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WO2025050069A1 - Insertion de gène programmable à l'aide d'enzymes d'intégration modifiées - Google Patents

Insertion de gène programmable à l'aide d'enzymes d'intégration modifiées Download PDF

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Publication number
WO2025050069A1
WO2025050069A1 PCT/US2024/044901 US2024044901W WO2025050069A1 WO 2025050069 A1 WO2025050069 A1 WO 2025050069A1 US 2024044901 W US2024044901 W US 2024044901W WO 2025050069 A1 WO2025050069 A1 WO 2025050069A1
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integration
engineered
enzyme
integration enzyme
atgrna
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PCT/US2024/044901
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English (en)
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Jesse COCHRANE
Sandeep Kumar
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Tome Biosciences, Inc.
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Publication of WO2025050069A1 publication Critical patent/WO2025050069A1/fr

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells

Definitions

  • the present disclosure describes integration enzymes engineered such that upon being introduced into a cell, the integration enzyme has increased stability (e.g., half-life) compared to a control integration enzyme not engineered to have increased stability.
  • the increase in stability extends the capacity of the integration enzyme to mediate integration.
  • the engineered integration enzyme includes an integration enzyme or fragment thereof, an at least first stabilization domain, and a nuclear localization signal.
  • the present disclosure provides integration enzymes engineered to have increased stability for use in site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE) (see lonnidi et al. doi: 10.1101/2021.11.01.466786; the entirety of lonnidi et al. is incorporated by reference), transposon-mediated gene editing, or other suitable gene editing or gene incorporation technology.
  • PASTE Programmable Addition via Site-Specific Targeting Elements
  • this disclosure features an engineered integration enzyme, comprising: (a) an integration enzyme or fragment thereof; (b) an at least first stabilization domain; and (c) a nuclear localization signal (NLS).
  • an integration enzyme or fragment thereof comprising: (a) an integration enzyme or fragment thereof; (b) an at least first stabilization domain; and (c) a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • the integration enzyme is a large serine integrase.
  • the method further comprises a second atgRNA.
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, wherein the at least first pair of atgRNAs have domains that are capable of guiding the gene editor polypeptide to the target DNA sequence;
  • the first atgRNA further includes a first RT template that comprises at least a portion of an at least first integration recognition site;
  • the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site, whereby the least first integration recognition site is integrated into the genome of the cell at the target sequence.
  • the method enhances integration efficiency of the donor polynucleotide template at the site-specifically integrated integration recognition site at the double-stranded target DNA sequence as compared to the integration efficiency of the donor polynucleotide template at the site-specifically integrated recognition site when using a method that does not comprise the engineered integration enzyme.
  • FIG. 1 shows a non-limiting illustration of a gene editor construct packaged within a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • FIG. 2 illustrates the donor polynucleotide (“donor template”) (synonymously, “cargo” or “payload” or “template polynucleotide”) packaged within a vector.
  • donor template synthetic polynucleotide
  • FIG. 3 illustrates integrase-mediated self-circularization of the donor template (template polynucleotide) within viral genome.
  • the circularized donor template is capable of being genomically incorporated into an orthogonal integrase target recognition site (i.e., “beacon”).
  • FIG. 4 shows non-limiting illustrations of a gene editor construct packaged within a lipid nanoparticle and an atgRNA, ngRNA, and donor template (i.e., template polynucleotide encoding a gene of interest) packaged within a vector.
  • GOI gene of interest.
  • PGI programmable gene insertion.
  • U6 U6 promoter.
  • atgRNA attachment site-containing guide RNA (atgRNA).
  • FIG. 5 shows non-limiting illustrations of a gene editor construct (e.g., mRNA encoding PE2-BxBl) and a nicking guide RNA (ngRNA) packaged within a lipid nanoparticle (LNP) and an atgRNA and donor template (i.e., template polynucleotide encoding a gene of interest) packaged within a vector.
  • a gene editor construct e.g., mRNA encoding PE2-BxBl
  • ngRNA nicking guide RNA
  • LNP lipid nanoparticle
  • an atgRNA and donor template i.e., template polynucleotide encoding a gene of interest
  • FIG. 6B shows non-limiting examples of sequences that enable self-circularization (e.g., LoxP AttP GT (SEQ ID NO: 568 and SEQ ID NO: 569); FRT AttP GT (SEQ ID NO: 570 and SEQ ID NO: 571); and AttB CC AttP GT (SEQ ID NO: 572 and SEQ ID NO: 573)).
  • GT indicates an AttP site with a GT dinucleotide.
  • AttB CC indicates an AttB site with a CC dinucleotide.
  • LoxP a LoxP recombinase recognition site.
  • FRT a FRT recombinase recognition site.
  • FIG. 7 shows a non-limiting illustration of recombinase/integrase-mediated intramolecular circularization products.
  • FIGs. 8A-8B show non-limiting illustrations of a ddPCR assay and intramolecular circularization ddPCR detection probes.
  • FIG. 8A shows a non-limiting illustration of the ddPCR strategy.
  • FIG. 8B shows non-limiting examples of the universal probe (SEQ ID NO: 574 and SEQ ID NO: 575) and an AttR probe (SEQ ID NO: 576 and SEQ ID NO: 577) that can be used in the assay shown in FIG. 8A.
  • FIG. 9 shows a non-limiting illustration of a pDNA genome and AAV transfection and screening protocol.
  • FIG. 10 shows data for circularization of AAV pDNA and packaged AAV genomic DNA with Bxb 1.
  • FIG. 11 shows data for Cre-, FLPe-, and Bxb 1 -mediated circularization of AAV pDNA confirmed by ddPCR.
  • FIG. 12 shows Cre-, FLPe-, and Bxb 1 -mediated circularization of packaged AAV confirmed by ddPCR
  • FIG. 13 shows percent circularization between the Bxb 1 -mediated attR scar ddPCR probe (“attR probe” described in FIG. 8B) and the universal ddPCR probe (“universal probe” described in FIG. 8B)
  • FIGs. 14A-14E shows analysis of AttP variants.
  • FIG. 14A shows a non-limiting schematic of AttP mutations tested for improving integration efficiency (SEQ ID NOS: 394 and 540-542, respectively, in order of appearance).
  • FIG. 14B shows integration efficiencies of wildtype and mutant AttP sites across a panel of AttB lengths.
  • FIG. 14C shows a non-limiting schematic of multiplexed integration of different cargo sets at specific genomic loci. Three fluorescent cargos (GFP, mCherry, and YFP) are inserted orthogonally at three different loci (ACTB, LMNB1, NOLC1) for in-frame gene tagging.
  • FIG. 14A shows a non-limiting schematic of AttP mutations tested for improving integration efficiency (SEQ ID NOS: 394 and 540-542, respectively, in order of appearance).
  • FIG. 14B shows integration efficiencies of wildtype and mutant AttP sites across a panel of AttB lengths.
  • FIG. 14C shows
  • FIG. 14D shows orthogonality of top 4 AttB/AttP dinucleotide pairs evaluated for GFP integration with PASTE at the ACTB locus.
  • FIG. 15 illustrates a schematic of single atgRNA and dual atgRNA approaches for beacon placement (“integration recognition site”).
  • FIG. 21A shows ddPCR data for percent in vivo beacon placement in the Nolcl locus of neonatal mice at 6 weeks post-delivery of a single dose of a mixture of two LNPs.
  • First LNP contained mRNA encoding a prime editing system and a first synthetic atgRNA (atgRNAl) at a 1 : 1 ratio.
  • Second LNP contained mRNA encoding a prime editing system and a second synthetic atgRNA (atgRNA2) at a 1 : 1 ratio.
  • Each of the first and second atgRNAs targeted the mouse Nolcl locus, encoded a portion of an integration recognition site (“beacon”), and together included a 6bp overlap.
  • the first and second LNPs were combined 1 : 1 as mixture and administered at either 1 mg/kg or 3 mg/kg.
  • LNP #F2 LNP formulation #F2.
  • Prime editing or CRISPR system Examples of prime editors can be found in the following: W02020/191153, W02020/191171, WO2020/191233,
  • an integrase or recombinase is directly linked or fused, for example by a peptide linker, which may be cleavable or non-cleavable, to the prime editor fusion protein (i.e., fused Cas9 nickase-reverse transcriptase) or Gene Writer protein.
  • a peptide linker which may be cleavable or non-cleavable, to the prime editor fusion protein (i.e., fused Cas9 nickase-reverse transcriptase) or Gene Writer protein.
  • Suitable linkers for example between the Cas9, RT, and integrase, may be selected from Table 3:
  • SpCas9 Streptococcus pyogenes Cas9
  • REC recognition
  • NUC nuclease
  • the RuvC domain is assembled from the three split RuvC motifs (RuvC I— III) and interfaces with the PI domain to form a positively charged surface that interacts with the 30 tail of the sgRNA.
  • the HNH domain lies between the RuvC II— III motifs and forms only a few contacts with the rest of the protein. Structural aspects of SpCas9 are described by Nishimasu et al., Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA, Cell 156, 935-949, February 27, 2014.
  • PAM-interacting domain The NUC lobe contains the PAM-interacting (PI) domain that is positioned to recognize the PAM sequence on the noncomplementary DNA strand.
  • the PI domain of SpCas9 is required for the recognition of 5’-NGG-3’ PAM, and deletion of the PI domain (A1099-1368) abolished the cleavage activity, indicating that the PI domain is critical for SpCas9 function and a major determinant for the PAM specificity.
  • RuvC domain- The NUC lobe contains the PAM-interacting (PI) domain that is positioned to recognize the PAM sequence on the noncomplementary DNA strand.
  • the PI domain of SpCas9 is required for the recognition of 5’-NGG-3’ PAM, and deletion of the PI domain (A1099-1368) abolished the cleavage activity, indicating that the PI domain is critical for SpCas9 function and a major determinant for the PAM specificity.
  • SpCas9 HNH nucleases have three catalytic residues, Asp839, His840, and Asn863 and cleave the complementary strand of the target DNA through a single-metal mechanism.
  • sgRNA:DNA recognition' The sgRNA guide region is primarily recognized by the REC lobe.
  • the backbone phosphate groups of the guide region interact with the RECI domain (Argl65, Glyl66, Arg403, Asn407, Lys510, Tyr515, and Arg661) and the bridge helix (Arg63, Arg66, Arg70, Arg71, Arg74, and Arg78).
  • the 20-hydroxyl groups of Gl, Cl 5, U16, and G19 hydrogen bond with Vai 1009, Tyr450, Arg447/Ile448, and Thr404, respectively.
  • nucleobases of G21 and U50 in the G21 :U50 wobble pair stack with the terminal C20:G10 pair in the guide:target heteroduplex and Tyr72 on the bridge helix, respectively, with the U50 04 atom hydrogen bonded with Arg75.
  • A51 adopts the syn conformation and is oriented in the direction opposite to U50.
  • the nucleobase of A51 is sandwiched between Phel 105 and U63, with its Nl, N6, and N7 atoms hydrogen bonded with G62, Glyl l03, and Phel 105, respectively.
  • Stem-loop recognition' Stem loop 1 is primarily recognized by the REC lobe, together with the PI domain.
  • the backbone phosphate groups of stem loop 1 (nucleotides 52, 53, and 59- 61) interact with the RECI domain (Leu455, Ser460, Arg467, Thr472, and Ile473), the PI domain (Lysl 123 and Lysl 124), and the bridge helix (Arg70 and Arg74), with the 20-hydroxyl group of G58 hydrogen bonded with Leu455.
  • A52 interacts with Phel 105 through a face-to-edge p-p stacking interaction, and the flipped U59 nucleobase hydrogen bonds with Asn77.
  • the single-stranded linker and stem loops 2 and 3 are primarily recognized by the NUC lobe.
  • the backbone phosphate groups of the linker (nucleotides 63-65 and 67) interact with the RuvC domain (Glu57, Lys742, and Lysl097), the PI domain (Thrl l02), and the bridge helix (Arg69), with the 20-hydroxyl groups of U64 and A65 hydrogen bonded with Glu57 and His721, respectively.
  • the C67 nucleobase forms two hydrogen bonds with Vail 100.
  • Stem loop 2 is recognized by Cas9 via the interactions between the NUC lobe and the non-Watson-Crick A68:G81 pair, which is formed by direct (between the A68 N6 and G81 06 atoms) and water-mediated (between the A68 Nl and G81 Nl atoms) hydrogen-bonding interactions.
  • the A68 and G81 nucleobases contact Serl351 and Tyrl356, respectively, whereas the A68:G81 pair interacts with Thrl358 via a water-mediated hydrogen bond.
  • the 20-hydroxyl group of A68 hydrogen bonds with His 1349, whereas the G81 nucleobase hydrogen bonds with Lys33.
  • Stem loop 3 interacts with the NUC lobe more extensively, as compared to stem loop 2.
  • the backbone phosphate group of G92 interacts with the RuvC domain (Arg40 and Lys44), whereas the G89 and U90 nucleobases hydrogen bond with Glnl272 and Glul225/Alal227, respectively.
  • the A88 and C91 nucleobases are recognized by Asn46 via multiple hydrogenbonding interactions.
  • Cas9 proteins smaller than SpCas9 allow more efficient packaging of nucleic acids encoding CRISPR systems, e.g., Cas9 and sgRNA into one rAAV (“all-in-one-AAV”) particle.
  • efficient packaging of CRISPR systems can be achieved in other viral vector systems (i.e., lentiviral, integration deficient lentiviral, hd-AAV, etc.) and non-viral vector systems (i.e., lipid nanoparticle).
  • Small Cas9 proteins can be advantageous for multidomain-Cas-nuclease-based systems for prime editing.
  • Cas9 proteins include Staphylococcus aureus (SauCas9, 1053 amino acid residues) and Campylobacter jejuni (CjCas9, 984 amino residues).
  • SerCas9 Staphylococcus aureus
  • CjCas9 Campylobacter jejuni
  • Staphylococcus lugdunensis (Siu) Cas9 as having genome-editing activity and provided homology mapping to SpCas9 and SauCas9 to facilitate generation of nickases and inactive (“dead”) enzymes (Schmidt et al., 2021, Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases. Nat Commun 12, 4219. doi.org/10.1038/s41467-021-24454-5) and engineered nucleases with higher cleavage activity by fragmenting and shuffling Cas9 DNAs.
  • the small Cas9s and nickases are useful in the instant invention.
  • the Cas9 proteins used herein may also include other “Cas9 variants” having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art.
  • Cas9 variants having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild
  • the crRNA-target strand heteroduplex is terminated by a stacking interaction with a conserved aromatic residue (Tyr410). This prevents base pairing between the crRNA and the target strand beyond nucleotides U20 and dA(-20), respectively. Beyond this point, the target DNA strand nucleotides re-engage the non-target DNA strand, forming a PAM-distal DNA duplex comprising nucleotides dC(-21)-dA(-27) and dG21*-dT27*, respectively. The duplex is confined between the REC2 and Nuc domains at the end of the central channel formed by the REC and NUC lobes.
  • the RuvC active site contains three catalytic residues (D917, E1006, and D1255). Structural observations suggest that both the target and non-target DNA strands are cleaved by the same catalytic mechanism in a single active site in Cpfl/Casl2a enzymes.
  • Another type V CRISPR is AsCpfl from Acidaminococcus sp BV3L6 (Yamano et al., Crystal structure of Cpfl in complex with guide RNA and target DNA, Cell 165, 949-962, May 5, 2016)
  • the nuclease comprises a Casl2f effector.
  • Small CRISPR- associated effector proteins belonging to the type V-F subtype have been identified through the mining of sequence databases and members classified into Casl2fl (Casl4a and type V-U3), Casl2f2 (Casl4b) and Casl2f3 (Casl4c, type V-U2 and U4).
  • Casl2fl Casl2fl
  • Casl4b Casl2f2
  • Casl4c type V-U2 and U4
  • protospacer adjacent sequence or “protospacer adjacent motif’ or “PAM” refers to an approximately 2-6 base pair DNA sequence (or a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-long nucleotide sequence) that is an important targeting component of a Cas9 nuclease.
  • PAM sequence is on either strand, and is downstream in the 5' to 3' direction of Cas9 cut site.
  • the PAM specificity can be modified by introducing one or more mutations, including (a) DI 135 V, R1335Q, and T1337R “the VQR variant”, which alters the PAM specificity to NGAN or NGNG, (b) D1135E, R1335Q, and T1337R “the EQR variant”, which alters the PAM specificity to NGAG, and (c) DI 135V, G1218R, R1335E, and T1337R “the VRER variant”, which alters the PAM specificity to NGCG.
  • the DI 135E variant of canonical SpCas9 still recognizes NGG, but it is more selective compared to the wild type SpCas9 protein.
  • Cas9 enzymes from different bacterial species can have varying PAM specificities and some embodiments are therefore chosen based on the desired PAM recognition.
  • Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN.
  • Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT.
  • Cas9 from Streptococcus thermophilis (StCas9) recognizes NNAGAAW.
  • Cas9 from Treponema denticola (TdCas) recognizes NAAAAC. These examples are not meant to be limiting.
  • Oh, Y. et al. describe linking reverse transcriptase to a Francisella novicida Cas9 [FnCas9(H969A)] nickase module.
  • FeCas9(H969A) Francisella novicida Cas9
  • nickase module By increasing the distance to the PAM, the FnCas9(H969A) nickase module expands the region of a reverse transcription template (RTT) following the primer binding site.
  • Prime editor fusion protein describes a protein that is used in prime editing.
  • Prime editing uses CRISPR enzyme that nicks or cuts only single strand of double stranded DNA, i.e., a nickase; and a nickase can occur either naturally or by mutation or modification of a nuclease that makes double stranded cuts.
  • Such an enzyme can be a catalytically-impaired Cas9 endonuclease (a nickase).
  • a nickase can be a Casl2a/b, MAD7, or variant thereof.
  • the nickase is fused to an engineered reverse transcriptase (RT).
  • the nickase is programmed (directed) with a primeediting guide RNA (pegRNA).
  • pegRNA primeediting guide RNA
  • the pegRNA both specifies the target site and encodes the desired edit.
  • the nickase is a catalytically-impaired Cas9 endonuclease, a Cas9 nickase, that is fused to the reverse transcriptase.
  • the Cas9 nickase part of the protein is guided to the DNA target site by the pegRNA, whereby a nick or single stranded cut occurs.
  • the reverse transcriptase domain then uses the pegRNA to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand.
  • the edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand.
  • the prime editor guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process (typically achieved with a nickase gRNA).
  • PEI refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a wild type MMLV RT having the following N-terminus to C-terminus structure: [NLS]-[Cas9(H840A)]- [linker] -[MMLV_RT(wt)] + a desired atgRNA (or PEgRNA).
  • the prime editors disclosed herein is comprised of PEI.
  • PE2 refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a variant MMLV RT having the following N-terminus to C-terminus structure:
  • PE3b refers to PE3 but wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence with mismatches to the unedited original allele that matches only the edited strand. Using this strategy, mismatches between the protospacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place.
  • a prime editing complex consists of a type II CRISPR PE protein containing an RNA-guided DNA-nicking domain fused to a reverse transcriptase (RT) domain and complexed with a pegRNA.
  • the pegRNA comprises (5’ to 3’) a spacer that is complementary to the target sequence of a genomic DNA, a nickase (e.g. Cas9) binding site, a reverse transcriptase template including editing positions, and primer binding site (PBS).
  • the PE-pegRNA complex binds the target DNA and the CRISPR protein nicks the PAM- containing strand.
  • the resulting 3' end of the nicked target hybridizes to the primer-binding site (PBS) of the pegRNA, then primes reverse transcription of new DNA containing the desired edit using the RT template of the pegRNA.
