WO2025224182A2 - Plate-forme de construction unique pour l'administration simultanée d'une machinerie d'édition de gènes et d'une cargaison d'acide nucléique - Google Patents

Plate-forme de construction unique pour l'administration simultanée d'une machinerie d'édition de gènes et d'une cargaison d'acide nucléique

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Publication number
WO2025224182A2
WO2025224182A2 PCT/EP2025/061088 EP2025061088W WO2025224182A2 WO 2025224182 A2 WO2025224182 A2 WO 2025224182A2 EP 2025061088 W EP2025061088 W EP 2025061088W WO 2025224182 A2 WO2025224182 A2 WO 2025224182A2
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nucleic acid
atgrna
acid construct
integration
hdad
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WO2025224182A3 (fr
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Yijun Zhang
Jonathan Douglas FINN
Sandeep Kumar
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Basecamp Research Ltd
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Basecamp Research Ltd
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Publication of WO2025224182A3 publication Critical patent/WO2025224182A3/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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • 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 [RNase]; Deoxyribonucleases [DNase]
    • 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 [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • 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
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • helper-dependent adenovirus can be used to efficiently deliver the components for site-specifically integrating an exogenous nucleic acid into a cellular genome at a desired target sequence. This avoids having to use multiple steps to deliver the reagents, which can negatively impact efficiency.
  • the present disclosure provides reagents for deliving the components for use in site-specific genetic engineering using Programmable Addition via Site- Specific Targeting Elements (PASTE) (see Ionnidi et al.; Nat.
  • the integrase may be directly linked, for example by a peptide linker, to the prime editor fusion or gene writer protein.
  • the nucleic acid construct described herein can be used to introduce, delete, or delete and introduce large pieces of DNA (as well as small pieces of DNA) to any genomic site in any organism.
  • a single nucleic acid construct is described herein that allows for incorporation of any template into any DNA locus using DNA delivery of a single component DNA. Additionally, a physical portion of the nucleic acid construct is capable of self-circularizing, forming a circular construct that contains a DNA template.
  • the nucleic acid construct can be packaged and delivered in any viral or non-viral delivery vector including a recombinant adenovirus, helper dependent adenovirus, AAV, HSV, annelovirus, retrovirus, lentivirus, DoggyboneTM DNA (dbDNATM), minicircle, plasmid, miniDNA, LNP, or nanoplasmid. Delivery of the nucleic acid construct can also be by fusosome or exosome, (See, e.g., WO2019222403 which is incorporated by reference herein). Delivery of nucleic acid construct can also be by VesiCas (See, e.g., US20210261957A1 which is incorporated by reference herein).
  • the present disclosure provides a nucleic acid construct, comprising: a polynucleotide encoding an integration enzyme, wherein the polynucleotide comprises an intron, whereby the intron prevents the expression of the integration enzyme in prokaryotic cells.
  • the nucleic acid construct further comprises: (a) a polynucleotide encoding a gene editor polypeptide; (b) a polynucleotide encoding a first attachment site-containing guide RNA (atgRNA), wherein the first atgRNA comprises a sequence encoding at least a portion of a first integration recognition site recognizable by the encoded integration enzyme; and (c) a polynucleotide encoding a second atgRNA, wherein the second atgRNA comprises a sequence encoding at least a portion of the first integration recognition site.
  • atgRNA attachment site-containing guide RNA
  • the first atgRNA and the second atgRNA have an overlap of 20 bp or less at the sequence encoding at least a portion of the first integration recognition site.
  • the nucleic acid construct further comprises a cargo polynucleotide linked to a polynucleotide encoding a second integration recognition site recognized by the encoded integrase, wherein the second integration recognition site is a cognate of the first integration recognition site.
  • the present disclosure further provides a nucleic acid construct, comprising: (a) a polynucleotide encoding a gene editor polypeptide; (b) a polynucleotide encoding a first attachment site-containing guide RNA (atgRNA), wherein the first atgRNA comprises a sequence encoding at least a portion of a first integration recognition site recognizable by the encoded integration enzyme; (c) a polynucleotide encoding a second atgRNA, wherein the second atgRNA comprises a sequence encoding at least a portion of the first integration recognition site; (d) a polynucleotide encoding an integration enzyme, wherein the polynucleotide comprises an intron; and (e) a cargo polynucleotide linked to a polynucleotide encoding a second integration recognition site, wherein the second integration recognition site is a cognate of the first integration recognition site.
  • atgRNA attachment site-containing guide RNA
  • the nucleic acid construct is part of a recombinant viral genome.
  • the viral construct is an adenoviral construct.
  • the adenoviral construct is a helper-dependent adenoviral (HDAd) construct.
  • the present disclosure further provides a method of making a helper-dependent adenovirus (HDAd) virion, comprising: (a) introducing into a cell line suitable for making HDAd: (i) the nucleic acid construct or the HDAd virion of the present disclosure and (ii) a helper virus (HV); (b) culturing the cell line under conditions suitable for encapsidating the HDAd genome into the HDAd virion capsid; (c) collecting the HDAd; and (d) purifying the HDAd, wherein the HDAd genome is not recombined in the HDAd virion.
  • a helper-dependent adenovirus virion comprising: (a) introducing into a cell line suitable for making HDAd: (i) the nucleic acid construct or the HDAd virion of the present disclosure and (ii) a helper virus (HV); (b) culturing the cell line under conditions suitable for encapsidating the HD
  • the present disclosure further provides an engineered cell line suitable for making HDAd, wherein the cell line is engineered to prevent the expression of an integration enzyme or to inactivate an integration enzyme.
  • the present disclosure further provides a method for site-specifically integrating an exogenous nucleic acid into a genome of a cell at a target sequence, the method comprising: (a) introducing into the cell a nucleic acid construct or the HDAd virion of the present disclosure; and culturing the cell under conditions sufficient to activate the first expression control sequence, thereby driving expression of the integration enzyme.
  • the present disclosure further provides an in vivo method for site-specifically integrating an exogenous nucleic acid into a cellular genome at a target sequence, the method comprising: administering to a subject the HDAd virion of present disclosure and an agent, wherein the agent activates the first expression control sequence, thereby allowing expression of the integration enzyme.
  • the present disclosure further provides an in vivo method for site- specifically integrating an exogenous nucleic acid into a cellular genome at a target sequence, the method comprising: administering to a subject the HDAd virion of present disclosure in the absence of an agent, wherein the agent inhibits the first expression control sequence, thereby allowing expression of the integration enzyme. 5.
  • FIG. 1 illustrates a single construct that contains a prime editor fusion protein or gene writer protein, the attachment site-containing guide RNA (atgRNA), a nickase guide RNA (ngRNA), an integrase, a recombinase, recombination target sites, integration target site, a DNA of interest, and flanking ITRs.
  • atgRNA attachment site-containing guide RNA
  • ngRNA nickase guide RNA
  • integrase a recombinase
  • recombination target sites integration target site
  • integration target site a DNA of interest
  • flanking ITRs flanking ITRs
  • FIG. 2 illustrates a single construct that contains a prime editor fusion protein or gene writer protein, the attachment site-containing guide RNA (atgRNA), a nickase guide RNA (ngRNA), an integrase, integration target sites, a DNA of interest, and flanking ITRs. Integrase expression leads to self-circularization of a subsequence of the single nucleic acid construct.
  • the integrase may be directly linked or fused to the prime editor protein or Gene Writer and expression driven from a single promoter.
  • Self-circularization occurs at an integrase recognition target sequence (attB2/attP2). Additionally, a DNA of interest contained within the self-circularized nucleic acid is capable of being integrated into a genomic locus of interest via the integrase at an orthogonal integration target site (i.e., cognate pairs (e.g., attP1/attB1)).
  • orthogonal integration target site i.e., cognate pairs (e.g., attP1/attB1)
  • Initial self- circularization, prior to genomic integration is achieved via the use of att integrase recognition target sites (i.e., attB2/attP2 and attP1/attB1) that are cognate pairs.
  • the orthogonal integrase sites display an integrase-mediated recombination rate difference to allow for template/cargo circularization prior to genomic integration.
  • FIGs. 3A-3E show multiplex and orthogonal gene insertion with PASTE.
  • FIG. 3A shows a schematic of AttP mutations tested for improving integration efficiency (SEQ ID NOS 394 and 540-542, respectively, in order of appearance).
  • FIG. 3B shows integration efficiencies of wildtype and mutant AttP sites across a panel of AttB lengths.
  • FIG. 3C shows a 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. 3A shows a schematic of AttP mutations tested for improving integration efficiency (SEQ ID NOS 394 and 540-542, respectively, in order of appearance).
  • FIG. 3B shows integration efficiencies of wildtype and mutant AttP sites across a panel of AttB lengths.
  • FIG. 3C shows a schematic of multiplex
  • FIG. 3D shows orthogonality of top 4 AttB/AttP dinucleotide pairs evaluated for GFP integration with PASTE at the ACTB locus.
  • FIGs. 4A-4E show additional characterization of AttP mutants for improved editing and multiplexing.
  • FIG. 4A shows AttP single mutants are characterized for PASTE EGFP integration at the ACTB locus.
  • FIG. 4B shows characterization of integration of a 5 kb payload at the ACTB locus with all 16 possible dinucleotides for AttB/AttP pairs between the atgRNA and minicircle.
  • FIG. 4C shows a schematic of the pooled AttB/AttP dinucleotide orthogonality assay. Each AttB dinucleotide sequence is cotransfected with a barcoded pool of all 16 AttP dinucleotide sequences and BxbINT, and relative integration efficiencies are determined by next generation sequencing of barcodes. All 16 AttB dinucleotides are profiled in an arrayed format with AttP pools.
  • FIG. 1 shows characterization of integration of a 5 kb payload at the ACTB locus with all 16 possible dinucleotides for AttB/AttP pairs between the atgRNA and minicircle.
  • FIG. 4C shows a schematic of the pooled AttB/AttP
  • FIG. 4D illustrates relative insertion preferences for all possible AttB/AttP dinucleotide pairs determined by the pooled orthogonality assay.
  • FIG. 4E shows orthogonality of BxbINT dinucleotides as measured by a pooled reporter assay. Each web logo motif shows the relative integration of different AttP sequences in a pool at a denoted AttB sequence with the listed dinucleotide.
  • FIG. 5 illustrates a schematic of single atgRNA and dual atgRNA approaches for beacon placement.
  • FIG. 5 illustrates a schematic of single atgRNA and dual atgRNA approaches for beacon placement.
  • FIG. 6 illustrates the six different C-terminus to N-terminus arrangements (C-to-N) of exemplary nucleic acid programmable DNA binding proteins (napDNAbp), the RT, and the integrase is be fused or linked.
  • FIG. 7 illustrates the extrachromosomal circular DNA (EccDNA) sensor assay to detect template circularization, beacon placement, and gene insertion.
  • AttP AttP (GT) for genome insertion.
  • AttB’-AG and AttP’-AG at both ends for circularization in presence of Bxb1.
  • EF1a promoter will drive NanoLuc and GFP expression. Screen for efficient di-nucleotides and configuration.
  • FIG. 8 illustrates transfection screening conditions for circularization detection and ACTB beacon placement and gene insertion.
  • FIG. 9 illustrates EccDNA ddPCR analysis.
  • FIG. 10 illustrates EccDNA ddPCR analysis with PE2, atgRNA, ngRNA components co-transfected.
  • FIG. 11 illustrates ACTB beacon placement analysis. [0029] FIG.
  • FIG. 12 illustrates EccDNA ACTB gene insertion analysis at a placed beacon.
  • FIG. 13 illustrates transfection screening conditions for circularization detection and LMNB beacon placement and gene insertion.
  • FIG. 14 illustrates in cell EccDNA circularization detection by GFP detection.
  • FIG. 15 illustrates EccDNA ddPCR analysis.
  • FIG. 16 illustrates EccDNA LMNB beacon placement analysis.
  • FIG. 17 illustrates LMNB gene insertion analysis at a placed beacon.
  • FIG. 18A shows an expression cassette of a helper dependent adenovirus (HDAd).
  • HDAd helper dependent adenovirus
  • PASTE components include: a promoter (e.g., a constitutive promoter) driving expression of a gene editor polypeptide (e.g., a nCas9-RT), an inducible promoter driving expression of a integration enzyme (e.g., a BxB1 integrase), a first U6 promoter driving expression of a first atgRNA, a second U6 promoter driving expression of a second atgRNA (where the first atgRNA and atgRNA encode a first integration recognition site), and a donor DNA sequence comprising at least a second integration recognition site, a promoter driving expression of a transgene.
  • a promoter e.g., a constitutive promoter
  • a gene editor polypeptide e.g., a nCas9-RT
  • an inducible promoter driving expression of a integration enzyme e.g., a BxB1 integrase
  • FIG. 18B shows a schematic identifying a first polynucleotide segment, a second polynucleotide segment, and a third polynucleotide segment.
  • the first polynucleotide segment includes a promoter driving expression of a gene editor polypeptide (“PE2”) and a first U6 promoter driving expression of a first atgRNA (atgR) and a second U6 promoter driving expression of a second atgRNA (atgF), where the atgR and atgF collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is integrated into the genome of the cell at the target sequence.
  • PE2 gene editor polypeptide
  • the second polynucleotide segment includes a promoter driving expression of an integration enzyme (e.g., a BxB1 integrase).
  • the third polynucleotide segment includes a transgene and a second integration recognition site that is a cognate of the first integration recognition site. After integration, the BxB1 mediates integration using the first and second integration recognition sites.
  • FIG. 18C shows a schematic identifying a first polynucleotide segment, a second polynucleotide segment, and a third polynucleotide segment.
  • the first polynucleotide segment includes a promoter driving expression of a gene editor polypeptide (“PE2”).
  • PE2 gene editor polypeptide
  • the first polynucleotide segment also includes a first U6 promoter driving expression of a first atgRNA (atgR), a second U6 promoter driving expression of a second atgRNA (atgF), a third U6 promoter driving expression of a third atgRNA (atgR), and a fourth U6 promoter driving expression of a fourth atgRNA (atgfF).
  • the first atgRNA and the second atgRNA collectively encode the entirety of a first integration recognition site and the third atgRNA and the fourth atgRNA collectively encode the entirety of a second integration recognition site.
  • the second polynucleotide segment includes a promoter driving expression of an integration enzyme (e.g., a BxB1 integrase).
  • the third polynucleotide segment includes a transgene and a third integration recognition site and a fourth integration recognition site.
  • the third integration recognition site is a cognate pair with the first integration recognition site
  • the fourth integration recognition site is a cognate pair with the second integration recognition site.
  • the BxB1 mediates integration using the first and third integration recognition sites as a first cognate pair and the second and fourth integration recognition sites as a second cognate pair.
  • HV helper virus
  • HV helper virus
  • P0 refers to the “rescue” of HDAd vector, which was conducted by transfecting HEK293Cre cells with linearized HDAd plasmid and then infecting cells with helper virus to start the amplification of HDAd.
  • P1 to P6 indicate the passage number after P0.
  • HDAd from cell lysate of previous passage and helper virus (HV) were used to co-infect HEK293Cre cells to amplify HDAd.
