WO2023205744A1 - Programmable gene insertion compositions - Google Patents

Programmable gene insertion compositions Download PDF

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Publication number
WO2023205744A1
WO2023205744A1 PCT/US2023/066024 US2023066024W WO2023205744A1 WO 2023205744 A1 WO2023205744 A1 WO 2023205744A1 US 2023066024 W US2023066024 W US 2023066024W WO 2023205744 A1 WO2023205744 A1 WO 2023205744A1
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linker
integration
gene editor
gene
reverse transcriptase
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PCT/US2023/066024
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French (fr)
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Jonathan Douglas FINN
Sandeep Kumar
Kaveh DANESHVAR
Christopher Libunao BARTOLOME
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Tome Biosciences, Inc.
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Publication of WO2023205744A1 publication Critical patent/WO2023205744A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
    • 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
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • Gene editor compositions are described herein that allow for site-specific integration recognition site placement within a genome (i.e., beacon placement), for example by Cas-reverse transcriptase activity, and subsequent genomic integration at the integrated integration recognition site via integrase activity of the gene editor composition.
  • the gene editor polypeptide can be comprised of a (i) DNA binding nickase, (ii) a reverse transcriptase, and (iii) an integration enzyme (i.e., an integrase or a recombinase), wherein at least any two elements of (i), (ii), or (iii) are linked via at least one C-terminal linker.
  • the linkers are peptide linkers that may be cleavable (i.e., ribosome skipping site, peptide cleavage site, etc.) or non-cleavable.
  • the gene editor polypeptide may be comprised of one or more cleavable and/or non-cleavable linkers at any given linkage position connecting at least any two elements of (i), (ii) or (iii).
  • a unique N-terminus to C-terminus configuration of elements (i), (ii), and (iii) is provided herein.
  • the polypeptide compositions described herein can be expressed from and/or delivered as a polynucleotide encoding the polypeptide. Further, an expression vector comprised of the polynucleotide encoding the polypeptides is described herein.
  • the present disclosure provides nucleic acid compositions, methods, and an overall platform for site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE) (see lonnidi et al.,' doi: 10.1101/2021.11.01.466786; the entirety of which is incorporated herein by reference), transposon-mediated gene editing, or other suitable gene editing, or gene incorporation technology.
  • PASTE Programmable Addition via Site-Specific Targeting Elements
  • this disclosure features a gene editor polypeptide, comprising: a DNA binding nuclease, optionally comprising a nickase activity; a reverse transcriptase; an integration enzyme; and wherein at least any two of elements (i), (ii), or (iii) are linked via at least a first C- terminal linker.
  • the gene editor polypeptide further comprises a second linker.
  • the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme.
  • the C-terminus of the integration enzyme is linked to the DNA binding nuclease, wherein the C-terminus of the DNA binding nuclease is linked to a reverse transcriptase.
  • the at least first linker, the second linker, or both is selected from a flexible linker, a semi-flexible linker, a rigid linker, a flexible-rigid linker.
  • the flexible linker is selected from: LI, L2, L5, L6, and L13.
  • the semi-flexible linker is selected from: L7 and L8.
  • the rigid linker is selected from: L3, L4, and L9.
  • the flexible-rigid linker is selected from LI 1 and L12.
  • the length of the at least first linker, the length of the second linker, or both is selected from short, medium, and long.
  • the short linker is selected from: LI, L3, L7, and L8.
  • the medium linker is selected from: L2, L4, L5, L6, L10, LI 1, L12, and L13.
  • the long linker is L9.
  • the at least first linker, the second linker, or both is a non-cleavable linker.
  • the non-cleavable linker is selected from: LI, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, and L13.
  • the at least first linker, the second linker, or both is a cleavable linker.
  • the cleavable linker is selected from: a self-cleavable linker, an enzyme cleavable linker, and chemically-induced cleavable linker.
  • the cleavable linker is a self-cleavable linker.
  • the self-cleavable linker is selected from P2A, E2A, F2A, and T2A.
  • the at least first linker is a non-cleavable linker and the second linker is a cleavable linker; the at least first linker is a cleavable linker and the second linker is a non-cleavable linker; the at least first linker is a cleavable linker and the second linker is a cleavable linker; or the at least first linker is a non-cleavable linker and the second linker is a non- cleavable linker.
  • the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L7, L10, or L12 links the DNA binding nuclease to the reverse transcriptase.
  • the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L8, L9, LIO, LI 2, or L13 links the reverse transcriptase to the integration enzyme.
  • the C-terminus of the integration enzyme is linked to the DNA binding nuclease by L13, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by LI 3.