  • PBS primer-binding site
  • the overall structure of the pegRNA is like that of a typical type II sgRNA with a reverse transcriptase template/primer binding site appended to the 3’ end. The structure leaves the PBS at the 3’ end of the pegRNA free to bind to the nicked strand complementary to the target which forms the primer for reverse transcription.
  • Guide RNAs of CRISPRs differ in overall structure. For example, while the spacer of a type II gRNA is located at the 5’ end, the spacer of a type V gRNA is located towards the 3 ’ end, with the CRISPR protein (e.g. Casl2a) binding region located toward the 5’ end. Accordingly, the regions of a type V pegRNA are rearranged compared to a type II pegRNA.
  • the overall structure of the pegRNA is like that of a typical type II sgRNA with a reverse transcriptase template/primer binding site appended to the 3’ end.
  • the pegRNA comprises (5’ to 3’) a CRISPR protein-binding region, a spacer which is complementary to the target sequence of a genomic DNA, a reverse transcriptase template including editing positions, and primer binding site (PBS).
  • a first integration recognition site e.g., any of the integration recognition sites described herein
  • a second integration recognition site e.g., any of the integration recognition sites described herein
  • a non-limiting example of a cognate pair include an attB site and an attP site, whereby a serine integrase mediates recombination between the attB site and the attP site.
  • an atgRNA comprises a reverse transcriptase template that encodes, partially or in its entirety, an integration recognition site (also referred to as an integration target recognition site) or a recombinase recognition site (also referred to as a recombinase target recognition site).
  • the integration target recognition site which is to be placed at a desired location in the genome or intracellular nucleic acid, is referred to as a “beacon,” a “beacon” site or an “attachment site” or a “landing pad” or “landing site.”
  • An integration target recognition site or recombinase target recognition site incorporated into the pegRNA is referred to as an attachment site containing guide RNA (atgRNA).
  • the primer binding site allows the 3’ end of the nicked DNA strand to hybridize to the atgRNA, while the RT template serves as a template for the synthesis of edited genetic information.
  • the atgRNA is capable for instance, without limitation, of (i) identifying the target nucleotide sequence to be edited and (ii) encoding new genetic information that replaces (or in some cases adds) the targeted sequence.
  • the atgRNA is capable of (i) identifying the target nucleotide sequence to be edited and (ii) encoding an integration site that replaces (or inserts/ deletes within) the targeted sequences.
  • the co-delivery system described herein includes a polynucleotide sequence encoding an attachment site-containing guide RNA (atgRNA) packaged in an LNP.
  • the co-delivery system described herein includes a vector comprising a polynucleotide sequence encoding an atgRNA.
  • the atgRNA comprises a domain that is capable of guiding the prime editor fusion protein to a target sequence, thereby identifying the target nucleotide sequence to be edited; and a reverse transcriptase (RT) template that comprises a first integration recognition site.
  • the atgRNA comprises a domain that is capable of guiding the prime editor fusion protein (or prime editor system) to a target sequence, thereby identifying the target nucleotide sequence to be edited; and a reverse transcriptase (RT) template that comprises at least a portion first integration recognition site.
  • a domain that is capable of guiding the prime editor fusion protein (or prime editor system) to a target sequence, thereby identifying the target nucleotide sequence to be edited
  • RT reverse transcriptase
  • the co-delivery system described herein includes a polynucleotide sequence encoding a first attachment site-containing guide RNA (atgRNA) and a polynucleotide nucleotide sequence encoding a second attachment site-containing guide RNA (atgRNA) packaged into the same LNP.
  • the co-delivery system described herein includes a polynucleotide sequence encoding a first attachment site-containing guide RNA (atgRNA) packaged into a first LNP and a polynucleotide nucleotide sequence encoding a second attachment site-containing guide RNA (atgRNA) packaged into a second LNP.
  • the co-delivery system described herein includes a vector comprising a polynucleotide sequence encoding a first attachment site-containing guide RNA (atgRNA), a polynucleotide sequence encoding a second atgRNA, or both.
  • atgRNA attachment site-containing guide RNA
  • the co-delivery system described herein includes a polynucleotide sequence encoding a first attachment site-containing guide RNA (atgRNA) packaged into a first LNP and a vector comprising a polynucleotide sequence encoding a second atgRNA.
  • atgRNA attachment site-containing guide RNA
  • the co-delivery system contains a first atgRNA and a second atgRNA
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, where the at least first pair of atgRNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to a target sequence
  • the first atgRNA further includes a first RT template that comprises at least a portion of the first integration recognition site
  • the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site
  • the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the first atgRNA’ s reverse transcriptase template encodes for a first single-stranded DNA sequence (i.e., a first DNA flap) that contains a complementary region to a second single-stranded DNA sequence (i.e., a second DNA flap) encoded by a second atgRNA comprising a second reverse transcriptase template.
  • the complementary region between the first and second single-stranded DNA sequences is comprised of more than 5 consecutive bases of an integrase target recognition site.
  • the complementary region between the first and second single-stranded DNA sequences is comprised of more than 10 consecutive bases of an integrase target recognition site.
  • the complementary region between the first and second single-stranded DNA sequences is comprised of more than 20 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 30 consecutive bases of an integrase target recognition site.
  • Use of two guide RNAs that are (or encode DNA that is) partially complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site-containing guide RNAs (atgRNAs).
  • RNAs that are (or encode DNA that is) full complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment sitecontaining guide RNAs (atgRNAs).
  • the first atgRNA upon introducing the nucleic acid construct into a cell, incorporates the first integration recognition site into the cell’s genome at the target sequence.
  • Table 9 includes atgRNAs, sgRNAs and nicking guides that can be used herein. Spacers are labeled in capital font (SPACER), RT regions in bold capital (RT REGION), AttB sites in bold lower case (attB site), and PBS in capital italics (PBS). Unless otherwise denoted, the AttB is for Bxbl.
  • the co-delivery system described herein contains an integrase and/or a recombinase.
  • the co-delivery system includes an integrase and/or a recombinase packaged in a LNP.
  • the co-delivery system includes a polynucleotide encoding an integrase and/or a recombinase.
  • the co-delivery system includes an integrase or a recombinase packaged in a vector (e.g., a viral vector).
  • the co-delivery system includes at least a first integrase (e.g., a first integrase and a second integrase) and/or at least a first recombinase (e.g., a first recombinase and a second recombinase).
  • a first integrase e.g., a first integrase and a second integrase
  • a first recombinase e.g., a first recombinase and a second recombinase
  • the integration enzyme (e.g., the integrase or recombinase) is selected from the group consisting of Dre, Vika, Bxbl, ⁇ pC31, RDF, cpBTl, Rl, R2, R3, R4, R5, TP901-1, Al 18, cpFCl, cpCl, MR11, TGI, cp370.1, W , BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, Concept!, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, cpRV, retrotransposases encoded by a Tcl/mariner family member including but not limited to retrotransposases encoded by
  • LSRs serine recombinases
  • embodiments can include any serine recombinase such as BceINT, SSCINT, SACINT, and INT10 (see lonnidi et al., 2021; Drag-and-drop genome insertion without DNA cleavage with CRISPR directed integrases.
  • the integration site can be selected from an attB site, an attP site, an attL site, an attR site, a lox71 site a Vox site, or a FRT site.
  • the Cre-lox system is referred to either as a control for programmable gene insertion or as a tool for a recombinase-mediated event separate and distinct from insertion of the donor polynucleotide template (or exogenous nucleic acid) into the integrated recognition site.
  • integrases, transposases and the like can depend on nuclear localization.
  • prokaryotic enzymes are adapted to modulate nuclear localization.
  • eukaryotic or vertebrate enzymes are adapted to modulate nuclear localization.
  • the invention provides fusion or hybrid proteins. Such modulation can comprise addition or removal of one or more nuclear localization signal (NLS) and/or addition or removal of one or more nuclear export signal (NES).
  • NLS nuclear localization signal
  • NES nuclear export signal
  • nuclear export signal (NES) of transposases affects the transposition activity of mariner-like elements Ppmarl and Ppmar2 of moso bamboo. Mob DNA. 2019 Aug 19; 10:35. doi: 10.1186/sl3100-019-0179-y).
  • the methods and constructs are used to modulate nuclear localization of system components of the invention.
  • the integrase used herein is selected from below (Table 10).
  • FIGs. 14A-14E shows analysis of effect of variant AttP sites on integration efficiency.
  • the integration enzyme is selected from one of the about 27,000 Serine integrases described in International Patent Publication No. WO 2023/070031 A2, which is hereby incorporated by reference in its entirety.
  • This disclosure features integration enzymes (also referred to integrases) engineered such that upon being introduced into a cell, the integration enzyme has increased stability (e.g., half- life) compared to a control integration enzyme not engineered to have increased stability.
  • An increase in stability includes an increase in half-life and a reduction in protein degradation rates, thereby extending the capacity of the integration enzyme to mediate integration.
  • ubiquitin proteosome system proteolysis by ubiquitin proteosome system (UPS) and autophagy.
  • proteins can be ubiquitinated on lysine residues, thereby leading to degradation.
  • Proteins can also have specific domains (degrons) that can be ubiquitinated, which also leads to protein degradation.
  • Degrons also referred to as degradation signals
  • Degrons are recognized and polyubiquitinated, targeting the protein for degradation.
  • Degrons can be present at both the N-terminus and/or C-terminus of a protein.
  • Polyubiquitinated protein is recognized by receptor subunits of the 26S proteosome (RpnlO (yeast)/p54 (drosophila), Rpnl3 and Rpnl).
  • the engineered integration enzymes is selected from the engineered integration enzymes described in FIGs. 23, 25, 28, 30B, 32A, 33, 35, and 40-42. In some embodiments, the engineered integration enzymes is selected from the engineered integration enzymes described in Table 26. In some embodiments, the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence listed in Table 26.
  • the engineered integration enzyme is encoded by a nucleic acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to a nucleic acid sequence listed in Table 26.
  • This disclosure features integration enzymes engineered to include a domain (e.g., a stabilization domain) that increases stability of the integration enzyme when compared to an integration enzyme not engineered to include the domain (e.g., the stabilization domain).
  • a domain e.g., a stabilization domain
  • the integration enzyme or fragment thereof and the stabilization domain are fused, thereby by creating a fusion protein.
  • an engineered integration enzyme comprises an integration enzyme or fragment thereof; an at least first stabilization domain; and a nuclear localization signal (NLS).
  • the integration enzyme or fragment thereof, the stabilization domain, and the NLS are fused, thereby by creating a fusion protein.
  • the engineered integration enzyme comprises an integration enzyme or fragment thereof comprising at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten) amino acid modification such that the combination of the at least two modifications increase the stability of the integration enzyme as compared to the integration enzyme not comprising the at least first amino acid modification.
  • the amino acid modification can be in a degron motif in the integration enzyme.
  • the amino acid modification can be a substitution, deletion, or insertion that increases the stability of the integration enzyme as compared to an integration enzyme not comprising the at least first amino acid modification.
  • the engineered integration enzyme is an integration enzyme described in Table 10 and comprises an amino acid modification that increases the stability of the integration enzyme as compared to an integration enzyme selected from Table 10 and not engineered to include the amino acid modification.
  • the amino acid modification can be a substitution, deletion, or insertion in an integration enzyme having an amino acid sequence of SEQ ID NO: 388 that increases the stability of the integration enzyme as compared to an integration enzyme having a sequence of SEQ ID NO: 388 that does not comprise the at least first amino acid modification.
  • the first amino acid modification is an amino acid substitution of L275V in SEQ ID NO: 388.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 378 and an amino acid modification in the sequence of SEQ ID NO: 378, where the amino acid modification increases the stability of the integration enzyme as compared to an integration enzyme having an amino acid sequence selected of SEQ ID NO: 378.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 382 and an amino acid modification in the sequence of SEQ ID NO: 382, where the amino acid modification increases the stability of the integration enzyme as compared to an integration enzyme having an amino acid sequence selected of SEQ ID NO: 382.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 383 and an amino acid modification in the sequence of SEQ ID NO: 383, where the amino acid modification increases the stability of the integration enzyme as compared to an integration enzyme having an amino acid sequence selected of SEQ ID NO: 383.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 384 and an amino acid modification in the sequence of SEQ ID NO: 384, where the amino acid modification increases the stability of the integration enzyme as compared to an integration enzyme having an amino acid sequence selected of SEQ ID NO: 384.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 385 and an amino acid modification in the sequence of SEQ ID NO: 385, where the amino acid modification increases the stability of the integration enzyme as compared to an integration enzyme having an amino acid sequence selected of SEQ ID NO: 385.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 386 and an amino acid modification in the sequence of SEQ ID NO: 386, where the amino acid modification increases the stability of the integration enzyme as compared to an integration enzyme having an amino acid sequence selected of SEQ ID NO: 386.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 387 and an amino acid modification in the sequence of SEQ ID NO: 387, where the amino acid modification increases the stability of the integration enzyme as compared to an integration enzyme having an amino acid sequence selected of SEQ ID NO: 387.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 388 and an amino acid modification in the sequence of SEQ ID NO: 388, where the amino acid modification increases the stability of the integration enzyme as compared to an integration enzyme having an amino acid sequence selected of SEQ ID NO: 388.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 389 and an amino acid modification in the sequence of SEQ ID NO: 389, where the amino acid modification increases the stability of the integration enzyme as compared to an integration enzyme having an amino acid sequence selected of SEQ ID NO: 389.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 390 and an amino acid modification in the sequence of SEQ ID NO: 390, where the amino acid modification increases the stability of the integration enzyme as compared to an integration enzyme having an amino acid sequence selected of SEQ ID NO: 390.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 391 and an amino acid modification in the sequence of SEQ ID NO: 391, where the amino acid modification increases the stability of the integration enzyme as compared to an integration enzyme having an amino acid sequence selected of SEQ ID NO: 391.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 392 and an amino acid modification in the sequence of SEQ ID NO: 392, where the amino acid modification increases the stability of the integration enzyme as compared to an integration enzyme having an amino acid sequence selected of SEQ ID NO: 392.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 393 and an amino acid modification in the sequence of SEQ ID NO: 393, where the amino acid modification increases the stability of the integration enzyme as compared to an integration enzyme having an amino acid sequence selected of SEQ ID NO: 393.
  • This disclosure also features engineered integration enzymes comprising a stabilization domain and an amino acid modification that increases the engineered integration enzymes stability.
  • the engineered integration enzyme comprises an integration enzyme as described in International Patent Publication No. WO 2023/070031A2, which is hereby incorporated by reference in its entirety, where the integration enzyme includes a stabilization domain and an amino acid modification, where the stabilization domain, the amino acid modification, or both, increase the stability of the integration enzyme as compared to an integration enzyme not engineered to have the amino acid modification and/or the stabilization domain.
  • the engineered integration enzyme is an integration enzyme described in Table 10 and comprises a stabilization domain and an amino acid modification in the same integration enzyme selected from Table 10, wherein the stabilization domain, the amino acid modification, or both, increase the stability of the integration enzyme as compared to an integration enzyme not engineered to have the amino acid modification and/or stabilization domain.
  • the engineered integration enzyme is a BxB 1 integration enzyme comprising a stabilization domain and an amino acid modification in the BxB 1 integration enzyme, wherein the stabilization domain, the amino acid modification, or both, increase the stability integration enzyme as compared to an integration enzyme not engineered to have the amino acid modification and/or stabilization domain.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 378-393, a stabilization domain, and an amino acid modification in the amino acid sequence, where the stabilization domain, the amino acid modification, or both, increase the stability of the engineered integration enzyme as compared to an integration enzyme not engineered to have the at amino acid modification and/or the stabilization domain.
  • the engineered integration enzyme comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 388, a stabilization domain and an amino acid modification in SEQ ID NO: 388, wherein the stabilization domain, the amino acid modification, or both, increase the stability of integration enzyme as compared to an integration enzyme not engineered to have the amino acid modification and/or stabilization domain.
  • This disclosure features a polynucleotide comprising a nucleic acid sequence encoding any of the engineered integration enzymes described herein (see Section 4.9.1, 4.9.2, and 4.9.3) or any of the engineered integration enzyme linked to the gene editor polypeptide or fusion proteins described herein (see Section 4.9.5).
  • the nucleic acid sequence encoding the engineered integration enzyme is codon optimized. In some embodiments, the codon optimization is performed using an algorithm.
  • the nucleic acid sequence of the engineered integration enzyme is optimized based on second structure such that the structure confers increased stability of the polynucleotide.
  • the optimization based on second structure relies on an algorithm to determine the optimal mRNA sequence.
  • a non-limiting example of an algorithm capable of being used to optimize the mRNA encoding any of the any of the engineered integration enzymes described herein is as described in Zhang et al. (Algorithm for Optimized mRNA Design Improves Stability and Immunogenicity, Nature 2023, doi.org/10.1038/s41586-023-06127-z), which is hereby incorporated by reference in its entirety. Zhang et al. refer to an algorithm called LinearDesign that optimizes both structural stability (via secondary structure) and codon usage.
  • the nucleic acid sequence of the engineered integration enzyme is optimized based on the LinearDesign algorithm.
  • This disclosure also features vector comprising the nucleic acid sequence encoding any of the engineered integration enzymes described herein (see Section 4.9.1, 4.9.2, and 4.9.3) or any of the engineered integration enzyme linked to the gene editor polypeptide or fusion proteins described herein (see Section 4.9.5).
  • This disclosure also features engineered integration enzymes (e.g., any of the engineered integration enzymes described herein (see, e.g., Section 4.9.1, 4.9.2, and 4.9.3) linked to a gene editor polypeptide (e.g., any of the gene editor polypeptides described herein).
  • the linked engineered integration enzyme-gene editor polypeptide can be used to incorporate an integration recognition site into the genome of a cell and for integrating a donor polynucleotide template into the genome.
  • the linker is a peptide fused in-frame between the engineered integration enzyme and the gene editor polypeptide.
  • the gene editor polypeptide comprises a DNA binding domain and a reverse transcriptase.
  • This disclosure also features a fusion protein comprising (a) a DNA binding domain, optionally comprising a nickase activity; (b) a reverse transcriptase; and (c) any of the engineered integration enzymes described herein (see, e.g., Sections 4.9.1, 4.9.2, and 4.9.3), wherein at least any two of elements (a), (b), or (c) are linked via at least a first C-terminal linker comprising.
  • the fusion protein comprises from N-terminus to C-terminus: (a), (b), and (c); (b), (a), and (c); (c), (a), and (b); (c), (b), and (a); (b), (c), and (a); and (b), (a), and (c).
  • the engineered integration enzyme is linked to a gene editor is described in FIG. 46.
  • Non-limiting examples of metrics that can be used as indication of protein stability include the following.
  • Protein Half-Life This is the time required for half of the amount of protein in a cell to be degraded. This can be used to measure protein turnover.
  • One non-limiting example of measuring protein half-life includes using pulse-chase experiments where the protein is labeled with a radioactive or other traceable isotope, then followed over time to see how rapidly it disappears.
  • Tm Melting Temperature
  • thermodynamic Stability This is the change in Gibbs free energy between the folded and unfolded state of the protein. It measures how much energy is required to unfold the protein, with a larger value indicating a more stable protein.
  • thermodynamic stability include equilibrium denaturation experiments, where the protein is exposed to varying concentrations of a denaturant and the degree of unfolding is monitored.
  • Protein Degradation Rate This method measures how quickly a protein is degraded in a cell. It can be quantified using a variety of methods, for example, without limitation: western blot analysis (with or without the use of protease inhibitors) to assess protein levels over time.
