissociation step.
4.2. Results of bio-layer interferometry (BLI) measurement of binding kinetics for TFRC- targeted capsids
[0118] Bio-layer interferometry assays corroborate the results obtained in cell culture experiments. Capsids that exhibit enhanced transduction of cells expressing human or cynomolgus macaque transferrin receptor likewise exhibit robust binding responses to immobilized human or cynomolgus macaque transferrin receptor (Fig 11). A capsid that was specific to human transferrin receptor in cell culture experiments likewise showed specific binding to human transferrin receptor (Fig 12). The parent capsid AAV9 exhibits no binding to human or cynomolgus macaque transferrin receptor (Fig 13). These trends are consistent across a range of capsid concentrations tested.
Sequences
[0119] The amino acid sequences of the parent capsids are defined as follows:
[0120] AAV3B capsid amino acid sequence SEQ ID NO: 246:
[0121] MAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLV LPGYKYLGPGNGLDKGEPVNEADAAALEHDKAYDQQLKAGDNPYLKYNHADAEF QERLQEDTSFGGNLGRAVFQAKKRILEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSG VGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADN NEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDN HYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQN DGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNG SQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQY LYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNN NSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNA ELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDR DVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFAS FITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPI GTRYLTRNL [0122] AAV5 capsid amino acid sequence SEQ ID NO: 247:
[0123] MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLP
GYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKL ADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDS KPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASG DWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWG YFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTS
TVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFF CLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNT
GGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEG ASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQP VNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPET GAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEME WELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL
[0124] AAV6 capsid amino acid sequence SEQ ID NO: 248:
[0125] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGL
VLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEF QERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSS GIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADN NEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASND NHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTT
NDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNN GSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQ
YLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNN NSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTA LDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRD VYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFI TQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIG
TRYLTRPL
[0126] AAV8 capsid amino acid sequence SEQ ID NO: 249:
[0127] MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGL
VLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEF QERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSST
GIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMAD
NNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGAT
NDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEV
TQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLN
NGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLID
QYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQN
NNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNA
DYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNR
DVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNS
FITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPI
GTRYLTRNL
[0128] AAV9 capsid amino acid sequence SEQ ID NO: 250:
[0129] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE
FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS
AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD
NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN
DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT
DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID
QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN
SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD
ADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQD
RDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKL
NSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPR PIGTRYLTRNL
[0130] The amino acid sequences of individual capsids are defined as follows (inserted
Peptide Sequence shown in bold):
[0131] CNSRCV420 capsid amino acid sequence SEQ ID NO: 251:
[0132] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID
QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD ADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAGSSHQWMQAQTGWVQNQGIL PGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPP TAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVN TEGVYSEPRPIGTRYLTRNL
[0133] CNSRCV421 capsid amino acid sequence SEQ ID NO: 252:
[0134] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD ADKVMITNEEEIKTTNPVATESYGQVATNHQSAQHMDSQTWQQAQTGWVQNQGIL PGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPP TAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVN
TEGVYSEPRPIGTRYLTRNL
[0135] CNSRCV422 capsid amino acid sequence SEQ ID NO: 253:
[0136] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVT DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD
ADKVMITNEEEIKTTNPVATESYGQVATNHQSAMELANPNPVQAQTGWVQNQGILP GMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNT EGVYSEPRPIGTRYLTRNL
[0137] CNSRCV423 capsid amino acid sequence SEQ ID NO: 254:
[0138] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVT DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD ADKVMITNEEEIKTTNPVATESYGQVATNHQSAYPYSKTETHQAQTGWVQNQGILP GMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNT
EGVYSEPRPIGTRYLTRNL
[0139] CNSRCV424 capsid amino acid sequence SEQ ID NO: 255:
[0140] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVT DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD
ADKVMITNEEEIKTTNPVATESYGQVATNHQSAMMFDATSPMQAQTGWVQNQGILP GMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNT EGVYSEPRPIGTRYLTRNL
[0141] CNSRCV425 capsid animo acid sequence SEQ ID NO: 256:
[0142] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE
FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT
DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD
ADKVMITNEEEIKTTNPVATESYGQVATNHQSANQYVKSLDSQAQTGWVQNQGILP GMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNT EGVYSEPRPIGTRYLTRNL
[0143] CNSRCV426 capsid animo acid sequence SEQ ID NO: 257:
[0144] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE
FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT
DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN
SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD
ADKVMITNEEEIKTTNPVATESYGQVATNHQSAPVYHVSDARQAQTGWVQNQGILP
GMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT
AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNT
EGVYSEPRPIGTRYLTRNL
[0145] CNSRCV427 capsid animo acid sequence SEQ ID NO: 258:
[0146] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE
FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS
AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD
NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN
DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT
DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID
QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN
SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD
ADKVMITNEEEIKTTNPVATESYGQVATNHQSAIDEYQRVKQQAQTGWVQNQGILP
GMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT
AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNT
EGVYSEPRPIGTRYLTRNL
[0147] CNSRCV428 capsid animo acid sequence SEQ ID NO: 259:
[0148] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE
FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS
AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD
NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN
DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT
DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID
QYLYYLSKTINGWQPLTSGPRSSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQ
RVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFG KQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQ
GILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPA DPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFA VNTEGVYSEPRPIGTRYLTRNL
[0149] CNSRCV429 capsid animo acid sequence SEQ ID NO: 260:
[0150] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE
FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS
AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD
NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN
DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT
DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID
QYLYYLSKTINGSGSREFMEGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVST
TVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGT
GRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPG
MVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTA
FNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTE GVYSEPRPIGTRYLTRNL
[0151] CNSRCV43 1 capsid animo acid sequence SEQ ID NO: 261 :
[0152] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE
FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS
AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD
NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN
DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT
DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID
QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN
SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD
ADKVMITNEEEIKTTNPVATESYGQVATNHQSAQLFFKDNMHVSAQAQTGWVQNQ
GILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPA DPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFA
VNTEGVYSEPRPIGTRYLTRNL
[0153] CNSRCV432 capsid animo acid sequence SEQ ID NO: 262:
[0154] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD ADKVMITNEEEIKTTNPVATESYGQVATNHQSAQRPSQKDAWPSAQAQTGWVQNQ GILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPA DPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFA
VNTEGVYSEPRPIGTRYLTRNL
[0155] CNSRCV433 capsid animo acid sequence SEQ ID NO: 263:
[0156] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD ADKVMITNEEEIKTTNPVATESYGQVATNHQSAHMDQPYFQSQAQTGWVQNQGILP GMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNT
EGVYSEPRPIGTRYLTRNL [0157] CNSRCV434 capsid animo acid sequence SEQ ID NO: 264:
[0158] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD ADKVMITNEEEIKTTNPVATESYGQVATNHQSAQKMQSHNWDQAQTGWVQNQGIL PGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPP TAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVN
TEGVYSEPRPIGTRYLTRNL
[0159] CNSRCV436 capsid animo acid sequence SEQ ID NO: 265:
[0160] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD ADKVMITNEEEIKTTNPVATESYGQVATNHQSAQSVSSYTKAQAQTGWVQNQGILPG MVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTA FNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTE
GVYSEPRPIGTRYLTRNL [0161] CNSRCV437 capsid animo acid sequence SEQ ID NO: 266:
[0162] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD ADKVMITNEEEIKTTNPVATESYGQVATNHQSAVKTTTQAYNQAQTGWVQNQGILP GMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNT
EGVYSEPRPIGTRYLTRNL
[0163] CNSRCV438 capsid animo acid sequence SEQ ID NO: 267:
[0164] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD ADKVMITNEEEIKTTNPVATESYGQVATNHQSAMMMQNPQPRQAQTGWVQNQGIL PGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPP TAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVN
TEGVYSEPRPIGTRYLTRNL [0165] CNSRCV439 capsid animo acid sequence SEQ ID NO: 268:
[0166] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL
VLPGYI<YLGPGNGLDI<GEPVNAADAAALEHDI<AYDQQLI<AGDNPYLI<YNHADAE
FQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSS
AGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD
NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSN
DNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT
DNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN
DGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLID
QYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNN
SEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVD
ADKVMITNEEEIKTTNPVATESYGQVATNHQSAIHDAHIIKMQAQTGWVQNQGILPG
MVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTA
FNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTE
GVYSEPRPIGTRYLTRNL

Claims

WHAT IS CLAIMED IS:
1. An adeno-associated virus (AAV) capsid protein comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 1-245 as shown in a single row in Tables 1 to 7.