  • FIG. 21 shows quality control data from HDAd virus production (HDAd-AIO (hF9) virus).
  • FIG. 22 shows restriction enzyme digestion of viral DNA from an HDAd production. Digestion patterns serve as an indication of intra-molecular recombination. Left panel shows DNA ladder. Middle panel shows agarose gel of digested viral DNA.
  • FIG. 23 shows PCR data of viral DNA from an HDAd production. PCR primers were designed to amplify individual components within the HDAd (see, e.g., FIGs. 18A-18B).
  • FIG.24 shows representative images of HDAd infection of HEK293A at 48 hours after infection. HEK293A were infected with HDAd at MOIs of 10, 100, and 1000. A no infection control was used as a negative control. [0044] FIGs.
  • FIG. 25A-25C shows data for intramolecular recombination between atgRNA attB and cargo attP.
  • FIG. 25A shows a schematic of an intramolecular recombination in a HDAd.
  • FIG. 25B shows a representative graphic summary of the outcome intramolecular recombination with the resulting deletion of the sequences encoding RT, Cas9, BxB1, rTA, and mScarlet.
  • FIG. 25C shows a sequence alignment of the query sequence (HDAd vector sequence without recombination “AdVG012 ref”) and the subject (HDAd vector sequence with recombination “AdVG012 UI vDNA”).
  • Figure discloses SEQ ID NOS 584-587, respectively, in order of appearance.
  • FIG. 26 shows a sequence alignment of the results of sanger sequencing of viral DNA and intramolecular recombination between atgRNA AttB (first integration recognition site) and cargo AttP (second integration recognition site).
  • Figure discloses SEQ ID NOS 588-589, and 426, respectively, in order of appearance.
  • FIGs. 27A-27C shows beacon placement, PGI, and beacon occupancy data in HEK293A cells infected with HDAd-AIO (hF9).
  • FIG. 27A shows beacon placement in HEK293A at days 3 and day 6 after infection with HDAd-AIO (hF9).
  • FIG. 27B shows PGI in HEK293A at days 3 and day 6 after infection with HDAd-AIO (hF9).
  • FIG. 27C shows beacon occupancy in HEK293A at days 3 and day 6 after infection with HDAd-AIO (hF9).
  • FIG. 28A-28B shows beacon placement and PGI data in HEK293-attB cells infected with HDAd-AIO (hF9).
  • FIG. 28A shows beacon placement in HEK293-attB at days 3 and day 6 after infection with HDAd-AIO (hF9).
  • FIG. 28B shows PGI in HEK293-attB at days 3 and day 6 after infection with HDAd-AIO (hF9).
  • FIG. 29A-29D shows PGI data in HEK293-lenti-AttB cells infected with HDAd-AIO (hF9).
  • FIG. 29A shows ddPCR data for PGI in HEK293-lenti-attB at days 3 and day 6 after infection with HDAd-AIO (hF9).
  • FIG. 29B shows raw ddPCR data for PGI primer set and Ref- RRE control.
  • FIG. 29C shows a schematic of an intramolecular recombination in a HDAd.
  • FIG. 29D shows raw ddPCR data for attB-attP recombination primers (see FIG. 29C) and the RNaseP Ref control.
  • FIG. 29A shows ddPCR data for PGI primer set and Ref- RRE control.
  • FIG. 30 shows a schematic that illustrates the competition between intramolecular recombination (i.e., recombination between the AttB and the AttP sites present in the nucleic acid construct) and recombination between an integrated AttB and the AttP site in the nucleic acid construct.
  • FIG. 31 shows the schematics of HDAd AIO plasmids with chimeric Bxb1-intron as compared to HDAd vector AdVG012, which include Bxb1 without intron.
  • the HDAd vector AdVG068 includes a Bxb1 with a chimeric intron, under the control of a Tet-off promoter.
  • FIGs. 32A and 32B show the recombination between attB site and attP site as measured by ddPCR, with FIG. 32A showing the percentage of AIO plasmid recombination and FIG. 32B showing raw ddPCR data with AttL recombination primers and the Nanoluc control. [0052] FIGs.
  • FIGs. 33A and 33B show the percentages of beacon placement (BP) and programmable gene insertion (PGI) achieved with HDAd vector AdVG102, with FIG. 33A showing the percentage BP and FIG. 33B showing the percentage PGI.
  • FIGs. 34A and 34B show the percentages of beacon placement (BP) and programmable gene insertion (PGI) achieved with HDAd vector AdVG092, with FIG. 34A showing the percentage BP and FIG. 34B showing the percentage PGI.
  • FIGs. 35A and 35B show the percentages of beacon placement (BP) and programmable gene insertion (PGI) achieved with HDAd vector AdVG093, with FIG. 35A showing the percentage BP and FIG. 35B showing the percentage PGI.
  • FIG. 35A and 35B show the percentages of beacon placement (BP) and programmable gene insertion (PGI) achieved with HDAd vector AdVG093, with FIG. 35A showing the percentage BP and FIG. 35B showing the percentage PGI.
  • FIG. 36 shows beacon placement (BP) efficiency with HDAd vectors AdVG102, AdVG092, and AdVG093.
  • FIG. 37 shows programmable gene insertion (PGI) efficiency with HDAd vectors AdVG102, AdVG092, and AdVG093.
  • FIG. 38 shows the Bxb1-shRNA design for producing the Bxb1 knockdown cell line. Figure discloses SEQ ID NOS 590-592, respectively, in order of appearance.
  • FIGs. 39A and 39B show the Bxb1 knockdown efficiency of 116 Bxb1-shRNA cell clones, with FIG. 39A showing the Bxb1 knockdown efficiency as determined by Western blot and FIG.
  • FIG. 40 shows the expression of the HDAd AIO reporter gene mScarlet in the 116 Bxb1-shRNA cell line and the control 116 cells without Bxb1-shRNA.
  • FIG. 41 shows the purity of virus with intact genome, measured as ratio of intact virus over total virus. The intact virus titer and the total virus titer were measured by ddPCR targeting mScarlet and Nanoluc respectively on PHH sample.
  • FIGs 42A and 42B show the toxicity and transduction efficiency with the HDAd AIO construct AdVG012 at different titers, with FIG.
  • FIGs 43A and 43B show the gene editing efficiency of HDAd AIO construct AdVG012, with FIG.43A showing the percentages of beacon placement (BP) and attL integration (PGI) and FIG. 43B showing the percentage occupancy. The percentage occupancy was calculated as PGI%/(BP%+PGI%). 6.
  • This disclosure features a nucleic acid construct, for example, helper-dependent adenovirus (HDAd), that can be used to deliver all the components for site-specifically integrating an exogenous nucleic acid into a cellular genome at a desired target sequence.
  • HDAd helper-dependent adenovirus
  • This avoids having to use multiple steps to deliver the reagnets, which can negatively impact efficiency.
  • intramolecular recombination is likely and has a high probability of disrupting HDAd virus production. Careful consideration is needed to avoid intramolecular recombination.
  • this disclosure features a nucleic acid construct (e.g., an HDAd vector) comprising: a first polynucleotide segment that includes a gene editor polynucleotide encoding a gene editor polypeptide, 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), where the first atgRNA and the second atgRNA collectively encode a first integration recognition site; a second polynucleotide segment comprising a polynucleotide sequence encoding an integration enzyme, and a third polynucleotide segment comprising a polynucleotide sequence encoding
  • Gene editor is a protein that that can be used to perform gene editing, gene modification, gene insertion, gene deletion, or gene inversion.
  • gene editor polynucleotide refers to polynucleotide sequence encoding the gene editor protein.
  • the gene editor comprises DNA- or RNA-targetable nuclease protein (i.e., Cas protein) wherein target specificity is mediated by a complexed nucleic acid (i.e., guide RNA).
  • the gene editor is a DNA-targetable or RNA-targetable protein in which target specificity is mediated by internal, conjugated, fused, or linked amino acids, such as within TALENs, ZFNs, or meganucleases.
  • the gene editor can demonstrate targeted nuclease activity, targeted binding with no nuclease activity, targeted nickase activity (or cleavase activity).
  • a gene editor comprising a targetable protein may be fused, linked, complexed, operate in cis or trans to one or more proteins or protein fragment motifs. Gene editors may be fused or linked to one or more integrase, recombinase, polymerase, telomerase, reverse transcriptase, or invertase. A gene editor can be an alpha editor fusion protein or a gene writer fusion protein. [0066] Base editors such as ADAR or ADAT may be delivered as cargo in various embodiments, as described herein. [0067] “Alpha editor protein or fusion protein” as used herein, is a gene editor protein.
  • Alpha editor system as used herein describes the components used in alpha editing and alpha editing and alpha editor are used interchangeably herein with the terms “prime editing or prime editor (PE).
  • Alpha editing uses a CRISPR protein, which has enzymatic activity that nicks or cuts only single strand of double stranded DNA, i.e., a nickase; the nickase can occur either naturally or by mutation or modification of a nuclease that makes double stranded cuts.
  • Prime editing (PE) and prime editing components (pegRNA) can be utilized as well.
  • the nickase is programmed (targeted) with an alpha-editing guide RNA (aeg RNA or a pegRNA).
  • the pegRNA both specifies the target site and encodes the desired edit.
  • Attachment site containing guide RNA (atgRNA) that both specifies the target and encodes for the desired integrase target recognition site are provided.
  • the nickase may be programmed (targeted) with an atgRNA.
  • the nickase is a catalytically impaired Cas9 endonuclease, i.e., a Cas9 nickase, that is fused to a reverse transcriptase.
  • the reverser transcriptase can also be provided in the alpha editor system split from the Cas9 nickase.
  • the Cas9 nickase part of the protein is guided to the DNA target site by the atgRNA (or pegRNA), whereby a nick or single stranded cut occurs.
  • the reverse transcriptase domain then uses the atgRNA (or 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 alpha editor (AE) guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process (typically achieved with a nickase gRNA).
  • cleavase e.g., cleavase I enzyme
  • an additional agent or agents may be added that improve the efficiency and outcome purity of the alpha or prime edit.
  • the agent may be chemical or biological and disrupt DNA mismatch repair (MMR) processes at or near the edit site (i.e., PE4 and PE5 and PEmax architecture by Chen et al. Cell, 184, 1-18, October 28, 2021; Chen et al. is incorporated herein by reference).
  • MMR DNA mismatch repair
  • the agent is a MMR-inhibiting protein.
  • the MMR-inhibiting protein is dominant negative MMR protein. In certain embodiments, the dominant negative MMR protein is MLH1dn. In particular embodiments, the MMR-inhibiting agent is incorporated into the multicomponent delivery method described herein. In some embodiments, the MMR-inhibiting agent is linked or fused to the alpha editor protein fusion, which may or may not have a linked or fused integrase. In some embodiments, the MMR-inhibiting agent is linked or fused to the Gene WriterTM protein, which may or may not have a linked or fused integrase. [0069] The alpha editor or gene editor system can be used to achieve DNA deletion and replacement.
  • the DNA deletion replacement is induced using a pair of atgRNAs or pegRNA that target opposite DNA strands, programming not only the sites that are nicked but also the outcome of the repair (i.e., PrimeDel by Choi et al. Nat. Biotechnology, October 14, 2021; Choi et al. is incorporated herein by reference and TwinPE by Anzalone et al. BioRxiv, November 2, 2021; Anzalone et al. is incorporated herein by reference).
  • the DNA deletion is induced using a single atgRNA.
  • the DNA deletion and replacement is induced using a wild type Cas9 alpha or prime editor (AE-Cas9 or PE-Cas9) system (i.e., PEDAR by Jiang et al. Nat. Biotechnology, October 14, 2021; Jiang et al. is incorporated herein by reference in its entirety).
  • the DNA replacement is an integrase target recognition site or recombinase target recognition site.
  • the constructs and methods described herein may be utilized to incorporate the pair of pegRNAs (or atgRNAs) used in PrimeDel, TwinPE (WO2021226558 incorporated by reference herein in its entirety), or PEDAR, the alpha editor fusion protein or Gene Writer protein, optionally a nickase guide RNA (ngRNA), an integrase, a nucleic acid cargo, and optionally a recombinase into a LNP delivery system or vector delivery system (e.g., AAV or Adenovirus).
  • the integrase may be directly linked, for example by a peptide linker, to the prime editor fusion or gene writer protein.
  • the prime editors can refer to a retrovirus or lentivirus reverse transcriptase such as a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (RT) fused to a CRISPR enzyme nickase such as a Cas9 H840A nickase, a Cas9nickase.
  • the prime editors can refer to a retrovirus or lentivirus reverse transcriptase such as a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (RT) fused to a cleavase.
  • the RT can be fused at, near or to the C-terminus of a Cas9nickase, e.g., Cas9 H840A. Fusing the RT to the C-terminus region, e.g., to the C-terminus, of the Cas9 nickase may result in higher editing efficiency.
  • a complex is called PEI.
  • the CRISPR enzyme nickase e.g., Cas9(H840A), i.e., a Cas9nickase
  • the CRISPR enzyme nickase instead of being a Cas9 (H840A), i.e., instead of being a Cas9 nickase, the CRISPR enzyme nickase instead can be a CRISPR enzyme that naturally is a nickase or cuts a single strand of double stranded DNA; for instance, the CRISPR enzyme nickase can be Casl2a/b. Alternatively, the CRISPR enzyme nickase can be another mutation of Cas9, such as Cas9(Dl0A).
  • a CRISPR enzyme such as a CRISPR enzyme nickase, such as Cas9 (wild type), Cas9(H840A), Cas9(Dl0A) or Cas 12a/b nickase can be fused in some embodiments to a pentamutant of M-MLV RT (D200N/ L603W/ T330P/ T306K/ W313F), whereby there can be up to about 45-fold higher efficiency, and this is called PE2.
  • a CRISPR enzyme nickase such as Cas9 (wild type), Cas9(H840A), Cas9(Dl0A) or Cas 12a/b nickase
  • a pentamutant of M-MLV RT D200N/ L603W/ T330P/ T306K/ W313F
  • the M-MLV RT comprise one or more of the mutations Y8H, P51L, S56A, S67R, E69K, Vl29P, L139P, Tl97A, H204R, V223H, T246E, N249D, E286R, Q2911, E302K, E302R, F309N, M320L, P330E, L435G, L435R, N454K, D524A, D524G, D524N, E562Q, D583N, H594Q, E607K, D653N, and L671P. Specific M-MLV RT mutations are shown in Table 1.
  • the reverse transcriptase can also be a wild-type or modified transcription xenopolymerase (RTX), avian myeloblastosis virus reverse transcriptase (AMV RT), Feline Immunodeficiency Virus reverse transcriptase (FIV-RT), FeLV-RT (Feline leukemia virus reverse transcriptase), HIV-RT (Human Immunodeficiency Virus reverse transcriptase).
  • RTX transcription xenopolymerase
  • AMV RT avian myeloblastosis virus reverse transcriptase
  • FV-RT Feline Immunodeficiency Virus reverse transcriptase
  • FeLV-RT FeLV-RT
  • HIV-RT Human Immunodeficiency Virus reverse transcriptase
  • the reverse transcriptase can be a fusion of MMuLV to the Sto7d DNA binding domain (see Ionnidi et al.; https://doi.org/10.1101/2021.11.01.466786).