  • the gene editor polypeptide is not comprised of aN-mostN-terminal reverse transcriptase.
  • the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by L13, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme by a cleavable linker.
  • the gene editor polypeptide further comprises of at least one nuclear localization signal.
  • this disclosure features a gene editor polypeptide comprising: from N- terminus to C-terminus: a DNA binding nuclease; a P2A linker, a reverse transcriptase; a mXTEN linker, and an integration enzyme.
  • this disclosure features a gene editor polypeptide comprising:
  • an integration enzyme from terminus to C-terminus: an integration enzyme, a mXTEN linker, a DNA binding nuclease; a mXTEN linker, and a reverse transcriptase.
  • this disclosure features a polynucleotide encoding any of the gene editor polypeptides described herein.
  • this disclosure features an expression vector comprising any of the polynucleotides described herein.
  • the expression vector comprises any of the polynucleotides described herein operably linked to an expression control element.
  • the expression control element is a promoter.
  • any of the polynucleotides described herein further comprise one or more sequences encoding an atgRNA, nicking gRNA (ngRNA), donor polynucleotide template, or donor polynucleotide template complement.
  • any of the expression vectors described herein further comprise one or more sequences encoding an atgRNA, nicking gRNA (ngRNA), donor polynucleotide template, or donor polynucleotide template complement.
  • the expression vector is recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, annelovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, or lipid nanoparticle.
  • this disclosure a system capable of site-specifically integrating an exogenous nucleic acid sequence into a genome of a cell, the system comprising: any the gene editor polypeptides described herein or any of the polynucleotides described herein.
  • this disclosure features a method of site-specifically integrating an exogenous nucleic acid sequence into a genome of a cell, the method comprising: incorporating an integration recognition site into a cell by delivering: any of the polynucleotides described herein; at least a first atgRNA comprising a first integration recognition site; optionally, a nicking gRNA; and integrating an exogenous nucleic acid sequence into the cellular genome by delivering into the cell concurrently with step (a): a donor polynucleotide template, wherein the donor polynucleotide template is comprised of the exogenous nucleic acid sequence positioned relative to a second integration recognition site that is a cognate pair of the first integration recognition site such that the exogenous nucleic acid sequence is integrated into the cellular genome at the first integration target recognition site when contacted with the integration enzyme and the first integration recognition site.
  • this disclosure features a method of site-specifically integrating an exogenous nucleic acid sequence into a target nucleic acid, the method comprising: incorporating an integration recognition site into a cell by delivering: any of the expression vectors described herein; and integrating an exogenous nucleic acid sequence into the cellular genome by integrating a donor polynucleotide template, wherein the donor polynucleotide template is comprised of the exogenous nucleic acid sequence positioned relative to a second integration recognition site that is a cognate pair of the first integration recognition site such that the exogenous nucleic acid sequence is integrated into the cellular genome at the first integration target recognition site when contacted with the integration enzyme and the first integration recognition site.
  • FIG. 1 illustrates an exemplary gene editor composition expression construct with identified linker positions.
  • Linker 1 position 1 (Pl).
  • Linker 2 position 2 (P2).
  • FIG. 2 illustrates an exemplary gene editor composition with identified linker positions.
  • Linker 1 position 1 (Pl).
  • Linker 2 position 2 (P2).
  • FIG. 3 illustrates non-cleavable peptide linkers used in the linker screen.
  • FIG. 4 illustrates linker screen experimental conditions and setup.
  • FIG. 5 illustrates a single plate linker screen with programmable gene insertion (i.e., precent of donor polynucleotide template encoding GFP inserted into ACTB locus) as a functional readout.
  • Linkers tested were selected from FIG. 3. Each column indicates the combination of linkers (e.g., Pl position/P2 position).
  • Dotted line indicates precent integration for a gene editor polypeptide having an mXTEN (i.e., L13) / mXTEN (L13) linker combination (e.g., Cas9- mXTEN-RT-mXTEN-Bxbl). * indicates significant increase compared to dotted line.
  • FIG. 6 illustrates a single plate linker screen with programmable gene insertion (i.e., precent of donor polynucleotide template encoding GFP inserted into ACTB locus) as a functional readout.
  • Linkers tested were selected from FIG. 3. Each column indicates the combination of linkers (e.g., Pl position/P2 position).
  • Dotted line indicates precent integration for a gene editor polypeptide having an mXTEN (i.e., L13) / mXTEN (L13) linker combination (e.g., Cas9- mXTEN-RT-mXTEN-Bxbl). * indicates significant increase compared to dotted line.