  • This disclosure features a system for site-specifically integrating a donor polynucleotide template into a mammalian cell genome at a target DNA sequence, comprising: an attachment site containing gRNA (atgRNA) comprising at least a portion of an at least first integration recognition site; a gene editor polypeptide comprising a DNA binding nickase domain linked to a reverse transcriptase domain capable of incorporating the integration recognition site into the target DNA sequence, any of the engineered integration enzymes described herein (e.g., an engineered integration enzyme comprising an integration enzyme and a stabilization domain (see Section 4.9.1) or an engineered integration enzyme comprising an integration enzyme comprising an amino acid modification that increases stability (see Section 4.9.2)); a donor polynucleotide template linked to a sequence that is an integration cognate of the integration recognition site present in the atgRNA, whereby the gene editor polypeptide site-specifically integrates the integration recognition site into the target DNA sequence, whereby the engineered integration enzyme integrates the
  • the first atgRNA comprises: (i) a domain that is capable of guiding the gene editor polypeptide to the target DNA sequence; and (ii) a reverse transcriptase (RT) template that comprises at least a portion of an at least first integration recognition site, whereby the at least portion of the at least first integration recognition site is integrated into the genome of the cell at the target sequence.
  • RT reverse transcriptase
  • the system also includes a second atgRNA.
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, wherein the at least first pair of atgRNAs have domains that are capable of guiding the gene editor polypeptide to the target DNA sequence;
  • the first atgRNA further includes a first RT template that comprises at least a portion of an at least first integration recognition site;
  • the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site, whereby the least first integration recognition site is integrated into the genome of the cell at the target sequence.
  • the engineered integration enzyme upon introducing the system into the cell, enhances integration efficiency of the donor polynucleotide template at the site-specifically integrated integration recognition site as compared to the integration efficiency of a system using a non-engineered integration enzyme to integrate donor polynucleotide template at the site-specifically integrated integration recognition site.
  • This disclosure also features a method for site-specifically integrating a donor polynucleotide template into a mammalian cell genome at a target DNA sequence, comprising: incorporating an integration recognition site into the genome by delivering into the cell: an attachment site containing guide RNA (atgRNA) comprising at least a portion of an at least first integration recognition site; and a gene editor polypeptide or polynucleotide encoding a gene editor polypeptide, wherein the gene editor polypeptide comprises a DNA binding nickase domain linked to a reverse transcriptase domain and is capable of incorporating the integration recognition site into the target DNA sequence; and optionally, a nicking gRNA; and integrating the donor polynucleotide template into the genome by delivering into the cell: any of the engineered integration enzymes described herein (e.g., an engineered integration enzyme comprising an integration enzyme and a stabilization domain (see Section 4.9.1) or an engineered integration enzyme comprising an integration enzyme comprising an integration enzyme compris
  • the first atgRNA comprises: (i) a domain that is capable of guiding the gene editor polypeptide to the target DNAsequence; and (ii) a reverse transcriptase (RT) template that comprises at least a portion of an at least first integration recognition site, whereby the at least portion of the at least first integration recognition site is integrated into the genome of the cell at the target sequence.
  • RT reverse transcriptase
  • the method also includes a second atgRNA.
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, wherein the at least first pair of atgRNAs have domains that are capable of guiding the gene editor polypeptide to the target DNA sequence;
  • the first atgRNA further includes a first RT template that comprises at least a portion of an at least first integration recognition site;
  • the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site, whereby the least first integration recognition site is integrated into the genome of the cell at the target sequence.
  • the method enhances integration efficiency of the donor polynucleotide template at the site-specifically integrated integration recognition site at the double-stranded target DNA sequence as compared to the integration efficiency of the donor polynucleotide template at the site-specifically integrated recognition site when using a method that does not comprise the engineered integration enzyme.
  • This disclosure features methods of delivering (e.g., co-delivery or dual delivery) a system capable of site-specifically integrating a template polynucleotide into the genome of a cell, where the methods include delivering to a (i) gene editor construct and a (ii) template polynucleotide, and (iii) at least a first attachment site-containing guide (atgRNA).
  • a system capable of site-specifically integrating a template polynucleotide into the genome of a cell
  • the methods include delivering to a (i) gene editor construct and a (ii) template polynucleotide, and (iii) at least a first attachment site-containing guide (atgRNA).
  • This disclosure also features a method for delivering a system capable of site-specifically integrating a template polynucleotide into the genome of a cell, where the method includes: delivering a lipid nanoparticle (LNP) comprising a gene editor polynucleotide (e.g., a gene editor polynucleotide construct) and a vector comprising a template polynucleotide and at least a first attachment site-containing guide RNA (atgRNA).
  • the first atgRNA comprises (i) a domain that is capable of guiding the prime editor system to a target sequence; and (ii) a reverse transcriptase (RT) template that comprises at least a portion of a first integration recognition site.
  • the RT template comprises the entirety of the first integration recognition site. In some embodiments, where the vector comprises a polynucleotide encoding a first atgRNA, the vector also includes a sequence encoding a nicking guide RNA (ngRNA).
  • ngRNA nicking guide RNA
  • This disclosure also features a method for delivering a system capable of site-specifically integrating a template polynucleotide into the genome of a cell, where the method includes: delivering a lipid nanoparticle (LNP) comprising a gene editor polynucleotide (e.g., a gene editor polynucleotide construct) and a vector comprising a template polynucleotide and a first attachment site-containing guide RNA (atgRNA) and a second attachment site-containing guide RNA (atgRNA).
  • LNP lipid nanoparticle
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, wherein the at least first pair of atgRNAs have domains that are capable of guiding the prime editor system to a target sequence, the first atgRNA further includes a first RT template that comprises at least a portion of the at least first integration recognition site; and the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the first atgRNA and second atgRNA include at least a 6bp overlap (e.g., 6bp of complementarity).
  • This disclosure also features a method for delivering a system capable of site-specifically integrating a template polynucleotide into the genome of a cell, where the method includes: delivering into a cell a lipid nanoparticle (LNP) comprising: (i) a gene editor polynucleotide (e.g., a gene editor polynucleotide construct), and (ii) a first attachment site-containing guide RNA (atgRNA); and a vector comprising: (i) a template polynucleotide, and (ii) a second atgRNA.
  • LNP lipid nanoparticle
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, wherein the at least first pair of atgRNAs have domains that are capable of guiding the prime editor system to a target sequence, the first atgRNA further includes a first RT template that comprises at least a portion of the a first integration recognition site; and the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the first atgRNA and second atgRNA include at least a 6bp overlap (e.g., 6bp of complementarity).
  • This disclosure also features a method for delivering a system capable of site-specifically integrating a template polynucleotide into the genome of a cell, where the method includes delivering: a lipid nanoparticle (LNP) comprising: (i) a gene editor polynucleotide (e.g., a gene editor polynucleotide construct), (ii) a first attachment site-containing guide RNA (atgRNA), and (iii) a second atgRNA; and a vector comprising (i) a template polynucleotide.
  • LNP lipid nanoparticle
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, wherein the at least first pair of atgRNAs have domains that are capable of guiding the prime editor system to a target sequence, the first atgRNA further includes a first RT template that comprises at least a portion of the at least first integration recognition site; and the second atgRNA further includes a second RT template that comprises at least a portion of the at least first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the first atgRNA and second atgRNA include at least a 6bp overlap (e.g., 6bp of complementarity).
  • This disclosure also features a method for delivering a system capable of site-specifically integrating a template polynucleotide into the genome of a cell, where the method includes delivering: a lipid nanoparticle (LNP) comprising: (i) a gene editor polynucleotide (e.g., a gene editor polynucleotide construct) and (ii) a first attachment site-containing guide RNA (atgRNA); and a vector comprising: (i) a template polynucleotide, and (ii) a nicking atgRNA.
  • LNP lipid nanoparticle
  • a gene editor polynucleotide e.g., a gene editor polynucleotide construct
  • atgRNA first attachment site-containing guide RNA
  • a vector comprising: (i) a template polynucleotide, and (ii) a nicking atgRNA.
  • the first atgRNA comprises (i) a domain that is capable of guiding the prime editor system to a target sequence; and (ii) a reverse transcriptase (RT) template that comprises at least a portion of a first integration recognition site.
  • the RT template comprises the entirety of the first integration recognition site.
  • the LNP and the first vector are delivered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks apart.
  • the LNP and the second vector are delivered a different times on the same day, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or 8 weeks apart.
  • the LNP and the first vector are delivered about 6 weeks apart.
  • This disclosure also features a method for delivering a system capable of site-specifically integrating a template polynucleotide into the genome of a cell, where the method includes delivering the system in vivo.
  • the system is delivered to a fetus or a neonate to site-specifically integrate in vivo a template polynucleotide into the genome of a cell.
  • Delivering the system to a fetus or a neonate provides advantages over delivering the system later in life (e.g., after the neonate phase ends), including: (i) fewer number of cells that need to be treated (e.g., in the adult, there are trillions of cells, but in a fetus, there are significantly fewer cells); (ii) developmental benefits: the early stage of development of a fetus or a neonate means that if a genetic disease is treated successfully, the individual could potentially develop normally, with significant reduction or even complete removal of any of the disease manifestations; (iii) preventing disease progression: in certain genetic conditions the physiological damage is irreversible damage and in some instances is exacerbated as the disease progresses, therefore, intervening at the fetal (or neonate) stage, it is possible to prevent or reduce the progression of the disease and potentially prevent any irreversible damage from occurring; (iv) higher cell turnover and cell division rate: in a developing fetus, cells are dividing rapidly as the
  • the method includes delivering an LNP and a first vector, the LNP and the first vector are delivered to a cell in vivo.
  • the in vivo cells are present in a fetus or a neonate.
  • the LNP is delivered between age 0 (day of birth) and age 7 days and the vector is delivered between age 5 weeks and age 7 weeks.
  • the LNP is delivered at about at 2 days and the vector is delivered at about age 6 weeks.
  • neonus refers to an unborn offspring.
  • nonate refers to a newborn infant, which includes the first 90 days of life. When referring to mice, neonate can refer to animals up to 10 days of age.
  • This disclosure also features a system capable of site-specifically integrating a template polynucleotide into the genome of a cell, the system comprising: a lipid nanoparticle (LNP) comprising a gene editor polynucleotide (e.g., a gene editor polynucleotide construct) and a vector comprising a template polynucleotide and at least a first attachment site-containing guide RNA (atgRNA).
  • the first atgRNA comprises (i) a domain that is capable of guiding the prime editor system to a target sequence; and (ii) a reverse transcriptase (RT) template that comprises at least a portion of a first integration recognition site.
  • the RT template comprises the entirety of the first integration recognition site. In some embodiments, where the vector comprises a polynucleotide encoding a first atgRNA, the vector also includes a sequence encoding a nicking guide RNA (ngRNA).
  • ngRNA nicking guide RNA
  • This disclosure also features a system capable of site-specifically integrating a template polynucleotide into the genome of a cell, the system comprising: a lipid nanoparticle (LNP) comprising a gene editor polynucleotide (e.g., a gene editor polynucleotide construct) and a vector comprising a template polynucleotide and a first attachment site-containing guide RNA (atgRNA) and a second attachment site-containing guide RNA (atgRNA).
  • LNP lipid nanoparticle
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, wherein the at least first pair of atgRNAs have domains that are capable of guiding the prime editor system to a target sequence, the first atgRNA further includes a first RT template that comprises at least a portion of the a first integration recognition site; and the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the first atgRNA and second atgRNA include at least a 6bp overlap.
  • This disclosure also features a system capable of site-specifically integrating a template polynucleotide into the genome of a cell, the system comprising: a lipid nanoparticle (LNP) comprising: (i) a gene editor polynucleotide (e.g., a gene editor polynucleotide construct), and (ii) a first attachment site-containing guide RNA (atgRNA); and a vector comprising: (i) a template polynucleotide, and (ii) a second atgRNA.
  • LNP lipid nanoparticle
  • a gene editor polynucleotide e.g., a gene editor polynucleotide construct
  • atgRNA first attachment site-containing guide RNA
  • a vector comprising: (i) a template polynucleotide, and (ii) a second atgRNA.
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, wherein the at least first pair of atgRNAs have domains that are capable of guiding the prime editor system to a target sequence, the first atgRNA further includes a first RT template that comprises at least a portion of the a first integration recognition site; and the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the first atgRNA and second atgRNA include at least a 6bp overlap.
  • This disclosure also features a system capable of site-specifically integrating a template polynucleotide into the genome of a cell, the system comprising: co-delivering: a lipid nanoparticle (LNP) comprising: (i) a gene editor polynucleotide (e.g., a gene editor polynucleotide construct), (ii) a first attachment site-containing guide RNA (atgRNA), and (iii) a second atgRNA; and a vector comprising (i) a template polynucleotide.
  • LNP lipid nanoparticle
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, wherein the at least first pair of atgRNAs have domains that are capable of guiding the prime editor system to a target sequence, the first atgRNA further includes a first RT template that comprises at least a portion of the a first integration recognition site; and the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the first atgRNA and second atgRNA include at least a 6bp overlap.
  • This disclosure also features a system capable of site-specifically integrating a template polynucleotide into the genome of a cell, the system comprising: a lipid nanoparticle (LNP) comprising: (i) a gene editor polynucleotide (e.g., a gene editor polynucleotide construct) and (ii) a first attachment site-containing guide RNA (atgRNA); and a vector comprising: (i) a template polynucleotide, and (ii) a nicking atgRNA.
  • LNP lipid nanoparticle
  • a gene editor polynucleotide e.g., a gene editor polynucleotide construct
  • atgRNA first attachment site-containing guide RNA
  • a vector comprising: (i) a template polynucleotide, and (ii) a nicking atgRNA.
  • the first atgRNA comprises (i) a domain that is capable of guiding the prime editor system to a target sequence; and (ii) a reverse transcriptase (RT) template that comprises at least a portion of a first integration recognition site.
  • the RT template comprises the entirety of the first integration recognition site.
  • the LNP comprising a gene editor polynucleotide construct is capable delivering to a cell cytoplasm the gene editor polynucleotide construct. In some embodiments, the LNP comprising a gene editor polynucleotide construct is capable delivering to a cell nucleus the gene editor polynucleotide construct. In some embodiments, the LNP comprises a gene editor protein and associated guide nucleic acids. In some embodiments, the LNP comprises a gene editor protein and associated guide nucleic acids that are capable of localizing to cell nucleus.
  • a gene editor polynucleotide construct is delivered to a cell by a fusosome. In some embodiments, a gene editor polynucleotide construct is delivered to a cell cytoplasm by a fusosome. In some embodiments, the fusosome comprises a gene editor protein and associated guide nucleic acids.
  • a gene editor polynucleotide construct is delivered to a cell by an exosome.
  • a gene editor polynucleotide construct is delivered to a cell cytoplasm by an exosome.
  • the exosome comprises a gene editor protein and associated guide nucleic acids.
  • the prime editor or Gene Writer protein fusion is incorporated (i.e., packaged) into LNP as protein.
  • associated atgRNA and optional ngRNAs may be co-packaged with gene editor proteins in LNP.
  • the gene editor polynucleotide construct comprises (a) a polynucleotide sequence encoding a prime editor fusion protein or a Gene WriterTM protein, (b) a polynucleotide sequence encoding an attachment site-containing guide RNA (atgRNA), (c) optionally, a polynucleotide sequence encoding a nickase guide RNA (ngRNA), (d) a polynucleotide sequence encoding an integrase, (e) and optionally, a polynucleotide sequence encoding a recombinase.
  • atgRNA attachment site-containing guide RNA
  • ngRNA nickase guide RNA
  • a polynucleotide sequence encoding an integrase e
  • a polynucleotide sequence encoding a recombinase optionally, a polynucleotide sequence encoding a recombina
  • the prime editor or Gene Writer protein fusion is expressed as a split construct.
  • the split construct in reconstituted in a cell.
  • the split construct can be fused or ligated via intein protein splicing.
  • the split construct can be reconstituted via protein-protein inter-molecular bonding and/or interactions.
  • the split construct can be reconstituted via chemical, biological, or environmental induced oligomerization.
  • the split construct can be adapted into one or more nucleic acid constructs described herein.
  • the systems described include a gene editor polynucleotide that is delivered to a cell using the methods described herein.
  • the gene editor polynucleotide is delivered as a polynucleotide (e.g., an mRNA).
  • the gene editor polynucleotide is delivered as a protein.
  • the gene editor polynucleotide or protein is packaged, and thereby vectorized, within a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the gene editor polynucleotide or protein is packaged in a LNP and is codelivered with a template polynucleotide (i.e., nucleic acid “cargo” or nucleic acid “payload”) packaged into a separate vector (e.g., a viral vector (e.g., an AAV or adenovirus)) or a second lipid nanoparticle (LNP).
  • a template polynucleotide i.e., nucleic acid “cargo” or nucleic acid “payload” packaged into a separate vector (e.g., a viral vector (e.g., an AAV or adenovirus)) or a second lipid nanoparticle (LNP).
  • a separate vector e.g., a viral vector (e.g., an AAV or adenovirus)
  • LNP second lipid nanoparticle
  • the gene editor polynucleotide is delivered to the cells as a polynucleotide.
  • the gene editor polynucleotide is delivered to the cells as an mRNA encoding the gene editor polynucleotide (e.g., the gene editor protein or the prime editor system).
  • the mRNA comprises one or more modified uridines.
  • the mRNA comprises a sequence where each of the uridines is a modified uridine.
  • the mRNA is uridine depleted.
  • the mRNA encoding the nickase comprises one or more modified uridines.
  • the mRNA encoding the reverse transcriptase comprises one or more modified uridines. In some embodiments, the mRNA encoding the nickase comprises one or more modified uridines, and the mRNA encoding the reverse transcriptase comprises one or more modified uridines. In some embodiments, where the integrase is encoded in an mRNA, the mRNA comprises modified uridines. In some embodiments, a modified uridine is a Nl-Methylpseudouridine-5’ -Triphosphate. In some embodiments, a modified uridine is a pseudouridine. In some embodiments, the mRNA comprises a 5’ cap. In some embodiments, the 5’ cap comprises a molecular formula of C32H43N15O24P4 (free acid).
  • the gene editor polynucleotide (e.g., a gene editor polynucleotide construct) comprises a polynucleotide sequence encoding a primer editor system (e.g., any of the prime editor systems described herein).
  • the prime editor system comprises a nucleotide sequence encoding a nickase (e.g., any of the Cas proteins or variants thereof (e.g., nickases) and nickases described herein, see Tables 4-8) and a nucleotide sequence encoding a reverse transcriptase (e.g., any of the reverse transcriptases described herein).
  • the nucleotide sequence encoding the nickase and the nucleotide sequence encoding the reverse transcriptase are positioned in the construct such that when expressed the nickase is linked to the reverse transcriptase.
  • the nickase is linked to the reverse transcriptase by in-frame fusion.
  • the nickase is linked to the reverse transcriptase by a linker.
  • the linker is a peptide fused in-frame between the nickase and reverse transcriptase.
  • the gene editor polynucleotide (e.g., a gene editor polynucleotide construct) further comprises a polynucleotide sequence encoding at least a first integrase (e.g., any of the integrases described herein, e.g., as described in Table 10 and also in Yamall et al., Nat. BiotechnoL, 2022, doi.org/10.1038/s41587-022-01527-4 and Durrant et al., Nat. BiotechnoL, 2022, doi.org/10.1038/s41587-022-01494-w, each of which are herein incorporated by reference in their entireties).
  • the linked nickase-reverse transcriptase are further linked to the first integrase.
  • the gene editor polynucleotide construct further comprises a polynucleotide sequence encoding at least a first recombinase (e.g., any of the recombinases described herein).
  • nucleotide sequence encoding the nickase and the nucleotide sequence encoding the reverse transcriptase are positioned in a construct such that when expressed the nickase is not linked to the reverse transcriptase.
  • a sequence encoding a self-cleaving peptide e.g., a P2A
  • a sequence encoding a self-cleaving peptide is positioned between the nucleotide sequence encoding the nickase and the nucleotide sequence encoding the reverse transcriptase such that when expressed the nickase is not linked to the reverse transcriptase (see, e.g., FIG. 34).