  • the fusion of MMuLV to the Sto7d DNA binding domain sequence is given in Table 2.
  • nicking guide RNA RNA
  • installer single nucleic acid construct
  • prime editors can be found in the following: WO2020/191153, WO2020/191171, WO2020/191233, WO2020/191234, WO2020/191239, WO2020/191241, WO2020/191242, WO2020/191243, WO2020/191245, WO2020/191246, WO2020/191248, WO2020/191249, each of which is incorporated by reference herein in its entirety.
  • the skilled person can incorporate the selected CRISPR enzyme, as part of the prime editor fusion or gene editor fusion, into a single nucleic acid construct (“installer”) described herein.
  • the prime editor protein (1) site- specifically targets a genomic locus and (2) performs a catalytic cut or nick. These steps are typically performed by a CRISPR-Cas.
  • the Cas protein may be substituted by other nucleic acid programmable DNA binding proteins (napDNAbp) such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or meganucleases.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • a Gene Writer can introduce novel DNA elements, such as an integration target site, into a DNA locus.
  • a Gene Writer protein comprises: (A) a polypeptide or a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain, and either (x) an endonuclease domain that contains DNA binding functionality or (y) an endonuclease domain and separate DNA binding domain; and (B) a template RNA comprising (i) a sequence that binds the polypeptide and (ii) a heterologous insert sequence. Examples of such Gene WriterTM proteins and related systems can be found in US20200109398, which is incorporated by reference herein in its entirety.
  • the prime editor or Gene Writer protein fusion or prime editor protein linked or fused to an integrase 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 single nucleic acid constructs described herein.
  • 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: Table 3. Table 3.
  • S equence (5’-3’) SEQ ID Amino acid sequence SEQ ID NO: NO: A - P2A GGAAGCGGAGCTACTAACTTC 5 GSGATNFSLLKQAG 13 AGCCTGCTGAAGCAGGCTGGC DVEENPGP GACGTGGAGG AGAACCCTGGACCT B - GGGGGAGGAGGTTCTGGAGGC 6 GGGGSGGGGSGGGG 14 (GGGS)3 GGAGG S CTCCGGAGGCGGAGGGTCA C - GGAGGTGGCGGGAGC 7 GGGGS 15 GGGGS D - CCCGCACCAGCGCCT 8 PAPAP 16 PAPAP E - GAGGCAGCTGCCAAGGAAGCC 9 EAAAKEAAAKEAAA 17 (EAAAK) GCTGCCAAGGAGGCGGCCGCA K 3 AAG Table 3.
  • S equence (5’-3’) SEQ ID Amino acid sequence SEQ ID NO: NO: F - XTEN AGTGGGAGCGAGACCCCTGGG 10 SGSETPGTSESATPES 18 ACTAGCGAGTCAGCTACACCCG AAAGC G - GGGGGGTCAGGTGGATCCGGC 11 GGSGGSGGSGGSGG 19 (GGS)6 GGAAGTGGCGGATCCGGTGGA SGGS TCTGGCGGCAGT H - GAAGCTGCTGCTAAG 12 EAAAK 20 EAAAK (GGGGS) GGCGGCGGCGGCAGCGGCGGC 4 GGCGGCAGCGGCGGCGGCGGC GGGGGGSGGGGSGGGG A GCGGCGGCGGCGGCAGC 543 SGGGGS 551 GGCGGCGCGAGCCCGGCGGGC PAS8 GGC 544 GGASPAGG 552 GGCGGCGCGAGCCCGGCGGCG PAS12 CCGGCCGGCGGGC 545 GGASPAAPAPAG 553 GCGGAAGCGGCGAAAGAAGCG
  • 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. 6 .2.
  • Type II CRISPR proteins [0079] The skilled person can incorporate a selected CRISPR enzyme, described below, as part of the prime editor fusion, into a single nucleic acid construct (“installer”) described herein.
  • SpCas9 Streptococcus pyogenes Cas9
  • REC recognition
  • NUC nuclease
  • the REC lobe can be divided into three regions, a long a helix referred to as the bridge helix (residues 60–93), the REC1 (residues 94–179 and 308–713) domain, and the REC2 (residues 180–307) domain.
  • the NUC lobe consists of the RuvC (residues 1–59, 718–769, and 909–1098), HNH (residues 775–908), and PAM-interacting (PI) (residues 1099–1368) domains.
  • the negatively charged sgRNA:target DNA heteroduplex is accommodated in a positively charged groove at the interface between the REC and NUC lobes.
  • 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.
  • REC lobe The REC lobe includes the REC1 and REC2 domains.
  • the REC2 domain does not contact the bound guide:target heteroduplex, indicating that truncation of REC lobe may be tolerated by SpCas9.
  • SpCas9 mutant lacking the REC2 domain (D175–307) retained ⁇ 50% of the wild-type Cas9 activity, indicating that the REC2 domain is not critical for DNA cleavage.
  • 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.
  • RuvC domain The RuvC nucleases of SpCas9 have an RNase H fold and four catalytic residues, Asp10 (Ala), Glu762, His983, and Asp986, that are critical for the two-metal cleavage of the noncomplementary strand of the target DNA.
  • the Cas9 RuvC domain has other structural elements involved in interactions with the guide:target heteroduplex (an end-capping loop between ⁇ 42 and ⁇ 43) and the PI domain/stem loop 3 ( ⁇ hairpin formed by ⁇ 3 and ⁇ 4).
  • HNH domain 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.
  • the backbone phosphate groups of the guide region interact with the REC1 domain (Arg165, Gly166, Arg403, Asn407, Lys510, Tyr515, and Arg661) and the bridge helix (Arg63, Arg66, Arg70, Arg71, Arg74, and Arg78).
  • the 20-hydroxyl groups of G1, C15, U16, and G19 hydrogen bond with Val1009, Tyr450, Arg447/Ile448, and Thr404, respectively.
  • RNA-guided DNA targeting: SpCas9 recognizes the guide:target heteroduplex in a sequence-independent manner.
  • the backbone phosphate groups of the target DNA interact with the REC1 (Asn497, Trp659, Arg661, and Gln695), RuvC (Gln926), and PI (Glu1108) domains.
  • the C2’ atoms of the target DNA form van der Waals interactions with the REC1 domain (Leu169, Tyr450, Met495, Met694, and His698) and the RuvC domain (Ala728).
  • the terminal base pair of the guide:target heteroduplex (G1:C20’) is recognized by the RuvC domain via end-capping interactions; the sgRNA G1 and target DNA C20’ nucleobases interact with the Tyr1013 and Val1015 side chains, respectively, whereas the 20-hydroxyl and phosphate groups of sgRNA G1 interact with Val1009 and Gln926, respectively.
  • the nucleobase of the flipped U44 is sandwiched between Tyr325 and His328, with its N3 atom hydrogen bonded with Tyr325, whereas the nucleobase of the unpaired G43 stacks with Tyr359 and hydrogen bonds with Asp364.
  • the 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 O4 atom hydrogen bonded with Arg75.
  • A51 adopts the syn conformation and is oriented in the direction opposite to U50.
  • Stem loop 1 is primarily recognized by the REC lobe, together with the PI domain.
  • the backbone phosphate groups of stem loop 1 interact with the REC1 domain (Leu455, Ser460, Arg467, Thr472, and Ile473), the PI domain (Lys1123 and Lys1124), and the bridge helix (Arg70 and Arg74), with the 20-hydroxyl group of G58 hydrogen bonded with Leu455.
  • A52 interacts with Phe1105 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 Lys1097), the PI domain (Thr1102), 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 Val1100.
  • 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 O6 atoms) and water-mediated (between the A68 N1 and G81 N1 atoms) hydrogen-bonding interactions.
  • the A68 and G81 nucleobases contact Ser1351 and Tyr1356, respectively, whereas the A68:G81 pair interacts with Thr1358 via a water-mediated hydrogen bond.
  • 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 Gln1272 and Glu1225/Ala1227, respectively.
  • the A88 and C91 nucleobases are recognized by Asn46 via multiple hydrogen- bonding 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.
  • 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.
  • Well characterized smaller Cas9 proteins include Staphylococcus aureus (SauCas9, 1053 amino acid residues) and Campylobacter jejuni (CjCas9, 984 amino residues).
  • Staphylococcus lugdunensis (Slu) 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
  • a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9.
  • the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is 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 the corresponding fragment of wild type Cas9.
  • a reference Cas9 e.g., a gRNA binding domain or a DNA-cleavage domain
  • the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.
  • the disclosure also may utilize Cas9 fragments that retain their functionality and that are fragments of any herein disclosed Cas9 protein.
  • the Cas9 fragment is at least 100 amino acids in length.
  • the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.
  • the prime editors disclosed herein may comprise one of the Cas9 variants described as follows, or a Cas9 variant thereof 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 variants. Table 4.
  • RRTKRTSRRR SEREKARKAM LKELFADEIN RVDPSFFIRL EESKFFLDDR ID margaretiae SENNRQRYTL FNDATFTDKD YYEKYKTIFH LRSALINSDE KFDVRLVFLA ILNLFSHRGH FLNASLKGDG DIQGMDVFYN DLVESCEYFE IELPRITNID NO: ATCC 43715 NFEKILSQKG KSRTKILEEL SEELSISKKD KSKYNLIKLI SGLEASVVEL 71) YNIEDIQDEN KKIKIGFRES DYEESSLKVK EIIGDEYFDL VERAKSVHDM EFM38267.1 GLLSNIIGNS KYLCEARVEA YENHHKDLLK IKELLKKYDK KAYNDMFRKM TDKNYSAYVG SVNSNIAKER RSVDKRKIED LYKYIEDTAL KNIPDDNKDK IEILEKIKLG EFLKKQLTAS NGVIPNQL
  • prime editors comprise type V or type VI CRISPR-Cas system enzymes. It will be appreciated that certain CRISPR enzymes exhibit promiscuous ssDNA cleavage activity and appropriate precautions should be considered. In certain embodiments, prime editors comprise a nickase or a dead CRISPR with nuclease function comprised in a different component.
  • Cas-equivalents further include those described in Makarova et al., “C2c2 is a single-component programmable RNA- guided RNA-targeting CRISPR effector,” Science 2016; 353(6299) and Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol.1. No.5, 2018, the contents of which are incorporated herein by reference.
  • One example of a nucleic acid programmable DNA-binding protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (i.e, Cas12a (Cpf1)).
  • Cas12a (Cpf1) is also a Class 2 CRISPR effector, but it is a member of type V subgroup of enzymes, rather than the type II subgroup. It has been shown that Cas12a (Cpf1) mediates robust DNA interference with features distinct from Cas9.
  • Cas12a (Cpf1) is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T- rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break.
  • Cpf1-family proteins Two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells.
  • Cpf1 proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p.949-962; the entire contents of which is hereby incorporated by reference. 6 .3.
  • Type V CRISPR proteins [0099]
  • prime editors used herein comprise the type V CRISPR family includes Francisella novicida U112 Cpf1 (FnCpf1) also known as FnCas12a.
  • FnCpf1 adopts a bilobed architecture with the two lobes connected by the wedge (WED) domain.
  • the N-terminal REC lobe consists of two a-helical domains (REC1 and REC2) that have been shown to coordinate the crRNA-target DNA heteroduplex.
  • the C-terminal NUC lobe consists of the C-terminal RuvC and Nuc domains involved in target cleavage, the arginine-rich bridge helix (BH), and the PAM- interacting (PI) domain.
  • the repeat-derived segment of the crRNA forms a pseudoknot stabilized by intra-molecular base-pairing and hydrogen-bonding interactions.
  • the pseudoknot is coordinated by residues from the WED, RuvC, and REC2 domains, as well as by two hydrated magnesium cations.
  • nucleotides 1–5 of the crRNA are ordered in the central cavity of FnCas12a and adopt an A-form-like helical conformation. Conformational ordering of the seed sequence is facilitated by multiple interactions between the ribose and phosphate moieties of the crRNA backbone and FnCpf1 residues in the WED and REC1 domains. These include residues Thr16, Lys595, His804, and His881 from the WED domain and residues Tyr47, Lys51, Phe182, and Arg186 from the REC1 domain.
  • FnCas12a-crRNA complex further reveals that the bases of the seed sequence are solvent exposed and poised for hybridization with target DNA.
  • Structural aspects of FnCpf1 are described by Swarts et al., Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a, Molecular Cell 66, 221-233, April 20, 2017. [0100]
  • Pre-crRNA processing Essential residues for crRNA processing include His843, Lys852, and Lys869.
  • R-loop formation The crRNA-target DNA strand heteroduplex is enclosed in the central cavity formed by the REC and NUC lobes and interacts extensively with the REC1 and REC2 domains.
  • the PAM-containing DNA duplex comprises target strand nucleotides dT0–dT8 and non-target strand nucleotides dA(8)*–dA0* and is contacted by the PI, WED, and REC1 domains.
  • the 5’-TTN-3’ PAM is recognized in FnCas12a by a mechanism combining the shape- specific recognition of a narrowed minor groove, with base-specific recognition of the PAM bases by two invariant residues, Lys671 and Lys613.
  • the duplex of the target DNA is disrupted by the side chain of residue Lys667, which is inserted between the DNA strands and forms a cation- ⁇ stacking interaction with the dA0–dT0* base pair.
  • the phosphate group linking target strand residues dT(-1) and dT0 is coordinated by hydrogen-bonding interactions with the side chain of Lys823 and the backbone amide of Gly826.
  • Target strand residue dT(-1) bends away from residue T0, allowing the target strand to interact with the seed sequence of the crRNA.
  • the non-target strand nucleotides dT1*–dT5* interact with the Arg692- Ser702 loop in FnCas12a through hydrogen-bonding and ionic interactions between backbone phosphate groups and side chains of Arg692, Asn700, Ser702, and Gln704, as well as main-chain amide groups of Lys699, Asn700, and Ser702.
  • Alanine substitution of Q704 or replacement of residues Thr698–Ser702 in FnCas12a with the sequence Ala-Gly3 (SEQ ID NO: 115) substantially reduced DNA cleavage activity, suggesting that these residues contribute to R-loop formation by stabilizing the displaced conformation of the nontarget DNA strand.
  • 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.
  • Target DNA cleavage FnCpf1 can independently accommodate both the target and non-target DNA strands in the catalytic pocket of the RuvC domain.
  • 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 Cpf1/Cas12a enzymes.
  • nuclease comprises a Cas12f 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 Cas12f1 (Cas14a and type V-U3), Cas12f2 (Cas14b) and Cas12f3 (Cas14c, type V-U2 and U4).
  • Exemplary CRISPR-Cas proteins and enzymes used in the Prime Editors herein include the following without limitation. Table 5.