2. The AAV capsid protein of claim 1, wherein the amino acid sequence is inserted into a parent capsid (“Parent Capsid”) at an insertion site (“Peptide Insertion Site”) as shown in a single row in Tables 1 to 7.
3. The AAV capsid protein of claim 1, wherein the amino acid sequence comprises a peptide sequence of any one of SEQ ID NOs: 1-245 as shown in a single row in Tables 1 to 7, and optionally wherein the parent capsid and/or the insertion site is/are as indicated in the same single row as shown in Tables 1 to 7.
4. The AAV capsid protein of any one of claims 1-3, wherein the amino acid sequence is inserted into a parental capsid, optionally wherein the parental capsid is selected from any one of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC-BBB.
5. An AAV capsid protein, wherein the AAV capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to or comprises a sequence set forth in SEQ ID NOs: 251-268 designated as CNSRCV420, CNSRCV421, CNSRCV422, CNSRCV423, CNSRCV424, CNSRCV425, CNSRCV426, CNSRCV427, CNSRCV428, CNSRCV429, CNSRCV431, CNSRCV432, CNSRCV433, CNSRCV434, CNSRCV436, CNSRCV437, CNSRCV438, or CNSRCV439.
6. The AAV capsid protein of any one of claims 1-5, wherein the AAV capsid protein interacts with transferrin receptor (TFRC), thereby enabling delivery of the AAV capsid protein to a cell or a tissue, optionally wherein interaction with TFRC enables the AAV capsid protein to cross a blood-brain barrier (BBB).
7. The AAV capsid protein of claim 6, wherein the AAV capsid protein interacts with both human and cynomolgus macaque TFRC, thereby enabling the AAV capsid protein to cross the BBB in both species.
8. The AAV capsid protein of claim 6 or claim 7, wherein the AAV capsid protein comprises a peptide sequence as shown in a single row in Tables 6 to 7.
9. The AAV capsid protein of any one of claims 6-8, wherein the amino acid sequence is inserted into a parental capsid, optionally wherein the parental capsid is selected from any one of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC-BBB.
10. An adeno-associated virus (AAV) capsid protein that interacts with both human and cynomolgus macaque TFRC, thereby enabling delivery of the AAV capsid protein to a cell or a tissue, optionally wherein interaction with TFRC enables the AAV capsid protein to cross a BBB.
11. A nucleic acid molecule encoding an AAV capsid protein of any one of claims 1-10.
12. A host cell comprising the nucleic acid molecule of claim 11.
13. A composition comprising: 1) an AAV capsid protein comprising of any one of claims 1-10; and 2) an expression construct comprising a coding sequence for a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and/or therapeutic payload.
14. The composition of claim 13, wherein the payload of interest is a therapeutic payload, and wherein the therapeutic payload comprises a DNA binding domain, optionally wherein the therapeutic payload comprises a fusion protein.
15. The composition of claim 13 or claim 14, wherein the payload of interest comprises a therapeutic protein, a zinc finger protein, a CRISPR-associated DNA binding protein, a TALE protein, an antibody, an enzyme, a regulatory RNA, a Bxbl serine recombinase, or a DNA recombinase protein.