  • nucleotide sequence encoding the nickase and the nucleotide sequence encoding the reverse transcriptase are on separate polynucleotides.
  • the nickase and/or the reverse transcriptase can be engineered such that when expressed they are linked to a binding domain, where the binding domain is part of a binding pair that enables recruitment of the reverse transcriptase to the genomic location of the nickase (e.g., nCas9).
  • a binding pair can be any pair of molecules wherein upon forming the binding pair the binding domains enable recruitment of the reverse transcriptase to the genomic location of the nickase. Once in sufficient proximity with each other the binding domains can dimerize. This dimerization (or other type of interaction) brings the nickase and RT into proximity with each other, thereby enhancing the reverse transcription of the RT template in the atgRNA.
  • Non-limiting examples of binding pairs that can aid recruitment of the RT to the genomic location of the nickase include without limitation: dimerizing leucine zippers, an antigen and a corresponding antigen binding domains (e.g., antibody), RNA aptamers and corresponding RNA aptamer binding proteins, affinity tags and corresponding affinity tag binding proteins.
  • dimerizing leucine zippers that can serve as a binding pair includes two binding domains: a Leucine Zip LZ1 domain having a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 568 and a Leucine Zip LZ2 domain having a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 569.
  • the Leucine Zip LZ1 domain is positioned either N- terminally or C-terminally to the nickase and the Leucine Zip LZ2 is positioned either N-terminally or C-terminally to the RT.
  • the Leucine Zip LZ1 domain is positioned either N-terminally or C-terminally to the RT and the Leucine Zip LZ2 domain is positioned either N- terminally or C-terminally to the Cas9.
  • the system and methods described herein include a construct that encodes a polypeptide having a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 576 (a Leucine Zip LZ1 domain positioned C-terminally to the nickase), and a construct that encodes a polypeptide having a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 577 (a Leucine Zip LZ2 domain positioned N-terminally to the RT).
  • the system and methods described herein include a construct that encodes a polypeptide having a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 578 (a Leucine Zip LZ1 domain positioned N-terminally to the nickase), and a construct that encodes a polypeptide having a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 577 (a Leucine Zip LZ2 domain positioned N-terminally to the RT).
  • dimerizing leucine zippers that can serve as a binding pair includes two binding domains: a EE1234L peptide having a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 570 and a RR1234L peptide having a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 571.
  • the EE1234L peptide is positioned either N-terminally or C-terminally to the nickase and the RR1234L peptide is positioned either N-terminally or C-terminally to the RT.
  • the EE1234L peptide is positioned either N-terminally or C-terminally to the RT and the RR1234L peptide is positioned either N-terminally or C-terminally to the Cas9.
  • the system and methods described herein include a construct that encodes a polypeptide having a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 579 (a EE1234L peptide positioned N-terminally to the nickase), and a construct that encodes a polypeptide having a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 580 (a RR1234L peptide positioned N-terminally to the RT).
  • an affinity tag is a SpyTag and the corresponding affinity tag binding protein is SpyCatcher, where upon binding of the SpyTag with the SpyCatcher the fusion protein is a covalently stabilized multi-protein complex.
  • the affinity tag e.g., SpyTag
  • the affinity tag binding protein e.g.,. SpyCatcher
  • the affinity tag binding protein e.g., SpyCatcher
  • the affinity tag binding protein is positioned either N-terminally or C-terminally to the nickase.
  • an antigen and corresponding antigen binding domain includes an antigen such as a peptide present within a tag or a tag itself and an antigen binding domain that binds specifically to the antigen.
  • an antigen is a GCN4 peptide within a SunTag and the antigen binding domain is an anti-GCN4 scFv.
  • the SunTag (GCN4 peptide) is positioned either N-terminally or C-terminally to the nickase and the anti-GCN4 antigen binding domain is positioned either N-terminally or C-terminally to the RT.
  • the SunTag (GCN4 peptide) is positioned either N-terminally or C-terminally to the RT and the anti-GCN4 antigen binding domain is positioned either N-terminally or C-terminally to the nickase.
  • the system and methods described herein include a construct that encodes a polypeptide having a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 574 (SunTag (GCN4 peptide) positioned C-terminally to the nickase), and a construct that encodes a polypeptide having a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 575 (anti-GCN4 antigen binding domain positioned N-terminally to the RT).
  • a construct includes a nucleotide sequence encoding a self-cleaving peptide (e.g., a P2A) positioned between the nucleotide sequence encoding the nickase and the nucleotide sequence encoding the RT.
  • a nucleotide sequence encoding a self-cleaving peptide e.g., a P2A
  • the nucleotide sequence encoding the RT is such that when expressed the nickase is not linked to the reverse transcriptase.
  • the construct includes the nucleotide sequences positioned 5 ’-3’: nucleotide sequence encoding the nickase, nucleotide sequence encoding the self-cleaving peptide (e.g., a P2A), and nucleotide sequence encoding the RT. In one embodiment, the construct includes the nucleotide sequences positioned 5 ’-3’: nucleotide sequence encoding the RT, nucleotide sequence encoding the selfcleaving peptide (e.g., a P2A), and nucleotide sequence encoding the nickase.
  • the construct that includes a nucleotide sequence encoding a selfcleaving peptide (e.g., a P2A) positioned between the nucleotide sequence encoding the nickase and the nucleotide sequence encoding the RT has an amino acid sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 581 or 582 (see FIG. 34).
  • This disclosure also features systems and methods that include supplementing with reverse transcriptase.
  • the system and methods include supplementation with a polynucleotide encoding a supplemental reverse transcriptase (i.e., a separate polynucleotide from the gene editor polynucleotide, which includes a polynucleotide encoding a reverse transcriptase).
  • Systems and methods that include supplementation with reverse transcriptase increase integration efficiency of the at least first integration recognition site into the genome of the cell when compared to systems and methods that do not include supplementation with reverse transcriptase.
  • These systems and methods refer to the supplemental or supplemental reverse transcriptase molecule as “supplementing” or “supplementation” with reverse transcriptase.
  • the supplemental reverse transcriptase is the same as the reverse transcriptase that is included in the gene editor polynucleotide. In some embodiments, the supplemental reverse transcriptase is different from the reverse transcriptase that is included in the gene editor polynucleotide.
  • the polynucleotide encoding the supplemental reverse transcriptase is an mRNA.
  • the supplemental reverse transcriptase is delivered concurrently with the one or more LNP, one or more atgRNA, and the template polynucleotide. In some embodiments, the supplemental reverse transcriptase is delivered to the cell prior to delivering the one or more LNP, one or more atgRNA, and the template polynucleotide to the cell. In some embodiments, the supplemental reverse transcriptase is delivered to the cell after delivering the one or more LNP, one or more atgRNA, and the template polynucleotide to the cell.
  • the method includes a ratio of 1 : 1, 1:2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10 of gene editor polynucleotide to polynucleotide encoding the supplemental reverse transcriptase.
  • Also provided herein are methods of increasing integration efficiency of the at least first integration recognition site into the genome of the cell where the method comprises delivering to the cell one or more LNPs, one or more vectors, one or more template polynucleotides, where the gene editor polynucleotide comprises a ratio of a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 of the nucleotide sequence encoding the nickase to the nucleotide sequence encoding the reverse transcriptase, and where the increase in integration efficiency is in comparison to methods that do not include supplementation with a polynucleotide encoding RT.
  • the system and methods described herein include supplementation with a polynucleotide encoding a reverse transcriptase, where the polynucleotide is in addition to the gene editor polynucleotide that also includes a polynucleotide encoding a reverse transcriptase.
  • a method for delivering a system capable of site-specifically integrating a template polynucleotide into the genome of a cell includes delivering one or more LNPs and one or more vectors, and also includes delivering a separate polynucleotide encoding a supplemental reverse transcriptase (RT).
  • RT supplemental reverse transcriptase
  • a method for delivering a system capable of site-specifically integrating a template polynucleotide into the genome of a cell comprises delivering the gene editor polynucleotide (e.g., a polynucleotide encoding a nickase and a reverse transcriptase either linked or not linked), one or more atgRNA, a template polynucleotide, and a polynucleotide encoding a supplemental reverse transcriptase.
  • the gene editor polynucleotide e.g., a polynucleotide encoding a nickase and a reverse transcriptase either linked or not linked
  • the method includes delivering into the cell a ratio of gene editor polynucleotide to polynucleotide encoding the supplemental RT of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:32, 1:34, 1:36, 1:38, or 1:40 or more.
  • the method includes delivering into the cell a gene editor polynucleotide and a polynucleotide encoding the supplemental RT, where the polynucleotide encoding the supplemental RT is delivered at 1.5 times (1.5X), 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 20X, 30X, 40X, 50X, 60X, 70X, 80X, 90X, or 100X or more the amount of the gene editor polynucleotide.
  • the method includes delivering into the cell a ratio of polynucleotide encoding the nickase to polynucleotide encoding the RT of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:32, 1:34, 1:36, 1:38, or 1:40 or more.
  • the method includes delivering into the cell a polynucleotide encoding the nickase to polynucleotide encoding the RT, where the polynucleotide encoding the RT is delivered at 1.5 times (1.5X), 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 20X, 30X, 40X, 50X, 60X, 70X, 80X, 90X, or 100X or more the amount of the nickase polynucleotide.
  • the systems and methods that include delivering of the gene editor polynucleotide supplemented with a polynucleotide encoding a supplemental RT increases integration efficiency of the at least first integration recognition site into the genome of the cell when compared to systems and methods that include delivering to the cell a gene editor polynucleotide not supplemented with a polynucleotide encoding supplemental reverse transcriptase.
  • the system and methods described herein include supplementation with a polynucleotide encoding a reverse transcriptase, where the system includes a gene editor polynucleotide split over two polynucleotides: a first polynucleotide encoding the nickase and a second polynucleotide encoding the reverse transcriptase.
  • the first and second polynucleotides can be delivered to the cell at a ratio of 1 : 1 and supplementation is achieved by adding a polynucleotide encoding a supplemental reverse transcriptase.
  • the supplementation comprises adding the polynucleotide encoding the supplemental reverse transcriptase at a ratio of 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 : 11, 1 : 12, 1 : 13, 1 : 14, 1 : 15, 1 : 16, 1 : 17, 1 : 18, 1 : 19, 1 :20, 1 :21, 1 :22, 1 :23, 1 :24, 1 :25, 1 :26, 1 :27, 1 :28, 1 :29, 1 :30, 1 :32, 1 :34, 1 :36, 1 :38, or 1 :40 (gene editor polynucleotide (e.g., the first and second polynucleotides delivered to the cell at a ratio of 1 : 1) to the polynucleotide encoding the supplemental reverse transcriptase).
  • a method of delivering to the cell the one or more atgRNA and the gene editor polynucleotide (where the nickase and the RT are encoded on different polynucleotides and delivered at a ratio 1 : 1 (nickase to RT)) and supplementing with a polynucleotide encoding a supplemental RT increases integration efficiency of the at least first integration recognition site into the genome of the cell when compared to methods that do not include supplementing with a polynucleotide encoding a supplemental RT.
  • the system and methods described herein include supplementation with a polynucleotide encoding a reverse transcriptase, where the system includes a gene editor polynucleotide split over two polynucleotides: a first polynucleotide encoding the nickase and a second polynucleotide encoding the reverse transcriptase, and supplementation is achieved by increasing the amount of the second polynucleotide encoding the reverse transcriptase.
  • supplementation of a supplemental reverse transcriptase refers to delivering a greater than 1 : 1 ratio of nickase to reverse transcriptase (e.g., a 1 :2 or greater ratio).
  • the gene editor polynucleotide delivered to the cell in a ratio 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 : 11, 1 : 12, 1 : 13, 1 : 14, 1 : 15, 1 : 16, 1 : 17, 1 : 18, 1 : 19, 1 :20, 1 :21, 1 :22, 1 :23, 1 :24, 1 :25, 1 :26, 1 :27, 1 :28, 1 :29, 1 :30, 1 :32, 1 :34, 1 :36, 1 :38, or 1 :40 of polynucleotide sequence encoding the nickase to the polynucleotide sequence encoding the reverse transcriptase.
  • a method of delivering to the cell the one or more atgRNA and the gene editor polynucleotide where the nickase and the RT are encoded on different polynucleotides and delivered at a ratio greater than 1 : 1 (nickase to RT) increases integration efficiency of the at least first integration recognition site into the genome of the cell when compared to methods comprising delivering to the cell a gene editor polynucleotide at a ratio of 1 : 1 (nickase to RT).
  • the system and methods described herein include supplementing with reverse transcriptase, where in supplementing comprises using controllable expression (e.g., inducible promoters, strong promoters) of a polynucleotide encoding a reverse transcriptase.
  • controllable expression e.g., inducible promoters, strong promoters
  • the polynucleotide encoding the nickase and the polynucleotide encoding the RT are on a dual promoter vector, where each polynucleotide is controlled by a different promoter.
  • the polynucleotide encoding the RT is operably linked to a stronger promoter than the polynucleotide encoding the nickase, whereby the RT is expressed at higher levels than the nickase.
  • the polynucleotide encoding the RT is operably linked to an inducible promoter, whereby inducing expression of the RT results in higher levels of the RT compared to the nickase.
  • the polynucleotide encoding the nCas9 is operably linked to an inducible promoter, whereby inducing expressing of the nCas9 results in lower levels of the nCas9 than the RT.
  • the systems and methods described herein include a vector that is capable of co-delivering a template polynucleotide, one or more attachment site-containing gRNA, one or more integrases, one or more recombinases, a gene editor polynucleotide, one or more integration recognition sites, one or more recombinase recognition sites, or a combination thereof.
  • Non-limiting examples of vectors that can be used in the methods or systems described herein include the vectors described in FIGs. 3-6.
  • the vector includes a polynucleotide sequence encoding an attachment site-containing guide RNA (atgRNA).
  • the polynucleotide sequence encoding the attachment site-containing guide RNA (atgRNA) is operably linked to a regulatory element (e.g., a U6 promoter) that is capable of driving expression of the atgRNA.
  • the atgRNA comprises (i) a domain that is capable of guiding the prime editor system to a target sequence; and (ii) a reverse transcriptase (RT) template that comprises at least a portion of a first integration recognition site.
  • the RT template comprises the entirety of the first integration recognition site.
  • the vector or the LNP includes a polynucleotide sequence encoding a nicking gRNA.
  • the vector includes a polynucleotide sequence encoding a first attachment site-containing guide RNA (atgRNA) and a polynucleotide sequence encoding a second attachment site-containing guide RNA (atgRNA).
  • atgRNA first attachment site-containing guide RNA
  • atgRNA second attachment site-containing guide RNA
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, wherein the at least first pair of atgRNAs have domains that are capable of guiding the prime editor system to a target sequence, the first atgRNA further includes a first RT template that comprises at least a portion of the a first integration recognition site; the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the first atgRNA and second atgRNA include at least a 6bp overlap.
  • the vector includes a template polynucleotide and a sequence that is an integration cognate of an integration recognition site site-specifically incorporated into the genome of a cell.
  • the vector includes a template polynucleotide and a second integration recognition site that is a cognate pair with the first integration recognition site site- specifically incorporated into the genome of the cell.
  • the sequence that is an integration cognate e.g., a second integration recognition site
  • the vector comprising a template polynucleotide is a recombinant adenovirus, a helper dependent adenovirus, an AAV, a lentivirus, an HSV, an annelovirus, a retrovirus, a DoggyboneTM DNA (dbDNA), a minicircle, a plasmid, a miniDNA, an exosome, a fusosome, or an nanoplasmid.
  • the vector is capable of localizing to the nucleus.
  • the template polynucleotide is delivered to the cytoplasm and localizes to the nucleus. In certain embodiments, the template polynucleotide is delivered to the cytoplasm by LNP. In certain embodiments, the donor template polynucleotide construct comprises a recognition sequence that is recognized by a DNA binding protein (DNA binding domain) or a transcription factor binding domain. In certain embodiments, the donor template polynucleotide construct is delivered to the nucleus by an integrase or recombinase.
  • the template polynucleotide is delivered to the mitochondria.
  • the donor template polynucleotide construct comprises a mitochondria targeting sequence.
  • the vector comprising a template polynucleotide is AAV.
  • the AAV contains a 5’ inverted terminal repeat (ITR).
  • the AAV contains a 3’ inverted terminal repeat (ITR).
  • the AAV contains a 5’ and a 3’ ITR.
  • the 5’ and 3’ ITR are not derived from the same serotype of virus.
  • the ITRs are derived from adenovirus, AAV2, and/or AAV5.
  • the vector comprising a template polynucleotide is single stranded AAV (ssAAV).
  • the vector comprising a donor template polynucleotide construct is self-complementary AAV (scAAV).
  • the template polynucleotide is capable of being integrated into a genomic locus that contains an integrase target recognition site or a recombinase target recognition site.
  • the template polynucleotide comprises at least one of the following: a gene, a gene fragment, an expression cassette, a logic gate system, or any combination thereof. In some embodiments, the template polynucleotide comprises at least one intron or exon.
  • the template polynucleotide further comprises at least one integrase target recognition site or a recombinase target integrase site.
  • at least one integrase target recognition site or a recombinase target integrase site is placed within the donor template vector inverted terminal repeat.
  • the delivery system (e.g., co-delivery system) includes a vector having a sub-sequence that is capable of self-circularizing to form a self-circular nucleic acid.
  • the vector comprises a physical portion or region of the vector that is capable of self-circularizing to form a circular construct.
  • sub -sequence refers to a portion of the vector that is capable of self-circularizing, where the sub-sequence is flanked by integration recognition sites or recombinase recognition sites positioned to enable selfcircularization.
  • self-circular nucleic acid refers to a double-stranded, circular nucleic acid construct produced as a result of recombination of a cognate pair of integrase or recombinase recognition sites present on the vector. Recombination occurs when the vector is contacted with an integrase or a recombinase under conditions that allow for recombination of the cognate pair of integrase or recombinase recognition sites.
  • the sub-sequence of the vector includes a first recombinase recognition site and a second recombinase recognition site, wherein the first and second recombinase recognition sites are capable of being recombined by a recombinase.
  • the sub-sequence of the vector includes a first recombinase recognition site, a second recombinase recognition site, and a second integration recognition site (e.g., the second integration recognition site is a cognate pair of the first integration recognition site), where the first and second recombinase recognition sites flank the integration recognition site.
  • the first recombinase recognition site, the second recombinase recognition, and a recombinase enable the self-circularizing and formation of the circular construct.
  • the sub-sequence of the vector includes a third integration recognition site and a fourth integration recognition site, wherein the third and fourth integration recognition sites are a cognate pair.
  • the subsequence of the vector includes the second integration recognition site, the third integration recognition site, the fourth integration recognition site, where the third and fourth integration recognition sites flank the second integration recognition site (where the second integration recognition site is a cognate pair of the first integration recognition site).
  • the third integration recognition site, the fourth integration recognition site, and an integrase enable self -circularization and formation of the circular construct.
  • the third integration recognition site and/or the fourth integration recognition sites cannot recombine with the first integration recognition site and/or the second integration recognition site due, in part, to having different central dinucleotides than the first and second integration recognition sites.
  • each integration recognition site or each pair of integration recognition is capable of being recognized by a different integrase. In some embodiments where the subsequence includes three or more integration recognition sites, each integration recognition site or each pair of integration recognition comprises a different central dinucleotide.
  • self-circularizing is mediated at the integration recognition sites or recombinase recognition sites. In some embodiments, the self-circularizing is mediated by an integrase or a recombinase.
  • the self-circular nucleic acid comprising the second integration recognition site upon introducing the vector into a cell and after selfcircularizing to form the self-circular nucleic acid, the self-circular nucleic acid comprising the second integration recognition site is capable of being integrated into the cell’s genome at the target sequence that contains the first integration recognition site.