  • NK2B42 NSAINLYNQK MHGAGSFKKL PKMKELYKQL LTEREEEFIE EYTDDEVLIT WP_029202018 SVHNYVSYLI DYLNSDKVES FFDTLRKSDG KEVFIKNDVS KTTMSNILFD NWSTIDDLIN HEYDSAPENV KKTKDDKYFE KRQKDLKKNK SYSLSKIAAL CRDTTILEKY IRRLVDDIEK IYTSNNVFSD IVLSKHDRSK KLSKNTNAVQ AIKNMLDSIK DFEHDVMLIN GSGQEIKKNL NVYSEQEALA GILRQVDHIY NLTRNYLTKK PFSTEKIKLN FNRPTFLDGW DKNKEEANLG ILLIKDNRYY LGIMNTSSNK AFVNPPKAIS NDIYKKVDYK LLPGPNKMLP KVFFATKNIA YYAPSEELLS KYRKGTHKKG DSFSIDDCRN L
  • Protospacer Adjacent Motif 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.
  • the PAM sequence is on either strand, and is downstream in the 5' to 3' direction of Cas9 cut site.
  • the canonical PAM sequence (i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5'-NGG-3' wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases.
  • N is any nucleobase followed by two guanine (“G”) nucleobases.
  • G guanine
  • Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms.
  • any given Cas9 nuclease may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes alternative PAM sequence.
  • the PAM specificity can be modified by introducing one or more mutations, including (a) D1135V, 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) D1135V, G1218R, R1335E, and T1337R “the VRER variant”, which alters the PAM specificity to NGCG.
  • 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.
  • Speptococcus thermophilis (StCas9) recognizes NNAGAAW.
  • Cas9 from Treponema denticola recognizes NAAAAC. These examples are not meant to be limiting. It will be further appreciated that non- SpCas9s bind a variety of PAM sequences, which makes them useful to expand the range of sequences that can be targeted according to the invention. Furthermore, non-SpCas9s may have other characteristics that make them more useful than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged into adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • 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.
  • 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 Such an enzyme 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 prime- editing guide RNA (pegRNA).
  • pegRNA prime- editing 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.
  • PE1 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.
  • the prime editors disclosed herein is comprised of PE1.
  • 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: [NLS]-[Cas9(H840A)]- [linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] + a desired atgRNA.
  • the prime editors disclosed herein is comprised of PE2.
  • the prime editors disclosed herein is comprised of PE2 and co-expression of MMR protein MLH1dn, that is PE4.
  • PE3 refers to PE2 plus a second-strand nicking guide RNA that complexes with the PE2 and introduces a nick in the non-edited DNA strand. The induction of the second nick increases the chances of the unedited strand, rather than the edited strand, to be repaired.
  • the prime editors disclosed herein is comprised of PE3.
  • the prime editors disclosed herein is comprised of PE3 and co-expression of MMR protein MLH1dn, that is PE5.
  • 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. 6 .6.
  • 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.
  • 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. Cas12a) binding region located toward the 5’ end.
  • 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).
  • the guide RNA e.g., atgRNA
  • guide RNA complex is capable of binding a DNA binding nickase selected from the group consisting of: Cas9-D10A, Cas9-H840A, Cas12a/b/c/d/e nickase, CasX nickase, SaCas9 nickase, and CasY nickase.
  • the nickase is linked or fused to one or more of a reverse transcriptase. In certain embodiments, the nickase is linked or fused to one or more of a reverse transcriptase and integrase. In certain embodiments, the nickase is linked or fused to one or more of an integrase. 6 .7.
  • Attachment Site-Containing Guide RNA [0121]
  • the term “attachment site-containing guide RNA” (atgRNA) and the like refer to an extended single guide RNA (sgRNA) comprising a primer binding site (PBS), a reverse transcriptase (RT) template sequence, and wherein the RT template encodes for an integration recognition site or a recombinase recognition site that can be recognized by a recombinase, integrase, or transposase.
  • the RT template comprises a clamp sequence and an integration recognition site.
  • an atgRNA may be referred to as a guide RNA.
  • An integration recognition site or recombinase target recognition site incorporated into the pegRNA is referred to as an attachment site containing guide RNA (atgRNA).
  • atgRNA guide RNA
  • cognate pair refers to a first integration recognition site (e.g., any of the integration recognition sites described herein) and a functionally symmetric second integration recognition site (e.g., any of the integration recognition sites described herein) that can be recombined.
  • Each of the first and second integration recognition sites is a “cognate”, or “integration cognate”, or “integration cognate site”, of the other member of the cognate pair.
  • a non-limiting example of a cognate pair are an attB site and an attP site, which are capable of recombination by the large serine integrase BxB1.
  • a single nucleic acid construct includes a first cognate pair (e.g., a first integration recognition site and a second integration recognition site) and a second cognate pair (e.g., a third integration recognition site and a fourth recognition site).
  • the first cognate pair and the second cognate pair have different central dinucleotides that enable recombination only with the other integration recognition site within the cognate pair.
  • 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” 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).
  • atgRNA guide RNA
  • 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 single nucleic acid construct i.e., “installer” contains a nucleotide sequence encoding an attachment site-containing guide RNA (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 gene editor polypeptide 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.
  • the single nucleic acid construct contains a contains a nucleotide sequence encoding a first attachment site-containing guide RNA (atgRNA) and a nucleotide sequence encoding a second attachment site-containing guide RNA (atgRNA).
  • the single nucleic acid construct 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 integration 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 integration 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 integration 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 integration 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 integration target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site-containing guide RNAs (atgRNAs).
  • use of two guide RNAs that are (or encode DNA that is) full complementarity to each other and comprised of consecutive bases of an integration target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site- containing guide RNAs (atgRNAs).
  • atgRNAs guide RNAs
  • 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.
  • the first pair of atgRNAs incorporate 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 Bxb1. Table 9.
  • the single nucleic acid construct i.e., “installer” contains an integrase or recombinase.
  • the single nucleic acid construct i.e., “installer” contains an integrase and a recombinase.
  • the single nucleic acid construct contains at least one integrase (e.g., at least two integrases) and at least one recombinase (e.g., at least two recombinases).
  • an integration enzyme e.g., an integrase or a recombinase
  • an integration enzyme is selected from the group consisting of Cre, Dre, Vika, Bxb1, ⁇ C31, RDF, FLP, ⁇ BTl, R1, R2, R3, R4, R5, TP901-1, A118, ⁇ FCl, ⁇ C1, MR11, TG1, ⁇ 370.l, W ⁇ , BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, ⁇ RV, retrotransposases encoded by a Tc1/mariner family member including but not limited to retrotransposases encoded by
  • Xu et al describes methods for evaluating integrase activity in E. coli and mammalian cells and confirmed at least R4, ⁇ C31, ⁇ BT1, Bxb1, SPBc, TP901-1 and W ⁇ integrases to be active on substrates integrated into the genome of HT1080 cells (Xu et al., 2013, Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome. BMC Biotechnol.2013 Oct 20;13:87. doi: 10.1186/1472-6750-13-87).
  • Durrant describes new large serine recombinases (LSRs) divided into three classes distinguished from one another by efficiency and specificity, including landing pad LSRs which outperform wild-type Bxb1 in episomal and chromosomal integration efficiency, LSRs that achieve both efficient and site- specific integration without a landing pad, and multi-targeting LSRs with minimal site-specificity. Additionally, embodiments can include any serine recombinase such as BceINT, SSCINT, SACINT, and INT10 (see Ionnidi et al., 2021; Drag-and-drop genome insertion without DNA cleavage with CRISPR directed integrases.
  • LSRs serine recombinases
  • the integration site can be selected from an attB site, an attP site, an attL site, an attR site, a Vox site, or a FRT site.
  • the single nucleic acid construct i.e., “installer” contains an integrase (e.g., any of the integreases described herein (e.g., any of the large serine integrases described herein).
  • the single nucleic acid construct contains a recombinase (e.g., any of the recombinases described herein).
  • the single nucleic acid construct i.e., “installer” contains a large serine integrase (e.g., any of the large serine integrases described herein) and a recombinase.
  • the single nucleic acid construct i.e., “installer” contains a BxB1 integrase and a flippase (e.g., FLP).
  • 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 Ppmar1 and Ppmar2 of moso bamboo. Mob DNA. 2019 Aug 19;10:35. doi:10.1186/s13100-019-0179-y).
  • the methods and constructs are used to modulate nuclear localization of system components of the invention.
  • the polynucleotide encoding the integrase comprises an intron, whereby the intron prevents the expression of the integration enzyme in prokaryotic cells.
  • the intron allows the plasmid preparation of the nucleic acid construct from prokaryotic cells.
  • the integrase used herein is selected from below. Table 10.
  • FIGs. 4A-4E shows analysis of effect of variant AttP sites on integration efficiency.
  • Table 11 D escription Forward Sequence (5’-3’) SEQ ID Reverse Sequence (5’-3’) SEQ ID NO: NO: Bxb1_AttP_ GTGGTTTGTCTGGTCAACCA SEQ ID TGGGTTTGTACCGTACACC SEQ ID GT_original CCGCGGTCTCAGTGGTGTAC NO: 394 ACTGAGACCGCGGTGGTTG NO: 473 _site GGTACAAACCCA ACCAGACAAACCAC Bxb1_AttP_ GTGGTTTGTCTGGTCAACCA 395 TGGGTTTGTACCGTACACC 474 CG_site CCGCGcgCTCAGTGGTGTAC ACTGAGCGCGCGGTGGTTG GGTACAAACCCA ACCAGACAAACCAC Table 11 Description Forward Sequence (5’-3’) SEQ ID Reverse Sequence (5’-3’) SEQ ID NO: NO: Bxb1_AttP_ GTGGTT
  • nucleic Acid Construct Design A single nucleic acid construct is described herein that allows for programmable gene insertion (PGI) (e.g., incorporation of any template into any DNA locus using DNA delivery of a single component DNA).
  • the nucleic acid construct contains a nucleotide sequence encoding an integrase, a nucleotide sequence encoding a prime editor fusion protein or a gene writer protein, a nucleotide sequence encoding at least a first attachment site-containing guide RNA (atgRNA), a DNA donor template (i.e., “cargo”), optionally a nucleotide sequence encoding a nickase guide RNA (ngRNA), and optionally a nucleotide sequence encoding a recombinase.
  • atgRNA a first attachment site-containing guide RNA
  • ngRNA nickase guide RNA
  • the nucleic acid construct contains a nucleotide sequence encoding an integrase, a nucleotide sequence encoding a prime editor fusion protein or a gene writer protein, a nucleotide sequence encoding at least a first attachment site-containing guide RNA (atgRNA), a DNA donor template (i.e., “cargo”), a nucleotide sequence encoding a nickase guide RNA (ngRNA), and optionally a nucleotide sequence encoding a recombinase.
  • atgRNA first attachment site-containing guide RNA
  • ngRNA nickase guide RNA
  • ngRNA nickase guide RNA
  • the nucleic acid construct contains a nucleotide sequence encoding an integrase, a nucleotide sequence encoding a prime editor fusion protein or a gene writer protein, a nucleotide sequence encoding at least a first attachment site-containing guide RNA (atgRNA), a DNA donor template (i.e., “cargo”), a nucleotide sequence encoding a nickase guide RNA (ngRNA), and a nucleotide sequence encoding a recombinase.
  • atgRNA first attachment site-containing guide RNA
  • ngRNA nickase guide RNA
  • ngRNA nickase guide RNA
  • the nucleic acid construct contains a nucleotide sequence encoding an integrase, a nucleotide sequence encoding a prime editor fusion protein or a gene writer protein, a nucleotide sequence encoding a first attachment site-containing guide RNA (atgRNA), a second attachment site-containing guide RNA (atgRNA), a DNA donor template (i.e., “cargo”), and a nucleotide sequence encoding a recombinase, where the first atgRNA and the second atgRNA are an at least first pair of atgRNAs.
  • the nucleic acid construct contains a nucleotide sequence encoding an integrase, a nucleotide sequence encoding a prime editor fusion protein or a gene writer protein, a nucleotide sequence encoding a first attachment site-containing guide RNA (atgRNA), a nucleotide sequence encoding a second attachment site-containing guide RNA (atgRNA), and a DNA donor template (i.e., “cargo”), where the first atgRNA and the second atgRNA are an at least first pair of atgRNAs.
  • atgRNA first attachment site-containing guide RNA
  • atgRNA a nucleotide sequence encoding a second attachment site-containing guide RNA
  • a DNA donor template i.e., “cargo”
  • the nucleic acid construct comprises: a nucleotide sequence encoding a prime editor fusion protein; a nucleotide sequence encoding at least a first attachment site-containing guide RNA (atgRNA); a nucleotide sequence encoding a recombinase; a nucleic acid cargo; and a nucleotide sequence encoding a nickase guide RNA (ngRNA).
  • atgRNA first attachment site-containing guide RNA
  • ngRNA nickase guide RNA
  • the nucleic acid construct comprises: a nucleotide sequence encoding a prime editor fusion protein, a nucleotide sequence encoding a first attachment site- containing guide RNA (atgRNA), a nucleotide sequence encoding a second attachment site- containing guide RNA (atgRNA), and a nucleotide sequence encoding a recombinase; a nucleic acid cargo; where the first atgRNA and the second atgRNA are an at least first pair of atgRNAs.
  • a single promoter drives expression of all the different nucleotide sequences on the single nucleic acid construct.
  • two or more promoters drive expression of the different nucleotide sequences on the single nucleic acid construct.
  • at least one promoter drives the expression of the prime editor fusion protein or the gene writer protein, atgRNA, optionally ngRNA, integrase (e.g., serine integrase), and optionally recombinase.
  • the promoter is an immediate early promoter such as a CMV promoter or a type III RNA polymerase III promoter such as a U6 promoter.
  • the promoter is any Pol II promoter.
  • the atgRNA and ngRNA are driven by any Pol III promoter.
  • the respective promoters used to drive the expression of the protein components, the atgRNA, and the ngRNA have different promoter expression strength, fidelity, selectivity, and/or tissue-specificity.
  • the integrase that is encoded in the nucleic acid construct is fused to the prime editor fusion protein or the Gene Writer protein optionally by a linker.
  • the recombinase that is encoded in the nucleic acid construct is fused to the prime editor fusion protein or the Gene Writer protein optionally by a linker.
  • the nucleic acid construct contains a 5’ inverted terminal repeat (ITR).
  • the nucleic acid construct contains a 3’ inverted terminal repeat (ITR). In some embodiments, the nucleic acid construct contains a 5’ and a 3’ inverted terminal repeat. In some embodiments, the 5’ and 3’ ITR are not derived from the same serotype of virus. In some embodiments, the ITRs are derived from Adenovirus, AAV2, AAV5, or both. [0148] In typical embodiments, the nucleic acid construct further comprises at least one integrase recognition target site (e.g., an integrase recognition site in the nucleic acid construct used to facilitate integration of all or part of the nucleic acid construct into an integrase recognition site incorporated into a cell genome).
  • integrase recognition target site e.g., an integrase recognition site in the nucleic acid construct used to facilitate integration of all or part of the nucleic acid construct into an integrase recognition site incorporated into a cell genome.
  • the at least one integrase recognition site is separate from the integration sequences encoded by the first atgRNA, second atgRNA, or both.