16. A method of delivering a payload of interest to a cell or a tissue, wherein a coding sequence for the payload of interest is encapsidated in an AAV capsid protein according to any one of claims 1-10, and wherein the AAV capsid protein interacts with TFRC.
17. The method of claim 16, wherein delivering the payload of interest to the cell or the tissue comprises crossing a BBB.
18. A method of activating, expressing, repressing, or modulating the expression of a therapeutically relevant gene of interest in a cell, comprising contacting the cell with a composition of any one of claims 13-15.
19. A method of treating a disease in a subject, comprising administering to the subject a composition of any one of claims 13-15.
20. Use of an AAV capsid protein of any one of claims 1-10, a nucleic acid construct of claim 11, or a host cell of claim 12 for the manufacture of a medicament in the method of any one of claims 16-19.
21. A method for identifying an AAV capsid variant that crosses the BBB or exhibits enhanced delivery to a cell or a tissue, comprising selecting for AAV capsids that interact with TFRC, optionally comprising selecting for AAV capsids that interact with both human and cynomolgus macaque TFRC.
22. A method for identifying an AAV capsid variant that crosses the BBB or exhibits enhanced delivery to a cell or a tissue, comprising selecting for AAV capsids that comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 1-245 as shown in a single row in Tables 1 to 7.
23. A targeting molecule that interacts with TFRC, thereby enabling delivery of the targeting molecule to a cell or a tissue, optionally wherein the interaction with TFRC enables the targeting molecule to cross the blood-brain barrier (BBB).
24. The targeting molecule of claim 23, wherein the targeting molecule interacts with both human and cynomolgus macaque TFRC.
25. The targeting molecule of claim 23 or claim 24, comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 1-245 as shown in a single row in Tables 1 to 7.
26. The targeting molecule of any one of claims 23-25, wherein the targeting molecule is fused or conjugated to an AAV, a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.
27. A composition comprising: 1) a targeting molecule of any one of claims 23-26; and 2) a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and/or therapeutic payload.
28. The composition of claim 27, wherein the payload of interest encodes or comprises a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.
29. A method of delivering a payload of interest to a cell or a tissue, wherein a coding sequence for the payload of interest is associated with a targeting molecule according to any one of claims 23-26, and wherein the targeting molecule interacts with TFRC.
30. The method of claims 29, wherein delivering the payload of interest to the cell or the tissue comprises crossing a BBB.
31. A method of delivering a therapeutically relevant gene or protein of interest to a cell, comprising contacting the cell with the composition of any one of claims 27-28.
32. A method of modulating the expression or activity of a therapeutically relevant gene of interest or protein in a cell, comprising contacting the cell with the composition of any one of claims 27-28.
33. A method of treating a disease in a subject, comprising administering to the subject the composition of any one of claims 27-28.
34. A method for identifying a targeting molecule that targets a cell or a tissue, optionally wherein the targeting molecule crosses a BBB, comprising selecting for targeting molecules that interact with TFRC, optionally comprising selecting for targeting molecules that interact with both human and cynomolgus macaque TFRC.
35. A method for identifying a targeting molecule that crosses the BBB or exhibits enhanced delivery to a cell or a tissue, comprising selecting for targeting molecules that comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 1-245 as shown in a single row in Tables 1 to 7.
PCT/US2025/028247 2024-05-07 2025-05-07 Peptides targeting tfrc for blood-brain barrier crossing Pending WO2025235682A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200354714A1 (en) * 2014-10-03 2020-11-12 University Of Massachusetts Novel high efficiency library-identified aav vectors
US20200370137A1 (en) * 2019-04-26 2020-11-26 Sangamo Therapeutics, Inc. Engineering aav

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200354714A1 (en) * 2014-10-03 2020-11-12 University Of Massachusetts Novel high efficiency library-identified aav vectors
US20200370137A1 (en) * 2019-04-26 2020-11-26 Sangamo Therapeutics, Inc. Engineering aav

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