  • the self-circular nucleic acid comprises one or more additional integration recognition sites that enable integration of an additional nucleic acid cargo.
  • the additional nucleic acid cargo includes a sequence that is a cognate pair with one or more of the additional integration recognition sites in the selfcircular nucleic acid.
  • integration of the self-circular nucleic acid into the genome of a cell results in integration of the one or more additional integration recognition sites into the genome along with the nucleic acid cargo.
  • the integrated one or more additional integration recognition sites serve as an integration recognition site (beacon) for placing the additional nucleic acid cargo.
  • the additional nucleic acid cargo is integrated into the cell’s genome.
  • the self-circularized nucleic acid comprises a DNA cargo
  • the DNA cargo is a gene or gene fragment.
  • the DNA cargo is an expression cassette.
  • the DNA cargo is a logic gate or logic gate system.
  • the logic gate or logic gate system may be DNA based, RNA based, protein based, or a mix of DNA, RNA, and protein.
  • the nucleic acid cargo is a genetic, protein, or peptide tag and/or barcode.
  • the system or methods described herein include a second vector.
  • the gene editor polynucleotide encodes a prime editor system comprising a nickase (e.g., any of the Cas proteins or variants thereof (e.g., nickases) and nickases described herein, see Tables 4-8) and a reverse transcriptase (e.g., any of the reverse transcriptase described herein)
  • the second vector comprises a polynucleotide sequence encoding an integrase (e.g., any of the integrases described herein, e.g., as described in Table 10 and also in Yamall et al., Nat.
  • the second vector comprises a polynucleotide sequence encoding at least a first recombinase.
  • the second vector comprises a polynucleotide sequence encoding at least a first recombinase. In some embodiments, where the gene editor polynucleotide encodes a prime editor system comprising a nickase, a reverse transcriptase, and an integrase, the second vector comprises a polynucleotide sequence encoding at least a second integrase.
  • the second vector includes a template polynucleotide and a sequence that is an integration cognate of an integration recognition site site-specifically incorporated into the genome of a cell.
  • the second vector includes a template polynucleotide and a second integration recognition site that is a cognate pair with the first integration recognition site site-specifically incorporated into the genome of the cell.
  • the sequence that is an integration cognate e.g., a second integration recognition site
  • the second vector is a vector selected from: adenovirus, AAV, lentivirus, HSV, annelovirus, retrovirus, DoggyboneTM DNA (dbDNA), minicircle, plasmid, miniDNA, exosome, fusosome, or nanoplasmid.
  • the polynucleotide sequence encoding the prime editor system is encoded on at least two different vectors.
  • a first vector comprises a polynucleotide sequence encoding a nickase and a second vector comprises a polynucleotide sequence encoding a reverse transcriptase. In such cases, the first vector and second are delivered concurrently.
  • the polynucleotide sequence(s) encoding the prime editor system is encoded on at least two (non-contiguous) polynucleotide sequences.
  • a first polynucleotide sequence encodes a nickase and a second polynucleotide sequence encodes a reverse transcriptase.
  • the first vector and second are delivered concurrently (e.g., in a first LNP).
  • LNPs Split Lipid Nanoparticles
  • the method includes co-delivering to a cell a first gene editor polynucleotide construct and a first attachment site-containing guide RNA (atgRNA) are packaged, and thereby vectorized, within the first LNP, and a second gene editor polynucleotide construct and a second attachment site containing guide RNR (atgRNA) are packaged, and thereby vectorized, within the second LNP, where the first atgRNA and the second atgRNA are an at least first pair of atgRNA.
  • the at least first pair of atgRNAs comprise domains that are capable of guiding the prime editor system to a target sequence.
  • the first atgRNA further includes a first RT template that comprises at least a portion of a first integration recognition site.
  • the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site.
  • the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the first atgRNA and second atgRNA include at least a 6bp overlap.
  • the method includes delivering a first LNP (e.g., a first LNP comprising a first gene editor polynucleotide construct and a first atgRNA) and a second LNP (e.g., a second LNP comprising a second gene editor polynucleotide construct and a second atgRNA), the first LNP and the second LNP are mixed prior to delivering to a cell.
  • a first LNP e.g., a first LNP comprising a first gene editor polynucleotide construct and a first atgRNA
  • a second LNP e.g., a second LNP comprising a second gene editor polynucleotide construct and a second atgRNA
  • the first LNP and the second LNP are mixed at a ratio of first LNP to second LNP of l:10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1, 1 :0.75, 0.75: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.
  • the first LNP and the second LNP are mixed at a ratio of 1 : 1.
  • a first LNP comprising a first gene editor polynucleotide construct and a first attachment site-containing guide RNA (atgRNAl) comprises a ratio of ratio of gene editor polynucleotide construct (e.g., mRNA) to atgRNAl of 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1, 1 :0.75, 0.75: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.
  • the first LNP comprises a ratio of mRNA to atgRNAl of 2: 1.
  • a second LNP comprising a second gene editor polynucleotide construct and a second attachment site-containing guide RNA (atgRNA2) comprises a ratio of gene editor polynucleotide construct (e.g., mRNA) to atgRNA2 of 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1, 1 :0.75, 0.75: 1, 2: 1, 3: 1, 4:1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.
  • the second LNP comprises a ratio of mRNA to atgRNA2 of 2: 1.
  • the method includes delivering a first LNP (e.g., a first LNP comprising a first gene editor polynucleotide construct and a first atgRNA) and a second LNP (e.g., a second LNP comprising a second gene editor polynucleotide construct and a second atgRNA)
  • the first LNP and the second LNP are mixed such that the ratio of gene editor polynucleotide construct (e.g., mRNA) to first atgRNA (atgRNAl) to second atgRNA (atgRNA2) is 1 :0.25:0.25, l :0.5:0.5, 1 :0.75:0.75, or 1 : 1 : 1.
  • the method of co-delivering to a cell a mixture of LNPs includes co-delivering three or more LNPs, four or more LNPs, five or more LNPs, six or more LNPs, seven or more LNPs, eight or more LNPs, nine or more LNPs, or ten or more LNPs.
  • a system capable of site-specifically integrating at least a first integration recognition site into the genome of a cell, the system comprising: a first gene editor polynucleotide construct and a first attachment site-containing guide RNA (atgRNA) are packaged, and thereby vectorized, within the first LNP, and a second gene editor polynucleotide construct and a second attachment site containing guide RNR (atgRNA) are packaged, and thereby vectorized, within the second LNP, where the first atgRNA and the second atgRNA are an at least first pair of atgRNA.
  • the at least first pair of atgRNAs comprise domains that are capable of guiding the prime editor system to a target sequence.
  • the first atgRNA further includes a first RT template that comprises at least a portion of a first integration recognition site.
  • the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site.
  • the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the first atgRNA and second atgRNA include at least a 6bp overlap.
  • the system comprises a first LNP (e.g., any of the first LNPs described herein) and a second LNP (e.g., any of the second LNPs described herein) at a ratio of first LNP to second LNP of 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1, 1 :0.75, 0.75: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9:1, or 10: 1.
  • the system comprise the first LNP and the second LNP at a ratio of 1 : 1.
  • the system comprises a first LNP having a ratio of a first gene editor polynucleotide construct to a first attachment site-containing guide RNA (atgRNAl) of 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1:4, 1 :3, 1 :2, 1 : 1, 1 :0.75, 0.75: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.
  • the system includes a first LNP having a ratio of mRNA (i.e., mRNA encoding the gene editor protein) to atgRNAl of 2: 1.
  • the system comprise a second LNP having a ratio of a second gene editor polynucleotide construct to a second attachment site-containing guide RNA (atgRNA2) of 1 : 10, 1 :9, 1 :8, 1 :7, 1:6, 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1, 1 :0.75, 0.75: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.
  • the system includes a second LNP having a ratio of mRNA (i.e., mRNA encoding the gene editor protein) to atgRNA2 of 2: 1.
  • the system comprises a ratio of gene editor polynucleotide construct (e.g., mRNA encoding the gene editor protein) to first atgRNA (atgRNAl) to second atgRNA (atgRNA2) of 1 :0.25:0.25, l :0.5:0.5, 1 :0.75:0.75, or 1 : 1 : 1.
  • gene editor polynucleotide construct e.g., mRNA encoding the gene editor protein
  • the system comprises a mixture of LNPs comprising three or more LNPs, four or more LNPs, five or more LNPs, six or more LNPs, seven or more LNPs, eight or more LNPs, nine or more LNPs, or ten or more LNPs.
  • a vector comprising a template polynucleotide and a sequence that is an integration cognate (i.e., cognate to an integration recognition site site-specifically incorporated into the genome of a cell) can be delivered to the cell concurrently with the split LNPs or after delivery of the split LNPs.
  • a vector that includes a template polynucleotide and a second integration recognition site that is a cognate pair with the first integration recognition site is delivered to the cell.
  • the sequence that is an integration cognate e.g., a second integration recognition site
  • a second integration recognition site enables integration of the template polynucleotide or portion thereof when contacted with an integrase and the site- specifically incorporated first integration recognition site.
  • the invention involves vectors, e.g. for delivering or introducing in a cell, but also for propagating these components (e.g. in prokaryotic cells).
  • a "vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • a vector is capable of replication when associated with the proper control elements.
  • the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, doublestranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • a ’’plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector wherein virally- derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g.
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors.” Vectors for and that result in expression in a eukaryotic cell can be referred to herein as “eukaryotic expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • Vector delivery e.g., plasmid, viral delivery:
  • the CRISPR enzyme for instance a Type V protein such as C2cl or C2c3, and/or any of the present RNAs, for instance a guide RNA
  • Effector proteins and one or more guide RNAs can be packaged into one or more vectors, e.g., plasmid or viral vectors.
  • the vector e.g., plasmid or viral vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the delivery is via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery may be either via a single dose, or multiple doses.
  • the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choice, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc.
  • Such a dosage may further contain, for example, a carrier (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, a pharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), a pharmaceutically-acceptable excipient, and/or other compounds known in the art.
  • a carrier water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.
  • a pharmaceutically-acceptable carrier e.g., phosphate-buffered saline
  • a pharmaceutically-acceptable excipient e.g., phosphate-buffered saline
  • the dosage may further contain one or more pharmaceutically acceptable salts such as, for example, a mineral acid salt such as a hydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and the salts of organic acids such as acetates, propionates, malonates, benzoates, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, gels or gelling materials, flavorings, colorants, microspheres, polymers, suspension agents, etc. may also be present herein.
  • Suitable exemplary ingredients include microcrystalline cellulose, carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin and a combination thereof.
  • the delivery is via an adenovirus, which may be at a single booster dose containing at least 1 x 10 5 particles (also referred to as particle units, pu) of adenoviral vector.
  • the dose preferably is at least about 1 x 10 6 particles (for example, about 1 x 10 6 - 1 x 10 11 particles), more preferably at least about 1 x 10 7 particles, more preferably at least about 1 x 10 8 particles (e.g., about 1 x 10 8 -l x 10 11 particles or about 1 x 10 9 -l x 10 12 particles), and most preferably at least about 1 x IO 10 particles (e.g., about 1 x 10 9 - 1 x IO 10 particles or about 1 x 10 9 -l x 10 12 particles), or even at least about 1 x IO 10 particles (e.g., about 1 x 10 10 -l x 10 12 particles) of the adenoviral vector.
  • the dose comprises no more than about 1 x 10 14 particles, preferably no more than about 1 x 10 13 particles, even more preferably no more than about 1 x 10 12 particles, even more preferably no more than about 1 x 10 11 particles, and most preferably no more than about 1 x IO 10 particles (e.g., no more than about 1 x 10 9 particles).
  • the dose may contain a single dose of adenoviral vector with, for example, about 1 x 10 6 particle units (pu), about 2 x 10 6 pu, about 4 x 10 6 pu, about 1 x 10 7 pu, about 2 x 10 7 pu, about 4 x 10 7 pu, about 1 x 10 8 pu, about 2 x 10 8 pu, about 4 x 10 8 pu, about 1 x 10 9 pu, about 2 x 10 9 pu, about 4 x 10 9 pu, about 1 x IO 10 pu, about 2 x IO 10 pu, about 4 x IO 10 pu, about 1 x 10 11 pu, about 2 x 10 11 pu, about 4 x 10 11 pu, about 1 x 10 12 pu, about 2 x 10 12 pu, or about 4 x 10 12 pu of adenoviral vector.
  • adenoviral vector with, for example, about 1 x 10 6 particle units (pu), about 2 x 10 6 pu, about 4 x 10 6 pu, about 1 x 10 7 pu, about 2 x
  • the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel, et. al., granted on Jun. 4, 2013; incorporated by reference herein, and the dosages at col 29, lines 36-58 thereof.
  • the adenovirus is delivered via multiple doses.
  • the delivery is via an AAV.
  • a therapeutically effective dosage for in vivo delivery of the AAV to a human is believed to be in the range of from about 20 to about 50 ml of saline solution containing from about 1 x 10 10 to about 1 x 10 50 functional AAV/ml solution. The dosage may be adjusted to balance the therapeutic benefit against any side effects.
  • the AAV dose is generally in the range of concentrations of from about 1 x 10 5 to 1 x 10 50 genomes AAV (sometimes referred to herein as “vector genomes” or “vg”), from about 1 x 10 8 to 1 x IO 20 genomes AAV, from about 1 x 10 10 to about 1 x 10 16 genomes, or about 1 x 10 11 to about 1 x 10 16 genomes AAV.
  • a human dosage may be about 1 x 10 13 genomes AAV.
  • concentrations may be delivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of a carrier solution.
  • the promoter used to drive nucleic acid-targeting effector protein coding nucleic acid molecule expression can include: AAV ITR can serve as a promoter: this is advantageous for eliminating the need for an additional promoter element (which can take up space in the vector). The additional space freed up can be used to drive the expression of additional elements (gRNA, etc.). Also, ITR activity is relatively weaker, so can be used to reduce potential toxicity due to over expression of nucleic acid-targeting effector protein. For ubiquitous expression, can use promoters: CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc.
  • promoters For brain or other CNS expression, can use promoters: SynapsinI for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc.
  • For liver expression can use Albumin promoter.
  • For lung expression can use SP-B.
  • For endothelial cells can use ICAM.
  • For hematopoietic cells can use IFNbeta or CD45.
  • Osteoblasts can use OG-2.
  • the promoter used to drive guide RNA can include: Pol III promoters such as U6 or Hl Use of Pol II promoter and intronic cassettes to express guide RNA Adeno Associated Virus (AAV).
  • Pol III promoters such as U6 or Hl Use of Pol II promoter and intronic cassettes to express guide RNA Adeno Associated Virus (AAV).
  • AAV Addeno Associated Virus
  • Nucleic acid-targeting effector protein and one or more guide RNA can be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus), U.S. Pat. No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No. 5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus.
  • AAV adeno associated virus
  • the route of administration, formulation and dose can be as in U.S. Pat. No. 8,454,972 and as in clinical trials involving AAV.
  • the route of administration, formulation and dose can be as in U.S. Pat. No. 8,404,658 and as in clinical trials involving adenovirus.
  • the route of administration, formulation and dose can be as in U.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.
  • Doses may be based on or extrapolated to an average 70 kg individual (e.g., a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species.
  • Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed.
  • the viral vectors can be injected into the tissue of interest.
  • the expression of nucleic acid-targeting effector can be driven by a cell-type specific promoter.
  • liver-specific expression might use the Albumin promoter and neuron-specific expression (e.g., for targeting CNS disorders) might use the Synapsin I promoter.
  • AAV is advantageous over other viral vectors for a couple of reasons: Low toxicity (this may be due to the purification method not requiring ultra centrifugation of cell particles that can activate the immune response) and Low probability of causing insertional mutagenesis because it doesn't integrate into the host genome.
  • AAV has a packaging limit of 4.5 or 4.75 Kb.
  • nucleic acid-targeting effector protein such as a Type V protein such as C2cl or C2c3
  • a promoter and transcription terminator have to be all fit into the same viral vector. Therefore embodiments of the invention include utilizing homologs of nucleic acid-targeting effector protein (such as a Type V protein such as C2cl or C2c3) that are shorter.
  • the AAV can be AAV1, AAV2, AAV5 or any combination thereof.
  • AAV8 is useful for delivery to the liver. The herein promoters and vectors are preferred individually.
  • Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and psi2 cells or PA317 cells, which package retrovirus.
  • Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line may also be infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.
  • Millington-Ward et al. (Molecular Therapy, vol. 19 no. 4, 642-649 April 2011) describes adeno-associated virus (AAV) vectors to deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site.
  • RNAi RNA interference
  • An injection of either 6.0 x 10 8 vp or 1.8 x IO 10 vp AAV were subretinally injected into the eyes by Millington-Ward et al.
  • the AAV vectors of Millington-Ward et al. may be applied to the system of the present invention, contemplating a dose of about 2 x 10 11 to about 6 x 10 11 vp administered to a human.
  • Dalkara et al. also relates to in vivo directed evolution to fashion an AAV vector that delivers wild-type versions of defective genes throughout the retina after noninjurious injection into the eyes' vitreous humor.
  • Dalkara describes a 7 mer peptide display library and an AAV library constructed by DNA shuffling of cap genes from AAV1, 2, 4, 5, 6, 8, and 9.
  • the rcAAV libraries and rAAV vectors expressing GFP under a CAG or Rho promoter were packaged and deoxyribonuclease-resistant genomic titers were obtained through quantitative PCR.
  • the libraries were pooled, and two rounds of evolution were performed, each consisting of initial library diversification followed by three in vivo selection steps.
  • P30 rho-GFP mice were intravitreally injected with 2 ml of iodixanol -purified, phosphate- buffered saline (PBS)-dialyzed library with a genomic titer of about 1. times.10. sup.12 vg/ml.
  • PBS phosphate- buffered saline
  • the AAV vectors of Dalkara et al. may be applied to the nucleic acid-targeting system of the present invention, contemplating a dose of about 1 x 10 15 to about 1 x 10 16 vg/ml administered to a human.
  • Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SW), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66: 1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol.
  • MiLV murine leukemia virus
  • GaLV gibbon ape leukemia virus
  • SW Simian Immuno deficiency virus
  • HAV human immuno deficiency virus
  • adenoviral based systems may be used.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
  • Adeno-associated virus vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No.
  • Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and yr2 cells or PA317 cells, which package retrovirus.
  • Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line may also be infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.
  • a host cell is transiently or non-transiently transfected with one or more vectors described herein.
  • a cell is transfected as it naturally occurs in a subject.
  • a cell that is transfected is taken from a subject.
  • Cells taken from a subject include, but are not limited to, hepatocytes or cells isolated from muscle, the CNS, eye or lung.
  • Immunological cells are also contemplated, such as but not limited to T cells, HSCs, B-cells and NK cells.
  • mRNA delivery methods and compositions that may be utilized in the present disclosure including, for example, PCT/US2014/028330, US8822663B2, NZ700688A, ES2740248T3, EP2755693A4, EP2755986A4, WO2014152940A1, EP3450553B1, BRI 12016030852A2, and EP3362461A1.
  • Expression of CRISPR systems in particular is described by W02020014577.
  • Each of these publications are incorporated herein by reference in their entireties. Additional disclosure hereby incorporated by reference can be found in Kowalski et al., “Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery,” Mol Therap., 2019; 27(4): 710-728.
  • the cell is derived from cells taken from a subject, such as a cell line.
  • a cell line A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHL231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep
  • a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences.
  • one or more vectors described herein are used to produce a nonhuman transgenic animal or transgenic plant.
  • the transgenic animal is a mammal, such as a mouse, rat, or rabbit.
  • the organism or subject is a plant.
  • the organism or subject or plant is algae. Methods for producing transgenic plants and animals are known in the art, and generally begin with a method of cell transfection, such as described herein.
  • the invention provides for methods of modifying a target polynucleotide in a prokaryotic or eukaryotic cell, which may be in vivo, ex vivo or in vitro.