  • the at least one integrase recognition site is a cognate pair with the integration sequences encoded by the first atgRNA, second atgRNA, or by a combination of the first atgRNA and second atgRNA.
  • the at least one integrase recognition site is specific for a BxB1, B. cereus (BceINTc or Bcec), N191352_143_72 stool sample from China (SscINTd or Sscd), N684346_90_69 stool sample from adult in China (SacINTd or Sacd).
  • the nucleic acid construct further comprises at least one recombinase recognition target site (e.g., one recombinase recognition site, two recombination recognition sites, three recombinase recognition sites, or four recombinase recognitions site, or more).
  • the at least one recombinase recognition site is specific for a FLP, a FLP mutant, Cre, or a Cre mutant.
  • the nucleic acid construct comprises two recombinase recognition sites where the two sites flank the nucleic acid cargo.
  • the two recombinase recognition sites are capable of self-circularizing to form a circular construct when contacted with a recombinase.
  • a circular cargo is generated in the target cell.
  • a linear cargo is generated in the target cell.
  • the nucleic acid construct further comprises at least one recombinase recognition target site and at least one integrase recognition target site.
  • the nucleic acid construct contains a nucleic acid cargo (i.e., “integration” cargo) of interest.
  • the nucleic acid cargo is one or more genes or gene fragments.
  • the nucleic acid cargo is at least one intron, at least one exon sequence, or a combination thereof. In some embodiments, the nucleic acid cargo is at least one intron fragment, at least exon fragment sequence, or a combination thereof. In some embodiments the nucleic acid cargo is an expression cassette. In some embodiments, the nucleic acid 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. In some embodiments, the nucleic acid cargo is DNA or RNA. In some embodiments, the nucleic acid cargo is a genetic, protein, or peptide tag and/or barcode.
  • the constructs and methods described herein may be utilized for monitoring a biological or biochemical cellular condition or circuits, such as pH via a marker.
  • the constructs and methods described herein may be utilized for recording, via writing directly to a genome or intracellular DNA element, cellular, environmental, chemical, or other cellular temporal or spatial related events.
  • the constructs and methods described herein may be utilized for recording, via writing directly to a genome or intracellular DNA element, cellular lineage information.
  • the genome to be programmably inserted into is eukaryotic or porkarytotic.
  • the genome is mammalian, nonmammalian, human, murine, or NHP.
  • constructs and methods describe herein may be utilized in agricultural settings for production of crops with improved properties or traits as well as to produce livestock, such as cattle, avian, or other species with improved or desirable features. 6 .10. Integrase- or Recombinase-Mediated Self-C1rcularization of a Subsequence of the Single Nucleic Acid Construct [0155]
  • the single nucleic acid construct comprises a sub-sequence of the nucleic acid construct that is capable of self-circularizing to form a self-circular nucleic acid.
  • the single nucleic acid construct comprises a physical portion or region of the nucleic acid construct that is capable of self-circularizing to form a circular construct.
  • sequence refers to a portion of the single nucleic acid construct that is capable of self-circularizing, where the subsequence is flanked by integrase recognition sites or recombinase recognition sites positioned to enable self-circularization.
  • 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 single nucleic acid construct. Recombination occurs when the single nucleic acid construct is contacted with an integrase or a recombinase under conditions that allow for recombination of the cognate pair or integrase or recombinase recognition sites.
  • the sub-sequence of the single nucleic acid construct 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 single nucleic acid includes a first recombinase recognition site, a second recombinase recognition site, and an integrase recognition site (e.g., a second integrase recognition site), where the first and second recombinase recognition sites flank the integrase recognition site.
  • the first recombinase recognition site, the second recombinase recognition, and a recombinase enable the self-circularizing and formation of the circular construct (see, e.g., FIG. 1).
  • the sub-sequence of the single nucleic acid construct includes a third integrase recognition site and a fourth integrase recognition site, wherein the third and fourth integrase recognition sites are a cognate pair.
  • the subsequence of the single nucleic acid construct includes the second integrase recognition site, the third integrase recognition site, the fourth integrase recognition site, where the third and fourth integrase recognition sites flank the second integrase.
  • the third integrase recognition site, the fourth integrase recognition site, and an integrase enable self -circularization and formation of the circular construct.
  • the third integrase recognition site and/or the fourth integrase recognition sites cannot recombine due, in part, to having different central dinucleotides with the first integrase recognition site and/or the second integrase recognition site.
  • each integrase recognition site or each pair of integrase recognition is capable of being recognized by a different integrase.
  • each integrase recognition site or each pair of integrase recognition comprises a different central dinucleotide.
  • self-circularizing is mediated at the integrase recognition sites or recombinase recognition sites. In some embodiments, the self-circularizing is mediated by an integrase or a recombinase.
  • the self-ciruclar nucleic acid comprising the second integrase recognition site is capable of being integrated into the cell’s genome at the target sequence that contains the first integrase recognition site.
  • the self-circular nucleic acid comprises one or more additional integrase 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 integrase recognition sites in the self-circular nucleic acid.
  • integration of the self-circular nucleic acid into the genome of a cell results in integration of the one or more integrase recognition sites into the genome along with the nucleic acid cargo.
  • the integrated one or more integrase recognition sites serve as an integrase recognition site (beacon) for placing the additional nucleic acid cargo.
  • the self-circular nucleic acid includes a second integrase recognition site that is capable of being integrated into a genomic locus that contains the first integrase recognition site (i.e., the first and second integrase recognition sites are a cognate pair). See, FIGs. 1-2.
  • the single nucleic acid construct comprises two recombinase recognition sites where the two sites flank the nucleic acid cargo. In such cases, the two recombinase recognition sites are capable of self-circularizing to form a self-circular nucleic acid when contacted with a recombinase.
  • FIG. 1 illustrates a non-limiting example of a single nucleic acid construct that includes two recombinase recognition sites capable of self-circularizing to form a circular construct (e.g., a self-circular nucleic acid) when contacted with a recombinase.
  • 101 and 102 are recombinase recognition sites present in the single nucleic acid construct.
  • the single nucleic acid construct also includes a sequence encoding a recombinase 103.
  • the recombinase 103 is expressed 104 and contacts 105 the recombinase recognition sites (101 and 102), thereby mediating self-circularization of a portion of the single nucleic acid construct and producing a self-circular nucleic acid 106.
  • the self-circular nucleic acid 106 includes a sequence 107 that is an integration cognate (e.g., a cognate pair) to the first integrase recognition sequence 108.
  • the self-circular nucleic acid is integrated into a genome at the incorporation sites of the first integrase recognition site.
  • integration of the self-circular nucleic acid into the genome is mediated by an integrase.
  • FIG. 1 illustrates a non-limiting example where the single nucleic acid construct also includes a sequence encoding an integrase 109.
  • the integrase 109 is expressed and integrates 110 the circular construct 106 into the first integrase recognition site 108 site-specifically incorporated into the genome.
  • the nucleic acid construct comprises two integrase recognition sites where the two sites flank the nucleic acid cargo. In such cases, the two integrase recognition sites are capable of self-circularizing to form a self-circular nucleic acid when contacted with an integrase.
  • FIG. 2 illustrates a non-limiting example of a single nucleic acid construct that includes two integration sequences capable of self-circularizing to form a circular construct (e.g., a self- circular nucleic acid) when contacted with a recombinase.
  • 201 and 202 are integrase recognition sites (e.g., the third and fourth integrase recognition sites) present in the single nucleic acid construct.
  • the single nucleic acid construct also includes a sequence encoding an integrase 203.
  • the integrase 203 is expressed 204 and contacts 205 the integrase recognition sites (201 and 202), thereby mediating self-circularization of a portion of the single nucleic acid construct and producing a self-circular nucleic acid 206.
  • the self-circular nucleic construct 206 includes a sequence 207 that is a cognate pair to the site-specifically incorporated integration sequence 208. As shown in FIG. 2, one embodiment uses the same integrase for both self-circularizing and integration of the self-circular nucleic acid.
  • the integrase 203 is expressed 204 and integrates 210 the self-circular nucleic acid 206 into the first integrase recognition site 208 site-specifically incorporated into the genome.
  • High efficiency and/or fast integrase recognition target sites allow for integrase- mediated template circularization to happen prior to integrase-mediated genomic integration at an integrase recognition target site within the genome (i.e. “beacon” or “landing pad”).
  • the integration rate can be altered by changing the dinucleotide used within the integrase recognition target site.
  • the integration rate can be altered by changing the integrase recognition target site sequence length.
  • the integration rate can be altered by changing the dinucleotide used within the integrase recognition target site and by changing the integrase recognition target site sequence length.
  • the attB/attP integrase recognition target site sequence length can be about 32-46 bp in length.
  • high efficiency and/or fast integrase target recognition is mediated by orthogonal integrases or recombinases.
  • a single nucleic acid construct includes a first cognate pair (e.g., a first integrase recognition site and a second integrase recognition site) and a second cognate pair (e.g., a third integrase recognition site and a fourth recognition site)
  • the first cognate pair and the second cognate pair are designed such that each cognate pair has a different integration rate.
  • the cognate pair with the faster integration rate recombines prior to the cognate pair with the slower integration rate.
  • the first cognate pair is represented by 207 and 208 and the second cognate pair is represented by 201 and 202.
  • the second cognate pair (i.e., 201 and 202) has a faster integration rate whereby self-circularization occurs prior to integration into the genome.
  • the self-circularizing is effected at an integrase or recombinase recognition target sequence. In typical embodiments, the self-circularizing is mediated by an integrase or a recombinase.
  • 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 DNA cargo contains one or more orthogonal recombinase recognition target site(s).
  • the DNA cargo contains one or more orthogonal integrase recognition target site(s).
  • the region that contains one or more orthogonal recombinase or integrase recognition target site(s) may be referred to as a multiple access site.
  • the additional one or more orthogonal recombinase or integrase target recognition site(s) contained within the inserted DNA cargo may be subsequently targeted via a recombinase or integrase to incorporate additional DNA cargo.
  • the DNA cargo may contain one or one or more orthogonal recombinase or integrase target recognition site(s).
  • each newly genomically incorporated DNA template, insert, or DNA cargo may contain at least one “embedded” or “nested” orthogonal recombinase or integrase target recognition site(s) it becomes possible to programmatically (spatially and temporally) access, introduce, delete, and modify a genomic- or DNA-locus of interest at the orthogonal recombinase or integrase target recognition site(s).
  • the self-circular nucleic acid is capable of being integrated into a genomic locus that contains an integrase or recombinase recognition site (i.e., “beacon” or “landing pad” site).
  • the self-circular nucleic acid contains the DNA cargo of interest.
  • the integrase or recombinase that mediates self-circularization is fused or linked to the prime editor protein fusion.
  • the nucleic acid construct that contains a nucleotide sequence encoding an integrase, encoding a prime editor fusion protein or a gene writer protein, a nucleotide sequence encoding one or more attachment site-containing guide RNA (atgRNA), optionally a nucleotide sequence encoding a nickase guide RNA (ngRNA), a nucleotide sequence encoding an integrase, a DNA cargo, and optionally a nucleotide sequence encoding a recombinase is vectorized.
  • an integration target recognition site is incorporated (i.e., beacon placement) into a human primary cell genome using a single atgRNA and a single nicking guide RNA (ngRNA).
  • an integration target recognition site is incorporated into a human primary cell genome using two atgRNAs (dual or paired or twin atgRNAs).
  • the nucleic acid construct comprises two atgRNAs.
  • the atgRNA 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 comprised of a reverse transcriptase template.
  • 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.
  • 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 complementary 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).
  • atgRNAs complementary, or twin attachment site-containing guide RNAs
  • 6 .11 Nucleic Acid Construct: Helper-Dependent Adenovirus (HDAd) [0176]
  • This disclosure features a nucleic acid construct or a set of nucleic acid constructs that includes all the components sufficient for programmable gene insertion.
  • the nucleic acid construct is a helper-dependent adenovirus.
  • one of the nucleic acid constructs in the set of nucleic acid constructs is a helper- dependent adenovirus.
  • the nucleic acid construct (e.g., a helper-dependent adenoviral vector) includes: (a) a first polynucleotide segment comprising: (i) a first expression control sequence; (ii) a gene editor polynucleotide encoding a gene editor polypeptide; and (iii) a polynucleotide sequence encoding a first attachment site-containing guide RNA (atgRNA), wherein the first atgRNA comprises a sequence encoding at least a portion of a first integration recognition site; and (iv) a polynucleotide sequence encoding a second atgRNA, wherein the second atgRNA comprises a sequence encoding at least a portion of the first integration recognition site, wherein the first expression control sequence is operably linked to the polynucleotide encoding the gene editor polypeptide; wherein the first atgRNA and the second atgRNA are operably linked to one or more additional expression control sequence
  • the set includes: (a) a first nucleic acid construct comprising (i) a first polynucleotide segment comprising: (1) a first expression control sequence; (2) a gene editor polynucleotide encoding a gene editor polypeptide; and (3) a polynucleotide sequence encoding a first attachment site-containing guide RNA (atgRNA), wherein the first atgRNA comprises a sequence encoding at least a portion of a first integration recognition site; and (4) a polynucleotide sequence encoding a second atgRNA, wherein the second atgRNA comprises a sequence encoding at least a portion of the first integration recognition site, wherein the first expression control sequence is operably linked to the polynucleotide encoding the gene editor polypeptide; wherein the first atgRNA and the second atgRNA are operably linked to one or more additional expression control sequences; (ii) a second
  • the third polynucleotide segment, the polynucleotide encoding the cargo, or both are flanked by the two integration recognition sites.
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNA, wherein the at least first pair of atgRNA 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 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 atgRNA collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is integrated into the genome of the cell at the target sequence.
  • the first integration recognition site is selected from attB, attB2, attP, or attP2 and the second integration recognition site is the cognate pair.
  • the nucleic acid construct e.g., a helper-dependent adenoviral vector
  • the nucleic acid construct includes: (a) a first polynucleotide segment comprising: (i) a first expression control sequence; (ii) a gene editor polynucleotide encoding a gene editor polypeptide; (iii) a polynucleotide sequence encoding a first atgRNA comprising a sequence encoding at least a portion of a first integration recognition site; (iv) a polynucleotide sequence encoding a second atgRNA comprising a sequence encoding at least a portion of the first integration recognition site; (v) a polynucleotide sequence encoding a third atgRNA comprising a sequence encoding at least
  • the set includes: (a) a first nucleic acid construct, comprising: (i) a first polynucleotide segment comprising: (1) a first expression control sequence; (2) a gene editor polynucleotide encoding a gene editor polypeptide; (3) a polynucleotide sequence encoding a first atgRNA comprising a sequence encoding at least a portion of a first integration recognition site; (4) a polynucleotide sequence encoding a second atgRNA comprising a sequence encoding at least a portion of the first integration recognition site; (5) a polynucleotide sequence encoding a third atgRNA comprising a sequence encoding at least a portion of a second integration recognition site; and (6) a polynucleotide sequence encoding a fourth atgRNA comprising a sequence encoding at least a portion of the second integration recognition site; wherein
  • 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 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 first integration recognition site is integrated into the genome of the cell at the target sequence.