  • FIG. 7 illustrates a single plate linker screen with programmable gene insertion (i.e., precent of donor polynucleotide template encoding GFP inserted into ACTB locus) as a functional readout.
  • Linkers tested were selected from FIG. 3. Each column indicates the combination of linkers (e.g., Pl position/P2 position).
  • Dotted line indicates precent integration for a gene editor polypeptide having an mXTEN (i.e., L13) / mXTEN (L13) linker combination (e.g., Cas9- mXTEN-RT-mXTEN-Bxbl). * indicates significant increase compared to dotted line.
  • FIG. 8 illustrates non-limiting examples of protein domain configurations that include a DNA binding nuclease (e.g., Cas9), a reverse transcriptase (RT), and an integration enzyme (e.g., Bxbl).
  • mXTEN refers to a L13 linker (SEQ ID NO: 558) (see FIG. 3).
  • P2A refers to a porcine teschovirus-1 2A peptide (SEQ ID NO: 13).
  • FIGs. 9A-9B show percent integration (i.e., percent PASTE) for various gene editor polypeptide configurations where the DNA binding nuclease, the RT, and the integration enzyme are linked via the indicated cleavable and non-cleavable linkers.
  • FIG. 9A shows percent integration as a functional indicator of programmable gene insertion (i.e., precent of donor polynucleotide template encoding GFP inserted into ACTB locus).
  • FIG. 9B shows percent integration as a functional indicator of programmable gene insertion (i.e., precent of donor polynucleotide template encoding GFP inserted into ACTB locus).
  • FIG. 10A-10E shows analysis of AttP variants.
  • FIG. 10A shows a non-limiting schematic of AttP mutations tested for improving integration efficiency (SEQ ID NOS: 394 and 540-542, respectively, in order of appearance).
  • FIG. 10B shows integration efficiencies of wildtype and mutant AttP sites across a panel of AttB lengths.
  • FIG. 10C shows a non-limiting schematic of multiplexed integration of different cargo sets at specific genomic loci. Three fluorescent cargos (GFP, mCherry, and YFP) are inserted orthogonally at three different loci (ACTB, LMNB1, NOLC1) for in-frame gene tagging.
  • FIG. 10A-10E shows analysis of AttP variants.
  • FIG. 10A shows a non-limiting schematic of AttP mutations tested for improving integration efficiency (SEQ ID NOS: 394 and 540-542, respectively, in order of appearance).
  • FIG. 10B shows integration efficiencies of wildtype and mutant AttP sites across
  • FIG. 10D shows orthogonality of top 4 AttB/AttP dinucleotide pairs evaluated for GFP integration with PASTE at the ACTB locus.
  • FIG. 11 illustrates a schematic of single atgRNA and dual atgRNA approaches for step 1 : beacon placement (“integration recognition site”) and step 2: gene insertion, thereby illustrating PASTE.
  • gene editor polypeptides comprising a DNA binding nuclease domain (e.g., comprising nickase activity), a reverse transcriptase (RT), and an integration enzyme used for programmable gene insertion/PASTE.
  • a DNA binding nuclease domain e.g., comprising nickase activity
  • RT reverse transcriptase
  • integration enzyme used for programmable gene insertion/PASTE.
  • the DNA binding nuclease, the RT, and the integration enzyme are linked (e.g., fused) via at least a first C-terminal linker.
  • the gene editor polynucleotide also includes a second C-terminal linker.
  • a linker in a gene editor polypeptide can serve as a spatial buffer that helps maintain the structural and functional integrity of each of the DNA binding nuclease domain, the RT, and the integration enzyme. As described herein, the linker enabled each of these domains to perform their respective roles without hindrance, thereby preserving their biological activities.
  • the gene editor polypeptide includes linkers designed to be cleavable, allowing for the selective separation of the fused proteins if desired.
  • this disclosure is based in part on the finding that gene editor polypeptides configured in various orientations and comprising different linkers and different combinations of linkers (e.g., a gene editor polypeptide that includes a first linker and a second linker where the first and second linkers are different types of linkers or is a linker with the same amino acid sequence) performed with varying efficiencies (e.g., varying percent integration of donor polynucleotide templates).
  • this disclosure identified preferred orientations and preferred linkers that when combined resulted in improved integration efficiency compared to the other orientation and linker combinations tested..
  • 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.
  • Such an enzyme or enzyme fusion may contain DNA or RNA targetable nuclease protein (i.e., Cas protein, ADAR, or AD AT), wherein target specificity is mediated by a complexed nucleic acid (i.e., guide RNA).