  • the method comprises sampling a cell or population of cells from a human or non-human animal or plant (including micro-algae) and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant (including micro-algae).
  • pathogens are often host-specific.
  • Fusariumn oxysporum f. sp. lycopersici causes tomato wilt but attacks only tomato
  • Plants have existing and induced defenses to resist most pathogens. Mutations and recombination events across plant generations lead to genetic variability that gives rise to susceptibility, especially as pathogens reproduce with more frequency than plants.
  • there can be non-host resistance e.g., the host and pathogen are incompatible.
  • Horizontal Resistance e.g., partial resistance against all races of a pathogen, typically controlled by many genes
  • Vertical Resistance e.g., complete resistance to some races of a pathogen but not to other races, typically controlled by a few genes.
  • Plant and pathogens evolve together, and the genetic changes in one balance changes in other. Accordingly, using Natural Variability, breeders combine most useful genes for Yield. Quality, Uniformity, Hardiness, Resistance.
  • the sources of resistance genes include native or foreign Varieties, Heirloom Varieties, Wild Plant Relatives, and Induced Mutations, e.g., treating plant material with mutagenic agents.
  • plant breeders are provided with a new tool to induce mutations. Accordingly, one skilled in the art can analyze the genome of sources of resistance genes, and in Varieties having desired characteristics or traits employ the present invention to induce the rise of resistance genes, with more precision than previous mutagenic agents and hence accelerate and improve plant breeding programs.
  • target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide.
  • target polynucleotides include a disease associated gene or polynucleotide.
  • a “disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non disease control.
  • a disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease.
  • the transcribed or translated products may be known or unknown, and may be at a normal or abnormal level.
  • the delivery system is packaged in one or more LNPs and administered intravenously.
  • the co-delivery system is packaged in one or more LNPs and administered intrathecally.
  • the co-delivery system is packaged in one or more LNPs and administered by intracerebral ventricular injection.
  • the co-delivery system is packaged in one or more LNPs and administered by intraci sternal magna administration.
  • the co-delivery system is packaged in one or more LNPs and administered by intravitreal injection.
  • lipidmucleic acid complexes including targeted liposomes such as immunolipid complexes
  • crystal Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos.
  • the LNP formulations are selected from LP01 (Cas No. 1799316-64-5), ALC-0315 (Cas No. 2036272-55-4), and cKK-E12 (Cas No. 1432494-65-9).
  • the LNP formulation is LP01.
  • the LNP formulation is ALC-0315.
  • the LNP formulation is cKK-E12.
  • LNP doses range from about 0.1 mg/kg to about 100 mg/kg (or any of the values or subranges therein). In some embodiments, LNP doses is about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1.0 mg/kg, 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 6 mg/kg, about7 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, or about 50 mg/kg or more
  • LNP doses of about 0.01 to about 1 mg per kg of body weight administered intravenously are contemplated.
  • Medications to reduce the risk of infusion-related reactions are contemplated, such as dexamethasone, acetaminophen, diphenhydramine or cetirizine, and ranitidine are contemplated.
  • Multiple doses of about 0.3 mg per kilogram every 4 weeks for five doses are also contemplated.
  • the charge of the LNP must be taken into consideration. As cationic lipids combined with negatively charged lipids to induce nonbilayer structures that facilitate intracellular delivery. Because charged LNPs are rapidly cleared from circulation following intravenous injection, ionizable cationic lipids with pKa values below 7 were developed (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011). Negatively charged polymers such as RNA may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge. However, at physiological pH values, the LNPs exhibit a low surface charge compatible with longer circulation times.
  • pH 4 e.g., pH 4
  • ionizable cationic lipids Four species of ionizable cationic lipids have been focused upon, namely l,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2- dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), l,2-dilinoleyloxy-keto-N,N-dimethyl- 3 -aminopropane (DLinKDMA), and l,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLinKC2-DMA).
  • DLinDAP 1,2- dilinoleyloxy-3-N,N-dimethylaminopropane
  • DLinKDMA 1,2- dilinoleyloxy-keto-N,N-dimethyl- 3 -aminopropane
  • LNP siRNA systems containing these lipids exhibit remarkably different gene silencing properties in hepatocytes in vivo, with potencies varying according to the series DLinKC2-DMA>DLinKDMA>DLinDMA»DLinDAP employing a Factor VII gene silencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011).
  • a dosage of 1 pg/ml of LNP in or associated with the LNP may be contemplated, especially for a formulation containing DLinKC2-DMA.
  • the LNP composition comprises one or more one or more ionizable lipids.
  • ionizable lipid has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties.
  • an ionizable lipid may be positively charged or negatively charged.
  • the one or more ionizable lipids are selected from the group consisting of 3-(didodecylamino)- N1 ,N1 ,4-tridodecyl- 1 -piperazineethanamine (KL 10), N1 -[2-(didodecylamino)ethyl]-N 1 ,N4,N4- tridodecyl-l,4-piperazinediethanami- ne (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza- octatriacontane (KL25), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-
  • the lipid nanoparticle may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cationic and/or ionizable lipids.
  • cationic and/or ionizable lipids include, but are not limited to, 3-(didodecylamino)-Nl,Nl,4-tridodecyl-l-piperazineethanamine (KL10), Nl-[2- (didodecylamino)ethyl]-Nl,N4,N4-tridodecyl-l,4-piperazinediethanami- ne (KL22), 14,25- ditridecyl- 15,18,21 ,24-tetraaza-octatriacontane (KL25), 1 ,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylamin
  • DOSPA (sperminecarboxamido)ethyl)-N,N-dimethyl- -ammonium trifluoracetate
  • DOGS dioctadecylamidoglycyl carboxyspermine
  • DODAP 1, 2-di oleoyl-3 -dimethylammonium propane
  • DODMA N,N-dimethyl-2,3-dioleyloxy
  • DMRIE N-(l,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide
  • lipids e.g., LIPOFECTIN.RTM. (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE.RTM. (including DOSPA and DOPE, available from GIBCO/BRL).
  • LIPOFECTIN.RTM including DOTMA and DOPE, available from GIBCO/BRL
  • LIPOFECTAMINE.RTM. including DOSPA and DOPE, available from GIBCO/BRL
  • KL10, KL22, and KL25 are described, for example, in U.S. Pat. No. 8,691,750.
  • the LNP composition comprises one or more amino lipids.
  • amino lipid and “cationic lipid” are used interchangeably herein to include those lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino head group (e.g., an alkylamino or dialkylamino head group).
  • a pH-titratable amino head group e.g., an alkylamino or dialkylamino head group.
  • the cationic lipid is typically protonated (i.e., positively charged) at a pH below the pKa of the cationic lipid and is substantially neutral at a pH above the pKa.
  • the cationic lipids can also be termed titratable cationic lipids.
  • the one or more cationic lipids include: a protonatable tertiary amine (e.g., pH-titratable) head group; alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and alkyl chains.
  • cationic lipids include, but are not limited to, DSDMA, DODMA, DOTMA, DLinDMA, DLenDMA, . gamma.
  • -DLenDMA DLin-K-DMA
  • DLin-K-C2- DMA also known as DLin-C2K-DMA, XTC2, and C2K
  • DLin-K-C3-DMA also known as DLin-K-C4-DMA
  • DLen-C2K-DMA y-DLen-C2-DMA
  • C12-200 cKK-E12, cKK-A12, cKK-012
  • DLin-MC2- DMA also known as MC2
  • DLin-MC3-DMA also known as MC3
  • Anionic lipids suitable for use in lipid nanoparticles include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N- dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.
  • Neutral lipids suitable for use in lipid nanoparticles include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, sterols (e.g., cholesterol) and cerebrosides.
  • the lipid nanoparticle comprises cholesterol.
  • Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains and cyclic regions can be used.
  • the neutral lipids used in the disclosure are DOPE, DSPC, DPPC, POPC, or any related phosphatidylcholine.
  • the neutral lipid may be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol.
  • amphipathic lipids are included in nanoparticles.
  • Exemplary amphipathic lipids suitable for use in nanoparticles include, but are not limited to, sphingolipids, phospholipids, fatty acids, and amino lipids.
  • the lipid composition of the pharmaceutical composition may comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof.
  • phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
  • a fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Particular amphipathic lipids can facilitate fusion to a membrane.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • elements e.g., a
  • Non-natural amphipathic lipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond.
  • an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide.
  • Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.
  • the LNP composition comprises one or more phospholipids.
  • the phospholipid is selected from the group consisting of 1,2-dilinoleoyl-sn- glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2- dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1 ,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-di-O- octadecenyl-sn-g
  • DLPC 1,2-dilino
  • phosphorus-lacking compounds such as sphingolipids, glycosphingolipid families, diacylglycerols, and P-acyloxyacids, may also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.
  • the LNP composition comprises one or more helper lipids.
  • helper lipid refers to lipids that enhance transfection (e.g., transfection of an LNP comprising an mRNA that encodes a site-directed endonuclease, such as a SpCas9 polypeptide).
  • site-directed endonuclease such as a SpCas9 polypeptide
  • the mechanism by which the helper lipid enhances transfection includes enhancing particle stability.
  • the helper lipid enhances membrane fusogenicity.
  • helper lipid of the LNP compositions disclosure herein can be any helper lipid known in the art.
  • helper lipids suitable for the compositions and methods include steroids, sterols, and alkyl resorcinols.
  • helper lipids suitable for use in the present disclosure include, but are not limited to, saturated phosphatidylcholine (PC) such as distearoyl-PC (DSPC) and dipalymitoyl-PC (DPPC), di oleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn- glycero-3 -phosphocholine (DLPC), cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate.
  • PC saturated phosphatidylcholine
  • DSPC distearoyl-PC
  • DPPC dipalymitoyl-PC
  • DOPE di oleoylphosphatidylethanolamine
  • DLPC 1,2-dilinoleoyl-sn- glycero-3 -phosphocholine
  • cholesterol 5-heptadecylresorcinol
  • cholesterol hemisuccinate hemisuccinate.
  • the LNP composition comprises one or more structural lipids.
  • structural lipid refers to sterols and also to lipids containing sterol moi eties. Without being bound to any particular theory, it is believed that the incorporation of structural lipids into the LNPs mitigates aggregation of other lipids in the particle.
  • Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof.
  • the structural lipid is a sterol.
  • sterols are a subgroup of steroids consisting of steroid alcohols.
  • the structural lipid is a steroid.
  • the structural lipid is cholesterol.
  • the structural lipid is an analog of cholesterol.
  • the lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids.
  • the LNP composition disclosed herein comprise one or more polyethylene glycol (PEG) lipid.
  • PEG-lipid refers to polyethylene glycol (PEG)-modified lipids. Such lipids are also referred to as PEGylated lipids.
  • PEG-lipids include PEG- modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG- CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan- 3-amines
  • a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG-lipid includes, but not limited to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG- 1,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA).
  • PEG-DMG 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol
  • PEG-DSPE 1,2-distearoyl-sn-
  • the PEG-lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
  • the lipid moiety of the PEG-lipids includes those having lengths of from about C. sub.14 to about C. sub.22, preferably from about C. sub.14 to about C. sub.16.
  • a PEG moiety for example a mPEG-NH.sub.2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.
  • the PEG-lipid is PEG2k-DMG.
  • the one or more PEG lipids of the LNP composition comprises PEG-DMPE.
  • the one or more PEG lipids of the LNP composition comprises PEG-DMG.
  • the ratio between the lipid components and the nucleic acid molecules of the LNP composition is sufficient for (i) formation of LNPs with desired characteristics, e.g., size, charge, and (ii) delivery of a sufficient dose of nucleic acid at a dose of the lipid component(s) that is tolerable for in vivo administration as readily ascertained by one of skill in the art.
  • a nanoparticle e.g., a lipid nanoparticle
  • a targeting moiety that is specific to a cell type and/or tissue type.
  • a nanoparticle may be targeted to a particular cell, tissue, and/or organ using a targeting moiety.
  • a nanoparticle comprises a targeting moiety.
  • targeting moieties include ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and antibodies (e.g., full-length antibodies, antibody fragments (e.g., Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, or F(ab')2 fragments), single domain antibodies, cam elid antibodies and fragments thereof, human antibodies and fragments thereof, monoclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies)).
  • the targeting moiety may be a polypeptide.
  • the targeting moiety may include the entire polypeptide (e.g., peptide or protein) or fragments thereof.
  • a targeting moiety is typically positioned on the outer surface of the nanoparticle in such a manner that the targeting moiety is available for interaction with the target, for example, a cell surface receptor.
  • a variety of different targeting moieties and methods are known and available in the art, including those described, e.g., in Sapra et al., Prog. Lipid Res. 42(5):439-62, 2003 and Abra et al., J. Liposome Res. 12: 1-3, 2002.
  • a lipid nanoparticle may include a surface coating of hydrophilic polymer chains, such as polyethylene glycol (PEG) chains (see, e.g., Allen et al., Biochimica et Biophysica Acta 1237: 99-108, 1995; DeFrees et al., Journal of the American Chemistry Society 118: 6101-6104, 1996; Blume et al., Biochimica et Biophysica Acta 1149: ISO- 184, 1993; Klibanov et al., Journal of Liposome Research 2: 321-334, 1992; U.S. Pat. No.
  • PEG polyethylene glycol
  • a targeting moiety for targeting the lipid nanoparticle is linked to the polar head group of lipids forming the nanoparticle.
  • the targeting moiety is attached to the distal ends of the PEG chains forming the hydrophilic polymer coating (see, e.g., Klibanov et al., Journal of Liposome Research 2: 321-334, 1992; Kirpotin et al., FEBS Letters 388: 115-118, 1996).
  • Standard methods for coupling the targeting moiety or moieties may be used.
  • phosphatidylethanolamine which can be activated for attachment of targeting moieties
  • derivatized lipophilic compounds such as lipid-derivatized bleomycin
  • Antibody- targeted liposomes can be constructed using, for instance, liposomes that incorporate protein A (see, e.g., Renneisen et al., J. Bio. Chem., 265: 16337-16342, 1990 and Leonetti et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451, 1990).
  • Other examples of antibody conjugation are disclosed in U.S. Pat. No.
  • targeting moieties can also include other polypeptides that are specific to cellular components, including antigens associated with neoplasms or tumors.
  • Polypeptides used as targeting moieties can be attached to the liposomes via covalent bonds (see, for example Heath, Covalent Attachment of Proteins to Liposomes, 149 Methods in Enzymology 111-119 (Academic Press, Inc. 1987)).
  • Other targeting methods include the biotin-avidin system.
  • a lipid nanoparticle includes a targeting moiety that targets the lipid nanoparticle to a cell including, but not limited to, hepatocytes, colon cells, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells (including primary tumor cells and metastatic tumor cells).
  • the targeting moiety targets the lipid nanoparticle to a hepatocyte.
  • the lipid nanoparticles described herein may be lipidoid-based.
  • the synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of polynucleotides (see Mahon et al., Bioconjug Chem. 2010 21 : 1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat. Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011 108: 12996-3001).
  • lipidoid formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse through the extracellular matrix into the blood stream. While a particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see e.g., Akinc et al., Mol Ther. 2009 17:872-879), use of lipidoid oligonucleotides to deliver the formulation to other cells types including, but not limited to, endothelial cells, myeloid cells, and muscle cells may not be similarly size-limited.
  • lipidoid formulations may have a similar component molar ratio.
  • Different ratios of lipidoids and other components including, but not limited to, a neutral lipid (e.g., diacylphosphatidylcholine), cholesterol, a PEGylated lipid (e.g., PEG-DMPE), and a fatty acid (e.g., an omega-3 fatty acid) may be used to optimize the formulation of the mRNA or system for delivery to different cell types including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc.
  • a neutral lipid e.g., diacylphosphatidylcholine
  • cholesterol e.g., a PEGylated lipid
  • PEG-DMPE PEGylated lipid
  • a fatty acid e.g., an omega-3 fatty acid
  • Exemplary lipidoids include, but are not limited to, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 (including variants and derivatives), DLin-MC3-DMA and analogs thereof.
  • lipidoid formulations for the localized delivery of nucleic acids to cells may also not require all of the formulation components which may be required for systemic delivery, and as such may comprise the lipidoid and the mRNA or system.
  • a system described herein may be formulated by mixing the mRNA or system, or individual components of the system, with the lipidoid at a set ratio prior to addition to cells.
  • In vivo formulations may require the addition of extra ingredients to facilitate circulation throughout the body.
  • a system or individual components of a system is added and allowed to integrate with the complex. The encapsulation efficiency is determined using a standard dye exclusion assays.
  • In vivo delivery of systems may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle PEGylation, degree of loading, oligonucleotide to lipid ratio, and biophysical parameters such as particle size (Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety).
  • particle size Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety.
  • small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids may result in significant effects on in vivo efficacy.
  • Formulations with the different lipidoids including, but not limited to penta[3-(l- laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401 :61 (2010)), C12-200 (including derivatives and variants), MD1, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA and DLin-MC3-DMA can be tested for in vivo activity.
  • the lipidoid referred to herein as "98N12-5" is disclosed by Akinc et al., Mol Ther. 2009 17:872-879).
  • the lipidoid referred to herein as "C12-200” is disclosed by Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869 and Liu and Huang, Molecular Therapy. 2010 669-670.
  • the LNPs of the present disclosure in which a nucleic acid is entrapped within the lipid portion of the particle and is protected from degradation, can be formed by any method known in the art including, but not limited to, a continuous mixing method, a direct dilution process, and an in-line dilution process. Additional techniques and methods suitable for the preparation of the LNPs described herein include coacervation, microemulsions, supercritical fluid technologies, phase-inversion temperature (PIT) techniques.
  • PIT phase-inversion temperature
  • the LNPs used herein are produced via a continuous mixing method, e.g., a process that includes providing an aqueous solution a nucleic acid described herein in a first reservoir, providing an organic lipid solution in a second reservoir (wherein the lipids present in the organic lipid solution are solubilized in an organic solvent, e.g., a lower alkanol such as ethanol), and mixing the aqueous solution with the organic lipid solution such that the organic lipid solution mixes with the aqueous solution so as to substantially instantaneously produce a lipid vesicle (e.g., liposome) encapsulating the nucleic acid molecule within the lipid vesicle.
  • a continuous mixing method e.g., a process that includes providing an aqueous solution a nucleic acid described herein in a first reservoir, providing an organic lipid solution in a second reservoir (wherein the lipids present in the organic lipid solution are solubilized in an organic solvent
  • the LNPs used herein are produced via a direct dilution process that includes forming a lipid vesicle (e.g., liposome) solution and immediately and directly introducing the lipid vesicle solution into a collection vessel containing a controlled amount of dilution buffer.
  • the collection vessel includes one or more elements configured to stir the contents of the collection vessel to facilitate dilution.
  • the amount of dilution buffer present in the collection vessel is substantially equal to the volume of lipid vesicle solution introduced thereto.
  • the LNPs are produced via an in-line dilution process in which a third reservoir containing dilution buffer is fluidly coupled to a second mixing region.
  • the lipid vesicle (e.g., liposome) solution formed in a first mixing region is immediately and directly mixed with dilution buffer in the second mixing region.
  • This disclosure is not limited to systems and methods described herein. Any delivery method that is capable of delivering the systems described herein can be used as long as it is capable of site-specifically integrating a template polynucleotide into the genome of a cell.
  • compositions, systems and methods for correcting or replacing genes or gene fragments (including introns or exons) or inserting genes in new locations comprises recombination or integration into a safe harbor site (SHS).
  • SHS safe harbor site
  • a frequently used human SHS is the AAVS1 site on chromosome 19q, initially identified as a site for recurrent adeno-associated virus insertion.
  • Another locus comprises the human homolog of the murine Rosa26 locus.