  • the third atgRNA and the fourth atgRNA are an at least second pair of atgRNAs, wherein the at least second pair of atgRNAs have domains that are capable of guiding the prime editor system to a target sequence; the third atgRNA further includes a third RT template that comprises at least a portion of the second integration recognition site; the fourth atgRNA further includes a fourth RT template that comprises at least a portion of the second integration recognition site, and the third atgRNA and the fourth atgRNAs collectively encode the entirety of the second integration recognition site, whereby the second integration recognition site is integrated into the genome of the cell at the target sequence.
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNAs and the third atgRNA and the fourth atgRNA are an at least second pair of atgRNAs, [0187] where 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 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 first integration recognition site is integrated into the genome of the cell at the target sequence, and where the at least second pair of atgRNAs have domains that are capable of guiding the prime editor system to a target sequence;
  • the third atgRNA further includes a third RT template that comprises at least a portion of the second integration recognition site;
  • the fourth atgRNA further includes a
  • the first integration recognition site is selected from attB, attB2, attP, or attP2 and the third integration recognition site is the cognate pair, thereby making a first cognate pair; and the second integration recognition site is selected from attB, attB2, attP, or attP2 and the fourth integration recognition site is the cognate pair, thereby making a second cognate pair.
  • the polynucleotide sequence encoding the third integration recognition site and the polynucleotide sequence encoding the fourth integration recognition site flank the third polynucleotide segment and are positioned such that when contacted with the integration enzyme, the first integration recognition site, and the second integration recognitions, only the third polynucleotide segment is recombined.
  • the first integration recognition site and the third integration recognition site are a first cognate pair are capable of being recombined by the first integration enzyme (e.g., any of the integration enzymes described herein (see Section 4.8)); and the second integration recognition site and the fourth integration recognition site are a second cognate pair capable of being recombined by a second integration enzyme (e.g., any of the integration enzymes described herein (see Section 4.8)).
  • the first integration recognition site or the second integration recognition site includes one or more modifications that prevent or reduce intramolecular recombination.
  • the one or more modifications include reducing the overlap length of the dual atgRNA.
  • a dual atgRNA approach to programmable gene insertion includes a first atgRNA and a second atgRNA that collectively encode an integration recognition site that is integrated into the target DNA sequence.
  • the first atgRNA and the second atgRNA have overlap, where the overlap includes sequence complementarity between the RT template (or reverse complement thereof) in the first atgRNA and the RT template (or the reverse complement thereof) in the second atgRNA.
  • the first atgRNA and the second atgRNA have at least 5 bp overlap (e.g., at least a 6 bp overlap, at least a 7 bp overlap, at least a 8 bp overlap, at least a 9 bp overlap, at least a 10 bp overlap, at least a 11 bp overlap, at least a 12 bp overlap, at least a 13 bp overlap, at least a 14 bp overlap, at least a 15 bp overlap, at least a 16 bp overlap, at least a 17 bp overlap, at least a 18 bp overlap, at least a 19 bp overlap, at least a 20 bp overlap, at least a 21 bp overlap, at least a 22 bp overlap, at least a 23 bp overlap, at least a 24 bp overlap, at least a 25 bp overlap, at least a 26 bp overlap, at least a 27 bp overlap, at least a 28
  • the first atgRNA and the second atgRNA have an overlap of 30 bp or less, such as an overlap of 29 bp or less, an overlap of 28 bp or less, an overlap of 27 bp or less, an overlap of 26 bp or less, an overlap of 25 bp or less, an overlap of 24 bp or less, an overlap of 23 bp or less, an overlap of 22 bp or less, an overlap of 21 bp or less, an overlap of 20 bp or less, an overlap of 19 bp or less, an overlap of 18 bp or less, an overlap of 17 bp or less, an overlap of 16 bp or less, an overlap of 15 bp or less, an overlap of 14 bp or less, an overlap of 13 bp or less, an overlap of 12 bp or less, an overlap of 11 bp or less, an overlap of 10 bp or less, an overlap of 9 bp or less, an overlap of 8 bp or less, an overlap of 30 bp or less,
  • the first atgRNA and second atgRNA have a 5 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 6 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 7 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 8 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 9 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 10 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 11 bp overlap.
  • the first atgRNA and second atgRNA have a 12 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 13 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 14 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 15 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 16 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 17 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 18 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 19 bp overlap.
  • the first atgRNA and second atgRNA have a 20 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 21 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 22 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 23 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 24 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 25 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 26 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 27 bp overlap.
  • the first atgRNA and second atgRNA have a 28 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 29 bp overlap. In some embodiments, the first atgRNA and second atgRNA have a 30 bp overlap. [0195] In some embodiments, the one or more modifications that reduce intramolecular recombination include asymmetrical overlap of the dual atgRNA. In some embodiments, a dual atgRNA approach to programmable gene insertion includes a first atgRNA and a second atgRNA that collectively encode an integration recognition site that is integrated into the target DNA sequence.
  • the first atgRNA and the second atgRNA have an asymmetrical overlap, for example, the first atgRNA and the second atgRNA have complementarity between the RT template (or reverse complement thereof) in the first atgRNA and the RT template (or the reverse complement thereof) in the second atgRNA but where the first RT template (or the reverse complement thereof) is longer than the second RT template (or the reverse complement thereof) or where the second RT template (or the reverse complement thereof) is longer than the first RT template (or the reverse complement thereof).
  • the first atgRNA and the second atgRNA have a symmetrical overlap.
  • the AttB (or the sequences encoding the entirety of an AttB) and AttP sites are placed at varying distances.
  • the AttB (or the sequences encoding the entirety of an AttB) is on a first nucleic acid construct and the second AttP is on a second nucleic acid construct.
  • the gene editor polynucleotide comprises a nucleotide sequence encoding a nickase and a nucleotide sequence encoding a reverse transcriptase.
  • the nucleotide sequence encoding the nickase and the nucleotide sequence encoding the reverse transcriptase are positioned in the gene editor polynucleotide 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 first integration enzyme is selected from: BxB1, Bcec, Sscd, Sacd, Int10, or Pa01. In some embodiments, the first integration enzyme is an integration enzyme disclosed in PCT Publication No. WO2023/070031, the disclosure of which is incorporated by reference in its entirety. [0203] In some embodiments, the second integration enzyme is selected from: BxB1, Bcec, Sscd, Sacd, Int10, or Pa01 but is different from the first integration enzyme. In some embodiments, the first integration enzyme is an integration enzyme disclosed in PCT Publication No. WO2023/070031, but is different from the first integration enzyme. [0204] In some embodiments, the first expression control sequence is a constitutive expression sequence.
  • the second expression control sequence is a controllable expression sequence.
  • the third expression control sequence is a constitutive expression control sequence.
  • the controllable expression control sequence is selected from: a tet-off expression control sequence or a tet-on expression control sequence.
  • controllable promoters and their corresponding agents that enable control (if necessary) in the following.
  • Tet-Off the presence of the drug turns gene expression off. Both are widely used in mammalian systems.
  • Steroid-responsive promoters These promoters respond to the presence of specific steroids.
  • An example is the MMTV (mouse mammary tumor virus) promoter, which is induced by glucocorticoids.
  • Ecdysone-inducible systems This system responds to the ecdysone, an insect hormone, or to a synthetic analog of ecdysone.
  • a mifepristone (RU486) inducible GeneSwitch system This system is activated by the synthetic steroid mifepristone.
  • Rheoswitch system This system responds to a synthetic small molecule ligand (RSL1).
  • Cumate switch This system is based on a bacterial operon that responds to cumate, a compound not normally present in mammalian cells, but can be used in mammalian cells.
  • Heat- shock promoters These promoters respond to a sudden increase in temperature. While these are not commonly used in mammalian cells due to the potential for cellular damage, they can be useful in certain contexts.
  • IPTG-inducible systems While IPTG is traditionally used with bacterial systems like the lac operon, there are modified versions of this system that can work in mammalian cells.
  • the one or more additional expression control sequences operably linked to the sequences encoding the atgRNAs are U6 promoters.
  • the cargo is a therapeutic agent.
  • the therapeutic agent is selected from a gene, a gene fragment, a nucleic acid therapeutic, and-based a protein-based therapeutic.
  • cargo include the genes, gene fragments, nucleic acid therapeutics, and protein-based therapeutics described herein (see Section 4.12). 6.11.1.
  • Helper-Dependent Adenoviruses [0210]
  • the nucleic acid construct comprises a viral vector. Different viral vectors each have their own set of advantages and limitations, which should be considered based on the specifics of the therapeutic application, including the target tissue, the size of the therapeutic gene, and the desired duration of expression.
  • the viral vector is an adenoviral vector.
  • the adenoviral vector is a helper-dependent adenoviral (HDAd) vector.
  • HDAd vectors can accommodate larger therapeutic gene sizes (up to 37 kb), which is a significant advantage over AAV vectors that have a smaller capacity (approximately 4.7 kb). This makes HD-Ad vectors suitable for diseases that require the delivery of larger genes.
  • HDAd vectors are efficient at transducing a wide range of cell types, including both dividing and non-dividing cells.
  • High-level transgene expression HDAd vectors can drive high levels of transgene expression, which can be beneficial in therapeutic situations where high levels of a protein are required.
  • the HDAd includes a stuffer sequence.
  • helper-dependent adenoviruses also known as “gutless” adenoviruses
  • all viral coding sequences are removed to minimize the host immune response and maximize the space available for the therapeutic gene.
  • the size of the adenovirus genome is important for its stability and packaging. If the genome is too small, it may not be efficiently packaged into viral particles, reducing the efficiency of the vector.
  • shuffer sequence refers to a non-coding DNA sequence used to maintain the genome size of HDAd vectors within an optimal range for viral packaging after the viral genes have been removed. In some cases, the “stuffer sequence” acts as DNA “fillers”.
  • the stuffer sequence has a length of about 500 nucleotides to about 10,000 nucleotides (e.g., about 500 nucleotides to about 9,000 nucleotides, about 500 nucleotides about 8,000 nucleotides, about 500 nucleotides about 7,000 nucleotides, about 500 nucleotides about 6,000 nucleotides, about 500 nucleotides about 5,000 nucleotides, about 500 nucleotides about 4,000 nucleotides, about 500 nucleotides about 3,000 nucleotides, about 500 nucleotides about 2,000 nucleotides, about 500 nucleotides about 1,000 nucleotides, about 1,000 nucleotides about 10,000 nucleotides, about 1,000 nucleotides about 9,000 nucleotides, about 1,000 nucleo
  • the stuffer sequence has a length of about 10,000 nucleotides to about 20,000 nucleotides (e.g., about 10,000 nucleotides to about 19,000 nucleotides, about 10,000 nucleotides to about 18,000 nucleotides, about 10,000 nucleotides to about 17,000 nucleotides, about 10,000 nucleotides to about 16,000 nucleotides, about 10,000 nucleotides to about 15,000 nucleotides, about 10,000 nucleotides to about 14,000 nucleotides, about 10,000 nucleotides to about 13,000 nucleotides, about 10,000 nucleotides to about 12,000 nucleotides, about 10,000 nucleotides to about 11,000 nucleotides, about 11,000 nucleotides to about 20,000 nucleotides, about 11,000 nucleotides to about 19,000 nucleotides, about 11,000 nucleotides to about 18,000 nucleotides, about 11,000 nucleotides
  • the length of the stuffer sequence is selected such that the HDAd vector is about 37,000 nucleotides, about 38,000 nucleotides, about 39,000 nucleotides, or about 40,000 nucleotides.
  • the HDAd vector includes a packaging signal ( ⁇ ). In some embodiments, the HDAd vector does not include a packaging signal ( ⁇ ).
  • the packaging signal ( ⁇ ) is selected from an adenovirus 2 packaging single ( ⁇ ) (Ad2 ⁇ ) and an adenovirus 5 packaging signal ( ⁇ ) (Ad5 ⁇ ).
  • the packaging signal ( ⁇ ) is an adenovirus 2 packaging single ( ⁇ ) (Ad2 ⁇ ). In some embodiments, the packaging signal ( ⁇ ) is an adenovirus 5 packaging signal ( ⁇ ) (Ad5 ⁇ ). 6 .11.2.
  • HDAd virion comprises: a HDAd genome comprising any of the nucleic acid constructs described herein (see Sections 4.9, Section 4.10, and Section 4.11.1).
  • This disclosure features a cell comprising any of the nucleic acid constructs described herein (see Sections 4.9, Section 4.10, and Section 4.11.1) or any of the HDAd virions described herein.
  • This disclosure features a method of making a helper-dependent adenovirus (HDAd) virion, comprising: (a) introducing into a cell line suitable for making HDAd: (i) any of HDAd genomes described herein comprising any of the nucleic acid constructs described herein (see Sections 4.9, Section 4.10, and Section 4.11.1); and (ii) a helper virus (HV); (b) culturing the cell line under conditions sufficient to encapsidate the HDAd genome into the HDAd virion capsid; (c) collecting the HDAd; and (d) purifying the HDAd, wherein the HDAd genome is intact (not recombined) in the HDAd virion.
  • HV helper virus
  • the introducing comprises two rounds of introducing (i) and (ii).
  • the second round of introducing is performed about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days after the first round of introducing. 6 .11.3.
  • This disclosure features a method of making a helper-dependent adenovirus (HDAd) virion, comprising: (a) introducing into a cell line suitable for making HDAd: (i) in a first step, any of HDAd genomes described herein comprising any of the nucleic acid constructs described herein (see Sections 4.9, Section 4.10, and Section 4.11.1); and (ii) in a second step, a helper virus (HV); (b) culturing the cell line under conditions sufficient to encapsidate the HDAd genome into the HDAd virion capsid; (c) collecting the HDAd; and (d) purifying the HDAd, wherein the HDAd genome is intact (not recombined) in the HDAd virion.
  • HV helper virus
  • the introducing comprises two rounds of introducing (i) and (ii), where the second round of introducing is performed about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days after the first round of introducing.
  • the method of making a helper-dependent adenovirus (HDAd) virion produces HDAd virion that is not recombined.
  • the cell line suitable for making the HDAd is selected from: HEK293, 911, AE1-2A, and LP-293.
  • the cell line suitable for making the HDAd comprises a cell line engineered to control expression of the polynucleotide encoding the integration enzyme.
  • the cell line is engineered to express an shRNA that inhibits the expression of the integration enzyme.
  • the cell line is engineered to express an antibody specific to the integration enzyme.
  • the cell line used to make the HDAd is engineered to include a BxB1 shRNA, whereby RNA encoding BxB1 is prevented from being transcribed by the BxB1 shRNA.
  • the cell line used to make the HDAd is engineered to include an anti-BxB1 antibody, for example, an anti-BxB1 nanobody.
  • the cell line used to make the HDAd is engineered to include more than one anti-BxB1 antibody, such as a first anti-BxB1 nanobody and a second anti-BxB1 nanobody.
  • the method of making the HDAd includes introducing a helper virus (HV) into the cell line suitable for making HDAd.
  • Helper viruses (HVs) provide all the necessary viral proteins in trans, for example, they are used to package the recombinant genome into viral particles during production. In some cases, the HV genomes are not packaged due to the preferential packaging of the HDAd genome, as its packaging signal is flanked by the ITRs.
  • Helper viruses used for the production of HDAds are typically E1-deleted adenoviruses, and the selection of a specific helper virus depends on various factors such as the type of cell line used for production and the desired tropism of the resultant HDAd.
  • the packaging signal is flanked by loxP site and is excised in HEK293Cre cells, resulting the packaging of only HDAd, not the helper virus.
  • the helper virus is AdNG163 of Ad5 serotype, the detailed information of which can be found at Palmer and Ng; “Improved system for helper- dependent adenoviral vector production.” Mol Ther.
  • the HV comprises a variant packaging signal ( ⁇ ).
  • the packaging signal ( ⁇ ) is selected from an adenovirus 2 packaging single (Ad2 ⁇ ) and an adenovirus 5 packaging signal (Ad5 ⁇ ), or variants thereof.
  • the packaging signal ( ⁇ ) is an adenovirus 2 packaging single (Ad2 ⁇ ) or a variant thereof.
  • the packaging signal ( ⁇ ) is an adenovirus packaging single (Ad5 ⁇ ) or a variant thereof.
  • producing high-quality, high-titer HDAd vectors includes the removal of the HV after production. In some cases, this is accomplished through Cre-LoxP or FLP-FRT recombinase systems, which allow selective excision of the packaging signal from the HV genome, thus preventing its packaging into viral particles. In some embodiments, removal of the HV after virus production is achieved by purifying the HDAd from the HV.
  • the culturing comprises culturing the cell line in the presence of an agent that controls the controllable promoter of the second expression control sequence, thereby preventing expression of the polynucleotide sequence encoding the at least first integration enzyme.
  • Non-limiting examples of agents and controllable promoters that enable this type of control over expression of the polynucleotide encoding the at least first integration enzyme include tetracycline and a tetracyline-ON promoter.
  • the gene expression switch is the X on switch system as described in Monteys et al., “Regulated control of gene therapies by drug- induced splicing”; Nature.2021 Aug; 596(7871):291-295, the disclosure of which is incorporated by reference in its entirety.
  • the culturing comprises culturing the cell line in the absence of an agent that controls the controllable promoter the second expression control sequence, thereby preventing expression of the polynucleotide sequence encoding the at least first integration enzyme.
  • agents and controllable promoters that enable this type of control over expression of the polynucleotide encoding the at least first integration enzyme include tetracycline and a tetracyline-OFF promoter.
  • the concentration of agent that controls the controllable promoter is added to the culture medium or to the subject at concentrations that enable sufficient control over the integration enzyme.
  • the concentration of doxycycline added to the culture medium or to the subject is optimized to a concentration that allows sufficient induction of the mRNA encoding the integration enzyme at the desired times (e.g., after incorporation of the first integration recognition site).
  • This disclosure also features any of the HDAd made using the methods of any of the methods describe herein. 6 .11.4.
  • This disclosure features methods for site-specifically integrating an exogenous nucleic acid into a cellular genome at a desired target sequence, the method comprising: introducing into the cell any of the nucleic acid constructs described herein (see Sections 4.9, Section 4.10, and Section 4.11.1) or any of the HDAd virions described herein (see Section 4.11.2 and and HDAd virions produced in Section 4.11.3), and culturing the cell line in a cell culture medium under conditions sufficient to induce expression of the second expression control sequence, thereby driving expression of the integration enzyme.
  • culturing the cell in a cell culture medium under conditions sufficient to induce expression of the second expression control sequence comprises culturing the cell line in the presence an agent that controls the controllable promoter the second expression control sequence is added to the medium, thereby allowing expression of the polynucleotide sequence encoding the at least first integration enzyme.
  • culturing the cell in a cell culture medium under conditions sufficient to induce expression of the second expression control sequence comprises culturing the cell in line in the absence of an agent that controls the controllable promoter the second expression control sequence is added to the culture medium, thereby allowing expression of the polynucleotide sequence encoding the at least first integration enzyme.
  • This disclosure features in vivo methods for site-specifically integrating an exogenous nucleic acid into a cellular genome at a desired target sequence, the method comprising: administering to a subject: any of the HDAd virions described herein (see Section 4.11.2 and and HDAd virions produced in Section 4.11.3), and an agent that controls the controllable promoter of the second expression control, thereby allowing expression of the polynucleotide sequence encoding the at least first integration enzyme.
  • This disclosure features in vivo method for site-specifically integrating an exogenous nucleic acid into a cellular genome at a desired target sequence, the method comprising: administering to a subject: any of the HDAd virion described herein (see Section 4.11.2 and and HDAd virions produced in Section 4.11.3) in the absence of an agent that controls the controllable promoter the second expression control sequence, thereby allowing expression of the polynucleotide sequence encoding the at least first integration enzyme. 6 .12. Genes and Targets [0238] This disclosure provides compositions, systems and methods for correcting or replacing genes or gene fragments (including introns or exons) or inserting genes in new locations.
  • such a method 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 H11 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.
  • 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).
  • ORFs open reading frame
  • 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.
  • Inherited Retinal Diseases ⁇ Stargardt Disease (ABCA4) ⁇ Leber congenital amaurosis 10 (CEP290) ⁇ X linked Retinitis Pigmentosa (RPGR) ⁇ Autosomal Dominant Retinitis Pigmentosa (RHO) Liver Diseases: ⁇ Wilson’s disease (ATP7B) ⁇ Alpha-1 antitrypsin (SERPINA1) Intellectual Disabilities: ⁇ Rett Syndrome (MECP2) ⁇ SYNGAP1-ID (SYNGAP1) ⁇ CDKL5 deficiency disorder (CDKL5) Peripheral Neuropathies: ⁇ Charcot-Marie-Tooth 2A (MFN2) Lung Diseases: ⁇ Cystic Fibrosis (CFTR) ⁇ Alpha-1 Antitrypsin (SERPINA1) Autoimmune diseases: ⁇ IgA Nephropathy (Berger’
  • 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 (SCD) 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).
  • ORFs open reading frame
  • recombination will be into safe harbor sites (SHS).
  • a frequently used human SHS is the AAVS1 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). In some embodiments, the site is the human H11 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.
  • 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 (Factor IX) 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 OTCD
  • 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.
  • 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 HCU. Homocystinuria is elevation of the amino acid, homocysteine (protein building block coming from our diet) in the urine or blood.
  • HCU 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
  • problems with converting homocysteine to the amino acid methionine 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.
  • a functional copy of a gene e.g., CBS
  • 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.
  • IgA immunoglobulin A
  • 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.
  • native cells e.g., B cells
  • 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.
  • native cells e.g., B cells
  • LN Lupus Nephritis
  • 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
  • MN is a kidney disease that affects the filters (glomeruli) of the kidney and can cause protein in the urine, as well as decreased kidney function and swelling. It can sometimes be called membranous glomerulopathy as well (these terms can be used interchangeably and mean the same thing).
  • 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. 6 .13.
  • Methods of treatment [0267] In another aspect, methods of treatment are presented. The method 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.
  • 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.
  • Methods of non-viral delivery of the single nucleic acid construct described herein include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic 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., Transfectam and Lipofectin).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, 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). 6 .13.1. Lipid Nanoparticle Delivery [0270]
  • the single nucleic acid construct is packaged in a LNP and administered intravenously.
  • the single nucleic acid construct is packaged in a LNP and administered intrathecally.
  • the single nucleic acid construct is packaged in a LNP and administered by intracerebral ventricular injection.
  • the single nucleic acid construct is packaged in a LNP and administered by intracisternal magna administration. In some embodiments, the single nucleic acid construct is packaged in a LNP and administered by intravitreal injection.
  • the preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., 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.
  • 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, acetampinophen, 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. [0273]
  • 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.
  • 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.
  • ionizable cationic lipids Four species of ionizable cationic lipids have been focused upon, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2- dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-keto-N,N-dimethyl- 3-aminopropane (DLinKDMA), and 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).
  • DLinDAP 1,2-dilineoyl-3-dimethylammonium-propane
  • DLinDMA 1,2- dilinoleyloxy-3-N,N-dimethylaminopropane
  • DLinKDMA 1,2-dilinoleyloxy-keto-N,N-di
  • the 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 ⁇ g/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. In some embodiments, an ionizable lipid may be positively charged or negatively charged. In principle, there are no specific limitations concerning the ionizable lipids of the LNP compositions disclosed herein.
  • the one or more ionizable lipids are selected from the group consisting of 3-(didodecylamino)- N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4- tridodecyl-1,4-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-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl 4-(di
  • the ionizable lipid may be selected from, but not limited to, an ionizable lipid described in International Publication Nos. WO2013086354 and WO2013116126.
  • the lipid nanoparticle may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cationic and/or ionizable lipids.
  • Such cationic and/or ionizable lipids include, but are not limited to, 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2- (didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-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-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)but
  • 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.
  • amino lipids of the LNP compositions disclosed herein.
  • 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.
  • a protonatable tertiary amine e.g., pH-titratable
  • alkyl chains wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds
  • ether, ester, or ketal linkages between the head group and alkyl chains e.g., 1, 2, or 3
  • Such 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, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2-DMA, C12-200, cKK-E12, cKK-A12, cKK-O12, DLin-MC2- DMA (also known as MC2), and 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 lipid-containing composition e.g., LNPs
  • 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.
  • Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).
  • 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. [0286] In some embodiments, 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), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O- octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2- cholesterylhemisuccino
  • 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).
  • helper lipids of the LNP compositions there are no specific limitations concerning the helper lipids of the LNP compositions disclosed herein. Without being bound to any particular theory, it is believed that the mechanism by which the helper lipid enhances transfection includes enhancing particle stability. In some embodiments, the helper lipid enhances membrane fusogenicity.
  • the helper lipid of the LNP compositions disclosure herein can be any helper lipid known in the art. Non- limiting examples of 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), dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn- glycero-3-phosphocholine (DLPC), cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate.
  • the helper lipid of the LNP composition includes cholesterol.
  • the LNP composition comprises one or more structural lipids.
  • structural lipid refers to sterols and also to lipids containing sterol moieties. 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 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, camelid antibodies and fragments thereof, human antibodies and fragments thereof, monoclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies)).
  • ligands 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
  • 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: 180- 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, or derivatized lipophilic compounds, such as lipid-derivatized bleomycin, can be used.
  • 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. 6,027,726.
  • Examples of 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).
  • 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, me
  • 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.2010267:9-21; Akinc et al., Nat. Biotechnol.
  • 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.
  • 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.
  • effective delivery to myeloid cells, such as monocytes, lipidoid formulations may have a similar component molar ratio.
  • 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-(1-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.200917:872-879).
  • LNPs 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.
  • 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, double- stranded, 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.
  • 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 C2c1 or C2c3, and/or any of the present RNAs, for instance a guide RNA, can be delivered using any suitable vector, e.g., plasmid or viral vectors, such as adeno associated virus (AAV), lentivirus, adenovirus or other viral vector types, or combinations thereof.
  • AAV adeno associated virus
  • 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.
  • one or more other conventional pharmaceutical ingredients such as preservatives, humectants, suspending agents, surfactants, antioxidants, anticaking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc. may also be present, especially if the dosage form is a reconstitutable form.
  • 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 105 particles (also referred to as particle units, pu) of helper- dependent adenoviral vector.
  • the dose preferably is at least about 1 x 106 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 -1 x 10 11 particles or about 1 x 10 9 -1 x 10 12 particles), and most preferably at least about 1 x 10 10 particles (e.g., about 1 x 10 9 -1 x 10 10 particles or about 1 x 10 9 -1 x 10 12 particles), or even at least about 1 x 10 10 particles (e.g., about 1 x 10 10 -1 x 10 12 particles) of the helper-dependent adenoviral vector.
  • 1 x 106 particles for example, about 1 x 10 6 -1 x 10 11 particles
  • 1 x 10 8 particles e.g., about 1 x 10 8 -1 x 10 11 particles or about 1 x 10 9 -1 x 10 12 particles
  • 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 10 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 10 10 pu, about 2 x 10 10 pu, about 4 x 10 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 helper-dependent 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
  • the helper-dependent adenovirus is delivered via multiple doses.
  • 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.
  • 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.
  • 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. [0311]
  • 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 messenger RNA
  • Examples of 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, BR112016030852A2, and EP3362461A1.
  • 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.
  • cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3
  • 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 non- human 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.
  • 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.
  • 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. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease.
  • 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.
  • Example 1 Single nucleic acid construct comprising PASTE components and a nucleic acid cargo of interest that is capable of recombinase-mediated subsequence circularization effects targeted integration of the cargo into a genomic locus
  • a single construct “installer” that contains a prime editor fusion protein, an attachment site-containing guide RNA (atgRNA), a nickase guide RNA (ngRNA), an integrase, a recombinase, recombination target sites, integration target site, a DNA of interest, and flanking ITRs is designed (FIG. 1).
  • Example 2 Single nucleic acid construct comprising PASTE components and a nucleic acid cargo of interest that is capable of integrase- mediated subsequence circularization effects targeted integration of the cargo into a genomic locus
  • a single construct “installer” that contains a prime editor fusion protein, an attachment site-containing guide RNA (atgRNA), a nickase guide RNA (ngRNA), an integrase, integration target sites, a DNA of interest, and flanking ITRs is designed (FIG. 2).
  • integrase expression and binding at integrase recognition sites leads to self-circularization of a subsequence of the single nucleic acid construct.
  • Stepwise control of self-circularization followed by genomic integration is achieved by use of central dinucleotide matched orthogonal integrase target recognition sites (i.e., attB/attP pairs) (FIG. 3D and FIG. 4D). Additionally, use of a kinetically fast attB/attP pair integrated into the single nucleic acid construct allows self-circularization prior to genomic integration.
  • FIG. 4C Screening of attB/attP pairs is achieved through a pooled attB/attP dinucleotide orthogonality assay (FIG. 4C) and relative insertion preferences for all attB/attP dinucleotide pairs results shown in FIG. 4E.
  • Improved genomic integration occurs via the selection of attP/attB mutant pairs (FIG. 3A) that demonstrate improved integration efficiency (FIGs. 3B-C and FIGs. 4A-4B).
  • a DNA of interest e.g., gene contained within the self-circularized nucleic acid integrates into a genomic locus of interest via the integrase via the attP1/attB1 sites.
  • Genomic integration occurs at an attB1 integrase recognition target site (i.e., “beacon”) placed via prime editing or gene writing. 7 .3.
  • Example 3 Single nucleic acid construct comprising PASTE components wherein an integrase is linked to a prime editor and a nucleic acid cargo of interest that is capable of integrase-mediated subsequence circularization effects targeted integration of the cargo into a genomic locus [0322]
  • an attachment site-containing guide RNA atgRNA
  • a nickase guide RNA ngRNA
  • an integrase integration target sites, a DNA of interest, and flanking ITRs
  • installer prime editor-integrase fusion (Cas9-RT-Integrase) expression and binding at integrase recognition sites (attP2/attB2) leads to self-circularization of a subsequence of the single nucleic acid construct.