  • Yet another SHS comprises the human Hl 1 locus on chromosome 22.
  • a complete gene may be prohibitively large and replacement of an entire gene impractical.
  • a method of the disclosure comprises recombining corrective gene fragments into a defective locus.
  • the methods and compositions can be used to target, without limitation, stem cells for example induced pluripotent stem cells (iPSCs), HSCs, HSPCs, mesenchymal stem cells, or neuronal stem cells and cells at various stages of differentiation.
  • stem cells for example induced pluripotent stem cells (iPSCs), HSCs, HSPCs, mesenchymal stem cells, or neuronal stem cells and cells at various stages of differentiation.
  • methods and compositions of the disclosure are adapted to target organoids, including patient derived organoids.
  • methods and compositions of the disclosure are adapted to treat muscle cells, not limited to cardiomyocytes for Duchene Muscular Dystrophy (DMD).
  • the dystrophin gene is the largest gene in the human genome, spanning ⁇ 2.3 Mb of DNA. DMD is composed of 79 exons resulting in a 14-kb full-length mRNA. Common mutations include mutations that disrupt the reading frame of generate a premature stop codon.
  • An aspect of DMD that lends it to gene editing as a therapeutic approach is the modular structure of the dystrophin protein. Redundancy in the central rod domain permits the deletion of internal segments of the gene that may harbor loss-of-function mutations, thereby restoring the open reading frame (ORFs).
  • the methods and systems described herein are used to treat DMD by site- specifically integrating in the genome a polynucleotide template that repairs or replaces all or a portion of the defective DMD gene.
  • MN Membranous Nephropathy
  • C3GN C3 glomerulonephritis
  • HCU Homocystinuria
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the most common cystic fibrosis (CF) mutation F508del removes a single amino acid.
  • recombining human CFTR into an SHS of a cell that expresses CFTR F508del is a corrective treatment path.
  • the methods and systems described herein are used to CF by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing CF. Proposed validation is detection of persistent CFTR mRNA and protein expression in transduced cells.
  • Sickle cell disease is caused by mutation of a specific amino acid — valine to glutamic acid at amino acid position 6.
  • SCD is corrected by recombination of the HBB gene into a safe harbor site (SHS) and by demonstrating correction in a proportion of target cells that is high enough to produce a substantial benefit.
  • the methods and systems described herein are used to sickle cell disease by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the disease.
  • validation is detection of persistent HBB mRNA and protein expression in transduced cells.
  • DMD Duchenne Muscular Dystrophy.
  • the dystrophin gene is the largest gene in the human genome, spanning ⁇ 2.3 Mb of DNA. DMD is composed of 79 exons resulting in a 14- kb full-length mRNA. Common mutations include mutations that disrupt the reading frame of generate a premature stop codon.
  • An aspect of DMD that lends it to gene editing as a therapeutic approach is the modular structure of the dystrophin protein. Redundancy in the central rod domain permits the deletion of internal segments of the gene that may harbor loss-of-function mutations, thereby restoring the open reading frame (ORFs).
  • recombination will be into safe harbor sites (SHS).
  • SHS safe harbor sites
  • a frequently used human SHS is the A4FS7 site on chromosome 19q, initially identified as a site for recurrent adeno-associated virus insertion.
  • the site is the human homolog of the e murine Rosa26 locus (pubmed. ncbi.nlm.nih.gov/18037879).
  • the site is the human Hl 1 locus on chromosome 22.
  • Proposed target cells for recombination include stem cells for example induced pluripotent stem cells (iPSCs) and cells at various stages of differentiation. In some cases, a complete gene may be prohibitively large and replacement of an entire gene impractical. In such instances, rescuing mutants by recombining in corrected gene fragments with the methods and systems described herein is a corrective option.
  • iPSCs induced pluripotent stem cells
  • correcting mutations in exon 44 (or 51) by recombining in a corrective coding sequence downstream of exon 43 (or 50), using the methods and systems described herein is a corrective option.
  • Proposed validation is detection of persistent DMD mRNA and protein expression in transduced cells.
  • F8 Factor VIII.
  • F8 A large proportion of severe hemophilia A patients harbor one of two types of chromosomal inversions in the F VIII gene.
  • the recombinase technology and methods described herein are well suited to correcting such inversions (and other mutations) by recombining of the FVIII gene into a SHS.
  • correcting factor VIII deficiency by recombining the FVIII gene into an SHS is a corrective path.
  • the methods and systems described herein are used to correct factor VIII deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the FIX deficiency. Proposed validation is detection of persistent FVIII mRNA and protein expression in transduced cells.
  • Factor 9 Hemophilia B, also called factor IX (FIX) deficiency is a genetic disorder caused by missing or defective factor IX, a clotting protein.
  • the methods and systems described herein are used to correct factor IX deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the FIX deficiency.
  • Proposed validation is detection of persistent FiX mRNA and protein expression in transduced cells.
  • Ornithine transcarbamylase deficiency is a rare genetic condition that causes ammonia to build up in the blood.
  • the condition - more commonly called OTC deficiency - is more common in boys than girls and tends to be more severe when symptoms emerge shortly after birth.
  • the methods and systems described herein are used to correct OTC deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the OTC deficiency or integrates a polynucleotide encoding a functional ornithine transcarbamylase enzyme.
  • Proposed validation is detection of persistent OTC mRNA and protein expression in transduced cells.
  • Phenylketonuria also called PKU, is a rare inherited disorder that causes an amino acid called phenylalanine to build up in the body. PKU is caused by a change in the phenylalanine hydroxylase (PAH) gene. This gene helps create the enzyme needed to break down phenylalanine.
  • PKU phenylalanine hydroxylase
  • the methods and systems described herein are used to correct PKU by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the PKU deficiency or integrates a polynucleotide encoding a functional phenylalanine hydroxylase (PAH) gene.
  • Proposed validation is detection of persistent PAH mRNA and protein expression in transduced cells.
  • Homocystinuria is elevation of the amino acid, homocysteine (protein building block coming from our diet) in the urine or blood.
  • Common causes of HCU include: problems with the enzyme cystathionine beta synthase (CBS), which converts homocysteine to the amino acid cystathionine (which then becomes cysteine) and needs the vitamin B6 (pyridoxine); and problems with converting homocysteine to the amino acid methionine.
  • CBS cystathionine beta synthase
  • pyridoxine pyridoxine
  • the methods and systems described herein are used to correct HCU by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the HCU or integrates a polynucleotide encoding a functional copy of a gene (e.g., CBS) able to reduce or prevent buildup of homocysteine in the urine.
  • Proposed validation is detection of persistent CBS mRNA and protein expression in transduced cells.
  • IgA Nephropathy (Berger’s disease). IgA nephropathy, also known as Berger's disease, is a kidney/autoimmune disease that occurs when an antibody called immunoglobulin A (IgA) builds up in the kidneys.
  • the methods and systems described herein are used to treat Berger’s disease by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of Berger’s disease.
  • ANCA vasculitis is an autoimmune disease affecting small blood vessels in the body. It is caused by autoantibodies called ANCAs, or Anti-Neutrophilic Cytoplasmic Autoantibodies. ANCAs target and attack a certain kind of white blood cells called neutrophils.
  • the methods and systems described herein are used to treat ANCA vasculitis by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of ANCA vasculitis.
  • Lupus is an autoimmune — a disorder in which the body’s immune system attacks the body’s own cells and organs.
  • the methods and systems described herein are used to treat SLE/LN by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of SLE/LN.
  • MN Membranous Nephropathy
  • the methods and systems described herein are used to treat MN by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of MN.
  • C3 glomerulonephritis C3GN.
  • C3 glomerulopathy is a group of related conditions that cause the kidneys to malfunction.
  • the major features of C3 glomerulopathy include high levels of protein in the urine (proteinuria), blood in the urine (hematuria), reduced amounts of urine, low levels of protein in the blood, and swelling in many areas of the body.
  • Affected individuals may have particularly low levels of a protein called complement component 3 (or C3) in the blood.
  • the methods and systems described herein are used to treat C3 glomerulopathy by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of C3 glomerulopathy.
  • methods of treatment comprises administering an effective amount of the pharmaceutical composition comprising the nucleic acid construct or vectorized nucleic acid construct described above to a patient in need thereof.
  • the system e.g., any of the systems described herein
  • the systems are delivered to a patient, thereby delivering to a cell in vivo.
  • DNA or RNA viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo).
  • Conventional viral based systems to be used herein could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
  • the co-delivery system described herein e.g., a gene editor construct packaged in a LNP and a donor template packaged in a vector
  • the co-delivery system described herein is administered intravenously.
  • the co-delivery system described herein e.g., a gene editor construct packaged in a LNP and a donor template packaged in a vector
  • the co-delivery system described herein e.g., a gene editor construct packaged in a LNP and a donor template packaged in a vector
  • the co-delivery system described herein e.g., a gene editor construct packaged in a LNP and a donor template packaged in a vector
  • the co-delivery system described herein e.g., a gene editor construct packaged in a LNP and a donor template packaged in a vector
  • Methods of non-viral delivery of the donor DNA template described herein include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM andLipofectinTM).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
  • mRNA delivery methods and compositions that may be utilized in the present disclosure including, for example, PCT/US2014/028330, US8822663B2, NZ700688A, ES2740248T3, EP2755693A4, EP2755986A4, WO2014152940A1, EP3450553B1, BRI 12016030852A2, and EP3362461A1.
  • Expression of CRISPR systems in particular is described by W02020014577.
  • Each of these publications are incorporated herein by reference in their entireties. Additional disclosure hereby incorporated by reference can be found in Kowalski et al., “Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery,” Mol Therap., 2019; 27(4): 710-728. 6.
  • Example 1 Delivery of gene editor polynucleotide sequence packaged in LNP and donor template packaged in AAV
  • a gene editor polynucleotide construct is packaged into a LNP (FIG. 1), wherein the gene editor polynucleotide sequence comprises a polynucleotide sequence encoding a prime editor protein linked to an integrase via peptide linker a polynucleotide sequence encoding an attachment site-containing guide RNA (atgRNA); a polynucleotide sequence encoding a nickase guide RNA (ngRNA).
  • atgRNA attachment site-containing guide RNA
  • ngRNA nickase guide RNA
  • a donor template polynucleotide construct is packaged in an AAV vector (FIG. 2).
  • Co-administration of the gene editor construct packaged LNP and the donor template packaged AAV co-delivers the gene editor construct to a cell cytoplasm and the donor template to a cell nucleus.
  • the direct activity of the associated integrase to the specific genomic site is guided.
  • Gene editor construct expression, with template co-delivery, results in integration of template “cargo” at a precisely defined target location.
  • Example 2 Delivery of gene editor polynucleotide sequence packaged in LNP and donor template capable of self-circularization packaged in AAV
  • a gene editor polynucleotide construct is packaged into a LNP (FIG. 1), wherein the gene editor polynucleotide sequence comprises a polynucleotide sequence encoding a prime editor protein linked to an integrase via peptide linker a polynucleotide sequence encoding an attachment site-containing guide RNA (atgRNA); a polynucleotide sequence encoding a nickase guide RNA (ngRNA).
  • atgRNA attachment site-containing guide RNA
  • ngRNA nickase guide RNA
  • a donor template polynucleotide construct is packaged in an AAV vector (FIG. 2).
  • Co-administration of the gene editor construct packaged LNP and the donor template packaged AAV co-delivers the gene editor construct to a cell cytoplasm and the donor template to a cell nucleus.
  • Integrase-mediated self-circularization of donor template occurs at integration target recognition sites within the AAV genome (FIG. 3).
  • an orthogonal integrase landing site i.e., distinct att site from att sites used for selfcircularization
  • Gene editor construct expression, with template co-delivery and integrase-mediated circularization of template results in integration of template “cargo” at a precisely defined target location.
  • Example 3 Delivery of gene editor polynucleotide sequence packaged in LNP and atgRNA, ngRNA, and donor template co-packaged in AAV
  • a gene editor polynucleotide construct is packaged into a LNP (FIG. 4), wherein the gene editor polynucleotide sequence comprises a polynucleotide sequence encoding a prime editor protein linked to an integrase via peptide linker.
  • a polynucleotide sequence encoding an attachment site-containing guide RNA (atgRNA), a polynucleotide sequence encoding a nicking guide RNA (ngRNA), and donor template are packaged in an AAV vector (FIG. 4).
  • Co-administration of the gene editor construct packaged LNP and the atgRNA, ngRNA, donor template packaged AAV co-delivers the gene editor construct to a cell.
  • Integrase- mediated self-circul arization of donor template occurs at integration target recognition sites within the AAV genome (FIG. 3).
  • an orthogonal integrase landing site i.e., distinct att site from att sites used for self-circularization
  • Gene editor construct expression, with atgRNA, ngRNA, and template co-delivery and integrase-mediated circularization of template results in integration of template “cargo” at a precisely defined target location.
  • Example 4 Delivery of gene editor polynucleotide sequence and ngRNA packaged in LNP and atgRNA and donor template co-packaged in AAV
  • a gene editor polynucleotide construct and a nicking guide RNA (ngRNA) are packaged into a LNP (FIG. 5), wherein the gene editor polynucleotide sequence comprises a polynucleotide sequence encoding a prime editor protein linked to an integrase via peptide linker.
  • a polynucleotide sequence encoding an attachment site-containing guide RNA (atgRNA) and donor template are packaged in an AAV vector (FIG. 5).
  • Example 5 Intramolecular circularization of plasmid and packaged AAV genomes
  • FIG. 7 A universal ddPCR probe capable of binding any linear or circularized AAV genome was designed, wherein the universal ddPCR probe is designed to only give signal upon cognate recombinase/integrase mediated circularization (FIGs. 8A-8B).
  • Circularization products are amplified by use of a circle junction PCR primer set that is designed to amplify only circular products due to primer direction constraints.
  • an attR scar quencher-fluorophore probe was designed.
  • a template reference primer set was designed and generated to quantify total template DNA (linear or circular confirmation) (FIGs. 8A-8B).
  • FIG. 10 demonstrates circularization of AAV pDNA and packaged AAV genomic DNA for both IX Bxbl and 2X Bxbl conditions (confirmed by use of attR ddPCR primer set). Further, replicates that lacked either Bxbl or AAV pDNA substrate demonstrated insignificant circularization. All three of the Cre-, FLPe-, and Bxbl -targeted AAV pDNA substrates demonstrated circularization upon cognate recombinase/integrase introduction, as confirmed by using the universal ddPCR probe (FIG. 11). Moreover, Cre-, FLPe-, and Bxbl-mediated circularization of packaged AAV DJ genomes substrates were demonstrated and confirmed using the universal ddPCR probe (FIG. 12).
  • the Bxbl-mediated attR scar probe provided similar percent circularization quantification compared to the universal probe.
  • Example 6 In vitro beacon placement in primary mouse hepatocytes and primary human hepatocytes using mRNA and AAV for co-delivery
  • This example assessed the efficiency of in vitro beacon placement in primary human hepatocytes using mRNA delivering of a polynucleotide encoding a gene editor polynucleotide construct and AAV to deliver the first and second atgRNA. See FIG. 15 for a non-limiting example of a dual atgRNA-mediated insertion of an integration recognition site.
  • the mRNA and AAV were delivered into the primary mouse hepatocytes (PMH) using (i) concurrent delivery (“co-dose”), (ii) AAV delivery followed by a “1- day delay” before delivery of the mRNA, or (iii) AAV delivery followed by a “2-day delay” before delivery of the mRNA.
  • Beacon placement was then assessed using next-generation sequencing of DNA isolated from cells subjected to the delivery conditions mentioned above.
  • the mRNA encoding the gene editor polynucleotide construct was delivered in various amounts per well: 2000 ng, 1000 ng, 500 ng, 250 ng, 125 ng, 62.5 ng, and 31.25 ng.
  • AAV encoding the first and second atgRNA see Table 12).
  • the primary mouse hepatocyte data is shown in FIG. 16 and the human primary hepatocyte data is shown in FIG. 17.
  • Example 7 In vivo beacon placement with mRNA + AAV guide
  • mice were administered AAV containing the first atgRNA (SEQ ID NO: 543; Table 12) and the second atgRNA (SEQ ID NO: 544) targeting the Nolcl locus at 3E11 to 1E12 vector genomes (vg) per animal two 2 weeks prior to administration of the mRNA containing the gene editing polynucleotide construct (see FIG. 18).
  • mRNA was delivered using various LNP formulations (e.g., LNP #F1 , LNP #F2, and LNP #F3) at concentrations ranging from 5 mg/kg to 0.5 mg/kg via intravenous injection (see FIG. 18).
  • LNP formulations e.g., LNP #F1 , LNP #F2, and LNP #F3
  • concentrations ranging from 5 mg/kg to 0.5 mg/kg via intravenous injection (see FIG. 18).
  • liver tissue was harvested, genomic DNA was isolated, and beacon efficiency was assessed by NGS. As shown in FIG. 18, three conditions resulted in vivo beacon placement efficiency greater than 10%.
  • this data provided proof-of-concept for successful in vivo beacon placement using AAV to deliver the first and second atgRNA and LNPs to deliver the mRNA encoding the gene editor polynucleotide construct.
  • Example 8 In vivo integration in mice using AAV to deliver the template polynucleotide and Adenovirus to deliver BxBl
  • In vivo integration efficiency in AttP mice was assessed using adenovirus to deliver an integrase (e.g., Bxbl) and an AAV to deliver the template polynucleotide.
  • adenovirus to deliver an integrase (e.g., Bxbl) and an AAV to deliver the template polynucleotide.
  • the adenovirus i.e., adenovirus containing polynucleotide encoding the integrase
  • the AAV i.e., AAV containing the template polynucleotide and an attB site
  • mice containing dual AttP sites integrated in to the Rosa26 locus (B6.RosaBxb-GT/GA; female, Strain# 036152).
  • the Rosa26 locus included a first AttP site comprising a GT dinucleotide and a second AttP site comprising a GA dinucleotide.
  • the AAV was a scAAV8 containing a vector having a template polynucleotide and a 38 bp GT AttB site.
  • the Adenovirus was an adenovirus-type 5 (Ad5) containing a polynucleotide encoding Bxbl (“Bxbl AdV”) (SEQ ID NO: 563; Table 14). Mice were administered the adenovirus and AAV according to the experimental details in Table 13.
  • this data establishes proof-of-concept for in vivo integration using an adenovirus to deliver and drive expression of Bxbl and an AAV to deliver the template polynucleotide to be integrated into a mammalian genome, in this case, the mouse genome.
  • the first LNP contained mRNA encoding a prime editing system and a first synthetic atgRNA (atgRNAl).
  • the mRNA and atgRNAl were included at 1 : 1 ratio in the first LNP.
  • the second LNP contained mRNA encoding a prime editing system and a second synthetic atgRNA (atgRNA2).
  • the mRNA and atgRNA2 were included at a 1 : 1 ratio in the second LNP.
  • Each of the first and second atgRNAs targeted the mouse Nolcl locus and each encoded a portion of an integration recognition site (a “beacon”).
  • the LNP mixture was administered to the neonatal mice (2-5 day old CD-I mice) according to the experimental details in Table 16.
  • liver samples either whole liver for groups 1-3 or liver punches from each lobe for groups 4-6 (see Table 13) were collected and genomic DNA was isolated. Beacon placement was detected using ddPCR and NGS.
  • Neonates were also assessed at six weeks after administration of the LNP mixture. Beacon placement was detected using ddPCR and NGS. As shown in FIG. 21 A., at six weeks post administration, ddPCR revealed about 4% beacon placement (in Nolcl alleles) for a 3mg/kg dose of the LNP mixture. Confirmation of beacon placement using NGS showed about 15% beacon placement (in Nolcl alleles) for a 3 mg/kg dose of the LNP mixture (see FIG. 2 IB). Assessment of the percent of integrated beacons that included the expected integration recognition site (“perfect beacon”) revealed that about 3.5% of beacons were comprised of perfect beacons (see FIG. 21C).