  • Stepwise control of self-circularization followed by genomic integration is achieved by use of central dinucleotide matched orthogonal integrase target recognition sites (i.e., attB/attP pairs) (FIG. 3D and FIG. 4D). Additionally, use of a kinetically fast attB/attP pair integrated into the single nucleic acid construct allows self-circularization prior to genomic integration. Screening of attB/attP pairs is achieved through a pooled attB/attP dinucleotide orthogonality assay (FIG. 4C) and relative insertion preferences for all attB/attP dinucleotide pairs results shown in FIG. 4E.
  • FIG. 4C pooled attB/attP dinucleotide orthogonality assay
  • FIG. 3A Improved genomic integration occurs via the selection of attP/attB mutant pairs (FIG. 3A) that demonstrate improved integration efficiency (FIG. 3B and FIG. 4B).
  • a DNA of interest e.g., gene contained within the self-circularized nucleic acid integrates into a genomic locus of interest via the integrase via the attP1/attB1 sites.
  • Genomic integration occurs at an attB1 integrase recognition target site (i.e., “beacon”) placed via prime editing mediated by the prime editor-integrase fusion.
  • FIG. 5 illustrates a schematic of single atgRNA and dual atgRNA approaches for beacon placement.
  • the single construct “installer” that contains a prime editor fusion protein linked to an integrase (FIG. 6), a first attachment site-containing guide RNA (atgRNA), a second attachment site-containing guide (atgRNA), an integrase, integration target sites, a DNA of interest, and flanking ITRs is designed.
  • the first atgRNA and the second atgRNAs collectively encode the entirety of the integration recognition site. 7 .4.
  • Example 4 Extrachromosomal circular DNA (EccDNA) sensor to evaluate template integrase-mediated circularization and programmable gene insertion within a ACTB beacon locus
  • EccDNA Extrachromosomal circular DNA
  • BxB1-mediated circularization of the EccDNA sensor which occurs at a attP’/attB’ target recognition site within the EccDNA sensor, orients the EF1 ⁇ promoter upstream of nanoluc and GFP, thereby allowing for dual reporter expression.
  • EccDNA circularization can also be confirmed by PCR amplification of the post-circularization attR’ scar using primers P1 and P2 as shown in FIG. 7.
  • Total EccDNA (linear and circularized) is quantified by primers P3 and P4 as shown in FIG. 7.
  • the EccDNA construct contains an orthogonal attP (GT central dinucleotide, see FIGs. 4A and 4D) to facilitate genomic insertion at a placed attB beacon site. Genomic integration of the EccDNA is verified using primers P5 and P6 (FIG. 7). [0327] A transfection screen was performed to confirm Bxb1-mediated EccDNA circularization (FIG. 8).
  • Plasmid expressed EccDNA sensor, prime editor protein, Bxb1, ACTB targeting atgRNA, and nicking guide RNA were transfected using Lipo3000 into HEK293T cells (200K cells in a 12-well plate). Cell samples were harvested 72 hours post transfection for circularization, beacon placement, and insertion analysis. [0328] As confirmed by ddPCR, transfection of both EccDNA sensor and Bxb1 resulted in confirmed intracellular circularization (FIG. 9). Circularization efficiency was >50% for Bxb1- containing samples tested at a 25,000-fold dilution, whereas equivalent samples that lacked BxB1 demonstrated ⁇ 1% circularization.
  • Beacon placement efficiency was >40% for samples containing the requisite beacon placement PE2/atgRNA/ngRNA components, however samples that also included Bxb1 demonstrated ⁇ 20% beacon placement. It is hypothesized that the drop in beacon placement efficiency is due an interaction between the plasmid-form atgRNA attB and the EccDNA AttP in the presence of BxB1.
  • FIG. 12 demonstrates programmable gene insertion of the EccDNA at the ACTB beacon locus was confirmed by ddPCR. 7 .5.
  • Example 5 Extrachromosomal circular DNA (EccDNA) sensor to evaluate template integrase-mediated circularization and p rogrammable gene insertion within a LMNB placed beacon
  • EccDNA Extrachromosomal circular DNA
  • a transfection screen was performed to confirm Bxb1-mediated EccDNA circularization and subsequent programmable gene insertion at a LMNB placed attB beacon site.
  • a linearized EccDNA sensor was tested in cell transfections (FIG. 13).
  • An EccDNA sensor called EccDNA-NC1 which lacks the attP’/B’ cognate pair was developed as a non-circularizing negative control.
  • LMNB targeting atgRNA and nicking guide RNA were transfected as synthetic RNAs (containing standard IDT chemical modifications).
  • Prime editor protein and Bxb1 effectors were transfected in plasmid form.
  • Transfection was conducted across 300,000 HEK293T cells in a 24-well plate format using Lipo3000 for plasmid delivery (PE2, BxB1, and EccDNA sensors) in conjunction with Lipo mRNAMAX for synthetic RNA delivery (atgRNA, ngRNA).
  • Cell samples were harvested 72 hours post transfection for circularization, beacon placement, and insertion analysis.
  • Intracellular circularization of the EccDNA sensor in the presence of BxB1 was confirmed via GFP expression (FIG. 14).
  • HDAds were produced using an HEK293Cre-P320 cells.
  • the HDAd vector used for HDAd virus production are as described in FIGs. 18A-18C.
  • the sequences of two exemplary HDAd are shown in Table 12 below.
  • the HDAd vector was transfected into the HEK293Cre-P320 using FectoVIR transfection reagent.
  • mScarlet expression indicates transfection of the HDAd vector.
  • HDAd Virus production was performed either in-house or at the University of Iowa Viral Vector core.
  • the HDAd plasmid was linearized by restriction enzyme and transfected into viral producer cell HEK293Cre cells (Passage 0, P0).
  • the cells were transduced with helper virus to package HDAd vector the following day and were harvested and lysed by freeze and thaw two days later.
  • the cell lysate and helper virus were used to co-infect new cells and amplify HDAd (P1). This step was repeated until HDAd reach high enough titer for large scale production.
  • the CsCl gradient and continuous centrifuge were performed to purify of HDAd vectors.
  • Quality control of HDAd vector production include measurement of titer (as measured by mScarlet expression in infected cells or nLuc in infected cells); ratio of intact versus recombined HDAd; helper virus titer; and ratio of helper virus (HV) to HDAd (see FIG. 21).
  • the intact all- in-one HDAd titer was measured by ddPCR with primer and probe targeting mScarlet gene.
  • All HDAd vector titer including intact and recombined HDAd was measured by ddPCR with Nanoluc- specific primer and probe. Because the recombination of all-in-one HDAd would delete the mScarlet-containing fragment, the mScarlet gene was used as the indicator of intact all-in-one HDAd genome DNA.
  • FIG. 21 shows that about 2% of the HDAd produced at the University of Iowa Viral Vector core was intact HDAd. [0338] The HDAd virus produced accordingly by the University of Iowa showed recombination. For these experiments, the HDAd was subject to restriction endonuclease digestion with various restriction enzymes (see Table 13). Table 13.
  • FIG. 23 shows a schematic of the HDAd construct and the AttB and AttP sites that recombined.
  • FIG. 25B shows a schematic of the HDAd vector with the deleted components in red.
  • FIG. 25C shows a sequence alignment of the query sequence (HDAd vector sequence without recombination “AdVG012 ref”) and the subject (HDAd vector sequence with recombination “AdVG012 UI vDNA”). The boxed sequences highlight the recombination and deletion of RT, Cas9, BxB1, tTA, and mScarlet.
  • FIG. 26 shows results of Sanger sequencing of viral DNA between atgRNA AttB (first integration recognition site) and cargo AttP (second integration recognition site). It is noted that the cargo sequence is included in the “all-in-one HDAd construct” and was packaged into HDAd vector as viral genomic DNA.
  • the cargo DNA is part of the HDAd vector genome DNA. 7 .7.
  • Example 7 Assessment of Beacon Placement and PGI using HDAd
  • the HDAd produced in Example 6 was assessed for both beacon placement and PGI in HEK293A cells.
  • the HDAd was added to the HEK293A cells at an MOI of 10, 100, or 1000.
  • the HDAd include atgRNAs having spacer sequences with complementarity to hF9, whereby the first integration recognition site is integrated into the hF9 locus.
  • Genomic DNA was harvested at day 3 and day 6 post infection. Beacon placement as shown in FIG. 27A was highest for the cells infected at an MOI 1000.
  • Example 8 Assessment of Beacon Placement and PGI using HDAd in HEK293-AttB cells [0347] The HDAd produced in Example 6 was assessed for both beacon placement and PGI in HEK293-AttB cells. [0348] For these experiments, the HDAd was added to the HEK293-AttB cells at an MOI of 10, 100, or 1000.
  • HEK293-AttB cells are HEK293 cells having an AttB sequence integrated into the genome of the HEK293 cellular genome.
  • Genomic DNA was harvested at day 3 and day 6 post infection. Beacon placement as shown in FIG. 28A was highest for the cells infected at an MOI 1000 at day 3 and highest for the cells infected at an MOI 100 at day 6. The MOI of 1000 also produced the highest levels of PGI for both time points (FIG.28B). Overall, this data shows that a higher MOI produced significantly more PGI despite being comparable with an MOI 100 for beacon placement. 7.9.
  • Example 9 Assessment of Beacon Placement and PGI using HDAd at a HEK293-lenti-attB site
  • the HDAd produced in Example 6 was assessed for both beacon placement and PGI in HEK293 comprising a lenti-AttB locus (HEK293-lentiAttB).
  • the HDAd was added to the HEK293-lentiAttB cells at an MOI of 10, 100, or 1000.
  • HEK293-lenti-AttB are HEK293 cells having an AttB sequence integrated into the genome of the HEK293 cells where the AttB was integrated via a lentivirus.
  • FIG. 29D shows the ddPCR data for ddPCR data.
  • the data shows AttB-AttP recombination in each sample but in varying amounts (FIG. 29D).
  • AttB 30 describes the competition between intramolecular recombination (i.e., recombination between the AttB and the AttP sites present in the nucleic acid construct) and recombination between an integrated AttB ant the AttP site in the nucleic acid construct.
  • Tetracyline inducibility of BxB1 reduces intramolecular recombination by ensuring that BxB1 is not expressed until after integration of the AttB site. 7.10.
  • Example 10 Assessment of HDAd AIO constructs with intron-containing Bxb1 [0354]
  • HDAd AIO plasmids with chimeric Bxb1-intron were tested for reducing the intramolecular recombination (i.e., recombination between the AttB and the AttP sites present in the nucleic acid construct).
  • the vector design is shown is FIG. 31.
  • AdVG068 includes a Bxb1 with a chimeric intron, under the control of a Tet-off promoter.
  • HDAd vectors AdVG092 (F22+R33), AdVG093 (F22+R29), and AdVG102 (F29+R29), includes a Bxb1 with a chimeric intron, under the control of a Tet-on promoter.
  • the sequences of HDAd vectors AdVG092, AdVG093, and AdVG102 and the Bxb1 with a chimeric intron are shown in Table 15 below.
  • 35A-B show the percentage of BP and PGI with AdVG102, AdVG092, and AdVG093 respectively.
  • high beacon placement efficiency up to 13%) was achieved and the percentage of beacon placement was higher for the group 3, when Dox was added 3 days after transfection instead of 6 hours.
  • FIGs. 33B, 34B, and 35B show that PGI was tightly regulated by the presence of Dox and there was no PGI in the absence of Dox.
  • neither BP nor PGI showed dose-dependent effect according to the concentration of Dox.
  • the overall PGI was low for the vectors tested, which may be due to limited DNA donor delivered to cell nucleus via transfection or low Bxb1 activity from TetOn promoter.
  • Example 11 Generation of Bxb1-shRNA cell line [0360] To further inactivate Bxb1 in HDAd producer cells, 116 Bxb1-shRNA cell line was generated.
  • the Bxb1-shRNA design is shown in FIG. 38 and the Bxb1-shRNA was expressed from human U6 promoter.
  • Lentiviral vectors containing shRNA were generated by GenScript. The selection marker was Puromycin.
  • HEK293Cre cells (116 cell) were transduced with lentivirus with Bxb1-shRNA (MOI 2, 50). The Bxb1 expression level and activity was determined by Western blot and DNA cargo circularization efficiency. Single cell clones were generated by serial dilution and cultured in 96 well plate.
  • 116 Bxb1-shRNA cell clones 2-2 and 2-4 showed the highest knockdown efficiency as determined by Western blot and DNA cargo circularization assay. Furthermore, the cell line derived from 116 Bxb1-shRNA cell clone 2-2 supported HDAd AIO amplification. The HDAd AIO reporter gene mScarlet increased along with each passage in the 116 Bxb1-shRNA cell line, but not the control 116 cells without Bxb1-shRNA (FIG. 40).
  • HDAd AIO vector AdVG012 was tested in primary human hepatocytes (PHHs). The virus was dosed at 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, and 0 MOI (based on intact titer, i.e., viruses with intact genome) per cell, respectively. The PHHs were collected five days after the infection. The sequence of HDAd vector AdVG012 is shown in Table 17 below.
  • Total virus titer was determined by ddPCR targeting NanoLuc gene, which was present in all viruses, including recombined viruses. The ratio of the intact virus over the total virus is the purity of virus with intact viral genome. As shown in FIG. 41, titering the total DNA extract showed about 2% pure virus out of total virus.
  • Primary human hepatocytes (PHHs) were seeded onto 96-well collagen-coated tissue culture plates and treated with AIO HDAD viral vector at doses of 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, and 0 particles (pure virus) per cell respectively, in addition to no virus control two days after plating.
  • Promega Cell Titer Glo 2 (CTG) assay was performed 5 days after viral transduction to assess cell viability. 100 ⁇ L of CTG reagent was added to each well after media aspiration and the level of luminescence was read on Promega Glomax plate reader. The reduction of luminescence in the CTG assay indicates toxicity. FIG. 42A indicates that the virus was well tolerated at lower doses. However, it reduced cell viability by approximately 60% at the highest dose tested (1000 MOI). [0366] The viral copy number per cell genome, as an estimation of virus transduction efficiency, was assessed using Bio-Rad QX200 ddPCR system. Total DNA was extracted from PHHs 5 days after viral transduction using Quick Extract and purified using SPRI magnetic bead cleanup.
  • FIG. 42B shows that more copies of virus were retained in cell with increased intact virus MOI. The average transduction efficiency was about 10%.
  • the percentage of beacon placement and attL integration was assessed by ddPCR normalized to hF9 reference assay.
  • FIGs. 43A and 43B show that successful editing was achieved with the HDAd AIO construct AdVG012 in primary human hepatocytes (PHHs).

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Abstract

La présente invention concerne des constructions d'acide nucléique (par exemple, des vecteurs HDAd), un ensemble de constructions d'acide nucléique (par exemple, un ou plusieurs vecteurs HDAd), des virions HDAd et un virus HDAd comprenant tous les composants pour intégrer spécifiquement un acide nucléique exogène dans un génome cellulaire à une séquence cible souhaitée. La présente divulgation concerne également leurs procédés d'utilisation.
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