  • this data demonstrated successful in vivo site-specific integration of an integration recognition site.
  • this data showed that a split LNP approach can be used for site- specifically integrating an integration recognition site in vivo in a mammalian genome, in this case neonatal mice.
  • the first LNP contained mRNA encoding a prime editing system and a first synthetic atgRNA (atgRNAl).
  • the mRNA and atgRNAl were included at different ratios (e.g., 1 :0.5, 1: 1, and 1 :2) ratio in the first LNP.
  • the second LNP contained mRNA encoding a prime editing system and a second synthetic atgRNA (atgRNA2).
  • the mRNA and atgRNA2 were included at different ratios (e.g., 1 :0.5, 1 : 1, and 1 :2) ratio in the second LNP.
  • the first and second atgRNAs targeted mouse Factor IX (“mF9”) locus and each encoded a portion of an integration recognition site (“beacon”). Similar to Example 9, atgRNAl and atgRNA2 together included a 6bp overlap and were combined 1 : 1 as mixture prior to administration.
  • the first atgRNA and second atgRNA are provide in Table 17, where the atgRNA include one or more 2’O-methyl modifications and one or more phosphorothioate linkages.
  • the LNP mixture was administered to female CD-I mice 6-8 weeks old according to the experimental details in Table 18.
  • **1:0.5:0.5 mRNA:atgRNAl 1:1; mRNA:atgRNA21:1; LNPs mixed 1:1
  • ***1:1:1 mRNA:atgRNAl 1:2; mRNA:atgRNA21:2; LNPs mixed 1:1
  • ddPCR revealed about 0.8% beacon placement (in mF9 alleles) following administration of a 1 :0.25:0.25 ratio of mRNA:atgRNAl :atgRNA2.
  • beacon placement using NGS showed about 14% beacon placement (in mF9 alleles) following administration of the 1 :0.25:0.25 ratio of mRNA:atgRNAl :atgRNA2 (see FIG. 22B). Similar to Example 9, an NGS-based assay was used to determined what percentages of the integrated beacons included the expected integration recognition site (“perfect beacon”). As shown in FIG. 22C, about 0.02% of the beacons placed in the mF9 locus were “perfect” beacons.
  • this data showed successful in vivo site-specific integration of an integration recognition site in adult mice.
  • this data showed that the ratio of mRNA to atgRNA is an important consideration in determining efficacy of in vivo site-specific integration of an integration recognition site.
  • Engineered integrases described in FIG. 23 were evaluated for ability to mediate programmable gene insertion, measured as Integration % (see FIG. 24B) or “Beacon Occupancy” (see FIG. 24C). Percent beacon placement, which precedes integration, is illustrated in FIG. 24 A.
  • the engineered integrases were introduced into primary human hepatocytes (PHH) at a high dose (750ng) and a low dose (250ng) along with an mRNA encoding a gene editor polypeptide (i.e., a Cas9 nickase fused to a reverse transcriptase (SEQ ID NO: 589)); an AAV expressing an atgRNA (the atgRNA comprising a spacer sequence with sequence complementarity to a sequence in the human factor IX locus, a scaffold, an RT template comprising an integration recognition site, and a primer binding site (See Table 19 below); and a donor polynucleotide comprising a cognate integration recognition site.
  • the AAV used to deliver the atgRNA also included the donor polynucleotide sequence.
  • the AAV sequence is AAVG048 (SEQ ID NO: 592; See Table 20).
  • Engineered integrases described in FIG. 25 were evaluated for their ability to mediate programmable gene insertion (as measured by “Occupancy %” (FIG. 26)).
  • the engineered integrases were introduced into primary human hepatocytes (PHH) at a high dose (1.3 pmol) and a low dose (0.2 pmol) along with a gene editor polypeptide (i.e., mRNA encoding a gene editor polypeptide (i.e., a Cas9 nickase fused to a reverse transcriptase)); an AAV expressing an atgRNA (the atgRNA comprising a spacer sequence with sequence complementarity to a sequence in the human factor IX locus, a scaffold, an RT template comprising an integration recognition site, and a primer binding site (see Table 19 above for atgRNA sequences); and a donor polynucleotide template comprising a cognate integration recognition site.
  • a gene editor polypeptide i.e., mRNA encoding a gene editor polypeptide (i.e., a Cas9 nickase fused to a reverse transcriptase)
  • the AAV used to deliver the atgRNA also included the donor polynucleotide template sequence.
  • the AAV sequence is AAVG048 (SEQ ID NO: 592; see Table 20 above). Genomic DNA was harvested 3 days after transduction. Data is shown in
  • Engineered integrases described in FIG. 23 comprising were evaluated for their ability to mediate programmable gene insertion (as indicated by “Occupancy %” (FIGs. 27A-27D)).
  • additional experiments were performed to assess engineered BxBl integrases having a c-terminal tag.
  • these engineered integrases were assessed for their ability to mediate programmable gene insertion (e.g., as indicated by “% Beacon Occupancy”).
  • the engineered integrases were introduced into a primary human hepatocyte (PHH) at amounts of 1000 ng, 500 ng, and 250 ng, along with a gene editor polypeptide (i.e., mRNA encoding a gene editor polypeptide (i.e., a Cas9 nickase fused to a reverse transcriptase)); an atgRNA comprising a spacer sequence with sequence complementarity to a sequence in the human factor IX locus, a scaffold, an RT template comprising an integration recognition site, and a primer binding site (see Table 19 above for atgRNA sequences); and a donor polynucleotide template comprising a cognate integration recognition site at 40 fmol, 20 fmol, and 10 fmol.
  • a gene editor polypeptide i.e., mRNA encoding a gene editor polypeptide (i.e., a Cas9 nickase fused to a reverse transcriptase)
  • the donor polynucleotide template was introduced into the cells as an AAV vector (PL 753 (SEQ ID NO: 593)). Genomic DNA was harvested 3 days after transduction. [0578] Data is shown in FIGs. 27A-27D. Overall, this data showed that a c-terminal tag reduced integration efficiency of BxBl.
  • the integration enzymes engineered to include stabilization domains were also assessed for their dose dependent impact on PGI. Additional engineered integration enzymes are described in FIG. 28 (see also Table 26).
  • the engineered integrases were introduced into a primary human hepatocyte (PHH) at a 250 ng along with a gene editor polypeptide (i.e., mRNA encoding a gene editor polypeptide (i.e., a Cas9 nickase fused to a reverse transcriptase)); an atgRNA comprising a spacer sequence with sequence complementarity to a sequence in the human factor IX locus, a scaffold, an RT template comprising an integration recognition site, and a primer binding site (see Table 19 above in Example 11 for atgRNA sequences); and a donor polynucleotide template comprising a cognate integration recognition site. Genomic DNA was harvested 3 days after transduction.
  • a gene editor polypeptide i.e., mRNA encoding a gene editor polypeptide (i.e., a Cas9 nickase fused to a reverse transcriptase)
  • an atgRNA comprising a spacer sequence with sequence complementar
  • FIGs. 29A-29B Data for programmable gene insertion (PGI) is shown in FIGs. 29A-29B. This data showed that an engineered BxBl comprising a stabilon domain resulted in increased programmable gene insertion (PGI) compared to the other BxB 1 polypeptides, including other engineered BxBl polypeptides.
  • a gene editor polypeptide i.e., mRNA encoding a gene editor polypeptide (i.e., a Cas9 nickase fused to a reverse transcriptase)
  • an atgRNA comprising a spacer sequence with sequence complementarity to a sequence in the human factor IX locus, a scaffold, an RT template comprising an integration recognition site, and a primer binding site (see Table 19 above for atgRNA sequences)
  • a donor polynucleotide template comprising a cognate integration recognition site.
  • Engineered integration enzymes were assessed for their ability to mediate PGI in pluripotent stem cells.
  • a non-limiting exemplary workflow is as described in FIG. 30A.
  • Engineered integration enzymes that were used in these experiments are as described in FIG. 30B (see also Table 26).
  • the engineered integrases were introduced into a pluripotent stem cell clone 52 or clone 17 along with a BxBl mRNA ; and a donor polynucleotide template comprising a cognate integration recognition site (PL1113 (SEQ ID NO: 594; see Table 22 below)).
  • PGI programmable gene insertion
  • FIG. 31A shows ddPCR data for PGI for clone 52 at day 3 and day 6.
  • Flow cytometry was used to assess GFP expression which indicates programmable gene insertion (PGI) (see FIG. 31B). This data shows that PL1323 and PL1325 yield % GFP+ cells by flow -50%.
  • Engineered integrases were also assessed for their ability to mediate programmable gene insertion in hematopoietic stem cells.
  • the engineered integrases were introduced into a hematopoietic stem cell along with a gene editor polypeptide (i.e., mRNA encoding a gene editor polypeptide (i.e., a Cas9 nickase fused to a reverse transcriptase)); an atgRNA comprising a spacer sequence a scaffold, an RT template comprising an integration recognition site, and a primer binding site; and a donor polynucleotide template comprising a cognate integration recognition site.
  • a gene editor polypeptide i.e., mRNA encoding a gene editor polypeptide (i.e., a Cas9 nickase fused to a reverse transcriptase)
  • an atgRNA comprising a spacer sequence a scaffold, an RT template comprising an integration recognition site, and a primer binding site
  • a donor polynucleotide template comprising a cognate integration recognition site.
  • the gene editor polypeptide i.e., mRNA encoding a gene editor polypeptide
  • the atgRNA were introduced into a cell using a first electroporation and the engineered integrase and the donor polynucleotide template (mini circle) were introduced into the cell using a second electroporation.
  • Engineered integrases used in these experiments are as shown in FIG. 32A
  • Genomic DNA was harvested 3 days after transduction.
  • Data for programmable gene insertion (PGI) is shown in FIG. 32B. This data shows that a BxB 1 integrase engineered to include a stabilon motif improves PGI by 3-5 fold. 6.17.
  • BxBl was engineered to include a stabilon domain or an Exin21 domain (see, e.g., engineered integration enzymes shown in FIG. 33 (see also Table 26)).
  • the engineered integration enzymes described in FIG. 33 were introduced into a HEK293 cell along with a gene editor polypeptide (i.e., mRNA encoding a gene editor polypeptide (i.e., a Cas9 nickase fused to a reverse transcriptase)); an atgRNA comprising a spacer sequence with sequence complementarity to a sequence in the human factor IX locus, a scaffold, an RT template comprising an integration recognition site, and a primer binding site (see Table 19 above for atgRNA sequences); and a donor polynucleotide template comprising a cognate integration recognition site.
  • the data is shown in FIG. 34.
  • the sub-set of engineered BxBl integrases shown in FIG. 35 were selected for further study.
  • BxBl integrases were engineered to include an amino acid modification with the aim that the modification would improve stability (e.g., half-life).
  • FIG. 36A shows degron motifs and lysine residues that are candidate for amino acid modifications that may increase the stability of a BxBl integrase.
  • mRNAs encoding BxBl were optimized with the aim of improving stability (i.e., half-life of the mRNA) upon being introduced into a cell.
  • FIGs. 37A-37B show first generations attempts at optimizing BxB 1 RNA structure using the linear design algorithm comparing non-optimized mRNA encoding BxB 1 in FIG. 37A with optimized mRNA encoding BxBl in FIG. 37B.
  • FIGs. 38A-38B show first generations attempts at optimizing an RNA structure for RNA encoding nCas9-RT using the LinearDesign algorithm comparing non-optimized mRNA encoding nCas9-RT in FIG. 38A with optimized mRNA encoding nCsa9-RT in FIG. 38B.
  • nCas9-RTs in FIGs. 38A-38B were tested for their ability to insert integration recognition sites into a genome at the human factor IX locus.
  • the nCas9-RTs in FIGs. 38A-38B were introduced into a cell along with an atgRNA comprising a spacer sequence with sequence complementarity to a sequence in the human factor IX locus, a scaffold, an RT template comprising an integration recognition site, and a primer binding site (see Table 19 above in Example 11 for atgRNA sequences).
  • Beacon placement data is shown in FIG. 39.
  • Fusion proteins comprising an nCas9-RT are fused with engineered integration enzymes (e.g., BxBl) and tested for their ability to mediate PGI.
  • engineered integration enzymes e.g., BxBl
  • fusion proteins included those as shown in FIG. 40 (see also Table 26). These fusions proteins are introduced into cells along with an atgRNA and a donor polynucleotide template. Genomic DNA is harvested three days after transduction.
  • Fusion proteins comprising a nCas9 and RT are fused with engineered integration enzymes (e.g., BxBl) in various combinations and tested for their ability to mediate PGI.
  • engineered integration enzymes e.g., BxBl
  • fusion proteins included those as shown in FIG. 41 (see also Table 26). These fusions proteins are introduced into cells along with an atgRNA and a donor polynucleotide template. Genomic DNA is harvested three days after transduction.
  • a non-limiting exemplary workflow is as described in FIG. 30A.
  • the engineered integrases were introduced into a pluripotent stem cell clone 52 or clone 17 along and a donor polynucleotide template comprising a cognate integration recognition site (PL1113 (SEQ ID NO: 591; see Table 22) or PL2134.
  • iPSC clones 52 and 17 include beacons site-specifically integrated into their genomes.
  • Genomic DNA was harvested 3 days after transduction.
  • Digital droplet PCR (ddPCR) data for programmable gene insertion (PGI) with PL1113 donor at day 3 is shown in FIG. 43 for the indicated engineered BxBl polypeptides.
  • Flow cytometry data for PGI with PL1113 donor at day 7 is shown in FIGs. 44A and 44B.
  • FIG. 44A provides a comparison between percent PGI between ddPCR data and flow cytometry data.
  • Flow cytometry was used to assess GFP expression, which indicates programmable gene insertion (PGI).
  • FIG. 45 PGI with PL2134 donor was also assessed and compared to PGI with PL1113. This data shows that PGI was higher with PL1113 than PL2134.
  • the engineered integration enzymes fused to a gene editor polypeptide are referred to herein as the all-in-one constructs.
  • mRNA encoding the engineered integrases from FIG. 46 were introduced into a primary human hepatocytes (line HU8403) along with a pair of atgRNA (AA089: atgRNAl is SEQ ID NO: 611 and atgRNA2 is SEQ ID NO: 612 (see Table 24)). Electroporation was performed as described elsewhere herein (see Example 22). mRNA was electroporated at either 187 fmol or 374 fmol. Controls included mRNA encoding nCas9-RT with and without a stabilization domain and a mRNA encoding RT-IRES-nCas9.
  • Genomic DNA was harvested 4 days after electroporation.
  • Digital droplet PCR (ddPCR) was used to assess beacon placement for each of the constructs tested (see FIG. 46).
  • ddPCR data for beacon placement revealed for the all-in-one mRNAs were slightly lower than the control (PL883) but were comparable.
  • the nCas9-RT-Bxbl orientation produced more efficient beacon placement compared to the Bxbl-nCas9-RT orientation (see FIG. 47).
  • mRNA encoding PL1931 (SEQ ID NO: 631 (amino acid) and SEQ ID NO: 632 (nucleic acid); see FIG. 46) was introduced into a primary human hepatocyte (line HU8403) along with a pair of atgRNA (AA115 (atgRNAl (SEQ ID NO: 613) and atgRNA2 (SEQ ID NO: 614); see Table below) using MessengerMax lipofection.
  • AA115 atgRNAl
  • SEQ ID NO: 614 atgRNA2
  • MessengerMax lipofection At day 0, 33,000 HU8403 cells were plated per well and MessengerMax, 0.15 pl atgRNA pair, and either 0.15 l or 0.3 pl of various concentrations of Bxbl mRNA (see FIG. 48).
  • beacon placement was similar between mRNA PL1931 and mRNA PL883 at equimolar input.
  • PGI in PHH was compared following lipofection with an mRNA encoding PL1931 (“all-in-one”) (SEQ ID NO: 631 (amino acid) and SEQ ID NO: 632 (nucleic acid)); see FIG. 46) or two mRNAs one encoding nCas9-RT and a second encoding BxB 1.
  • mRNA was introduced into PHH line HU8403 along with a pair of atgRNA (AA115) using MessengerMax lipofection.
  • BxBl was introduced at 210 fmol (or -125 ng) (note specific constructs included: PL1323 (cmyc-NLS-no stabilon), PL1325 (SV40-NLS- Stabilon), and PL 1709 (SV40-exin21 -stabilon).
  • a 3 lipoplex approach was used: guides 0.15pl per well (20 mM) with 0.15 pl MessengerMax (or 30 pl MessengerMax); BxBl mRNA at 250 ng with 0.15 pl MessengerMax (or 30 pl MessengerMax) per well; and nCas9- RT mRNA PL883 (SEQ ID NO: 617) fixed at 400 ng or 187 fmol with 0.15 pl MesserngerMax (or 30 pl MessengerMax) per well.
  • Genomic DNA was harvested 4 days after lipofection.
  • Digital droplet PCR (ddPCR) was used to assess PGI. As shown in FIG. 49, PGI was around 1% for each of the conditions tested. Looking closer at each PGI event, FIG. 50 and FIG.
  • FIG. 51 show data for total edits (AttB + AttL). Analysis of whether MessengerMax volume impacted beacon placement revealed that increased MessengerMax volume decreased beacon placement but increased integration (FIG. 52). Analysis of the 3 lioplex approach in FIG. 53, revealed a 20-30% hit to beacon placement ratio observed with integration samples (3 lipo) compared to beacon only samples (2 lipo). This data shows that mRNAs encoding a BxBl-stabilon result in increased beacon occupancy compared to mRNA encoding BxBl without a stabilon (see PL1323 (SEQ ID NO: 618 (amino acid) and SEQ ID NO: 619 (nucleic acid)).
  • BxB 1 was engineered to include a stabilon peptide motif and/or an Exin21 peptide motif. See FIGs. 54, 57, 59, 61, and 63, see also Table 27.
  • the engineered integration enzyme constructs described in FIG. 54, 61, and 63 were introduced into HEK293 mediating integration of a donor polynucleotide (cargo) having an integration site cognate to the integration site in the target genome. Recombination site attL was produced and percent integration was measured by ddPCR. The data is provided in FIG. 55 and FIG. 64. The results showed that various engineered bxbl integrases showed significant improved percent integration as compared to others. Percent integration was measured by ddPCR.
  • beacon placement was performed in neonatal mice. Beacon placement was conducted using methods such as those already described in this text. For example, see Example 9. Next, we performed integration of cargo into the genome of the cell using methods previously described herein this document. The results showed that various Bxbl integrases displayed significant beacon conversion compared to others. See FIG. 56. Beacon conversion was measured using ddPCR.
  • PGI as measured by beacon conversion mediated by the engineered integration enzymes was assessed in primary human hepatocytes (PHH, line HU8403). Data is shown in FIG. 58 and FIG. 60. See also Table 26 and 27. Results showed that various engineered BxBl constructs resulted in increased integration of the donor polynucleotide compared to the other engineered BxB 1 polypeptides.
  • FIG. 62A shows percent integration resulting in recombination site attL.
  • FIG. 62B shows percent integration resulting in recombination site attR.

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Abstract

La présente divulgation concerne des compositions comprenant des enzymes d'intégration modifiées et leurs procédés d'utilisation. Dans certains modes de réalisation, l'enzyme d'intégration modifiée comprend un domaine de stabilisation qui augmente la stabilité de l'enzyme d'intégration par comparaison avec des enzymes d'intégration ne comprenant pas le domaine de stabilisation. Dans d'autres modes de réalisation, l'enzyme d'intégration modifiée comprend au moins une première modification d'acide aminé qui augmente la stabilité de l'enzyme d'intégration par comparaison avec une enzyme d'intégration qui ne comprend pas la modification d'acide aminé.
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