  • Such an enzyme or enzyme fusion may be a DNA/RNA targetable protein, wherein target specificity is mediated by internal, conjugated, fused, or linked amino acids, such as within TALENs, ZFNs, or meganucleases.
  • 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, or targeted nickase activity (or cleaves 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 a prime editor fusion protein or a gene writer fusion protein.
  • Prime editing uses CRISPR enzyme 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.
  • the nickase is programmed (directed) with a prime-editing guide RNA (pegRNA).
  • pegRNA prime-editing guide RNA
  • attachment site containing guide RNA that both specifies the target and encodes for the desired integrase target recognition site.
  • the nickase may be programmed (directed) with an atgRNA.
  • 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 atgRNA (pegRNA), whereby a nick or single stranded cut occurs.
  • the reverse transcriptase domain then uses the atgRNA (pegRNA) to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand.
  • the edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand.
  • the prime editor (PE) guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process (typically achieved with a nickase gRNA).
  • Other enzymes that can be used to nick or cut only a single strand of double stranded DNA includes a cleavase (e.g., cleavase I enzyme).
  • an additional agent or agents may be added that improve the efficiency and outcome purity of the 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.
  • the dominant negative MMR protein is MLHldn.
  • the MMR-inhibiting agent is incorporated into the single nucleic acid construct design described herein that encodes any of the gene editor polypeptides described herein.
  • the MMR-inhibiting agent is linked or fused to the prime editor protein fusion, which may or may not have a linked or fused integrase.
  • the MMR-inhibiting agent is linked or fused to the Gene WriterTM protein, which may or may not have a linked or fused integrase.
  • the prime editor or gene editor system can be used to achieve DNA deletion and replacement.
  • the DNA deletion replacement is induced using a pair of atgRNA 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 a/.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 prime editor (PE-Cas9) system (i.e., PED AR 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 used in PrimeDel, TwinPE (WO2021226558 incorporated by reference herein in its entirety), or PED AR, the prime 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 single nucleic acid construct described herein.
  • 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(D10A).
  • a CRISPR enzyme such as a CRISPR enzyme nickase, such as Cas9 (wild type), Cas9(H840A), Cas9(D10A) 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(D10A) 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, V129P, L139P, T197A, 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
  • Feline leukemia virus reverse transcriptase 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 lonnidi et al.
  • PE3, PE3b, PE4, PE5, and/or PEmax which a skilled person can incorporate into a single nucleic acid construct described herein, involves nicking the non-edited strand, potentially causing the cell to remake that strand using the edited strand as the template to induce HR.
  • the nicking of the non-edited strand can involve the use of a nicking guide RNA (ngRNA).
  • ngRNA nicking guide RNA
  • Prime editors or gene editors can be found in the following: W02020/191153, W02020/191171, WO2020/191233,
  • the prime editor protein Prior to RT-mediated edit incorporation, 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
  • meganucleases meganucleases
  • 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 reconstituted via nanobody binding ALFA-tagged proteins.
  • the split construct can be adapted into one or more single nucleic acid polynucleotides.
  • 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 die Cas9, RT, and integrase, may be selected from Table 3:
  • 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 nucleic acid constructs 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 RECI (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.
  • the REC lobe includes the RECI 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 [0074] PAM-interacting domain'.
  • the NUC lobe contains the PAM-interacting (PI) domain that is positioned to recognize the PAM sequence on the noncomplementary DNA strand.
  • the PI domain of SpCas9 is required for the recognition of 5’-NGG-3’ PAM, and deletion of the PI domain (A1099-1368) abolished the cleavage activity, indicating that the PI domain is critical for SpCas9 function and a major determinant for the PAM specificity.
  • RuvC domain- The RuvC nucleases of SpCas9 have an RNase H fold and four catalytic residues, AsplO (Ala), Glu762, His983, and Asp986, that are critical for the two-metal cleavage of the noncomplementary strand of the target DNA.
  • AsplO AsplO
  • 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 a42 and a43) and the PI domain/stem loop 3 (P hairpin formed by P3 and [34).
  • SpCas9 HNH nucleases have three catalytic residues, Asp839, His840, and Asn863 and cleave the complementary strand of the target DNA through a single-metal mechanism.
  • sgRNA:DNA recognition' The sgRNA guide region is primarily recognized by the REC lobe.
  • the backbone phosphate groups of the guide region interact with the RECI domain (Argl65, Glyl66, Arg403, Asn407, Lys510, Tyr515, and Arg661) and the bridge helix (Arg63, Arg66, Arg70, Arg71, Arg74, and Arg78).
  • the 20-hydroxyl groups of Gl, Cl 5, U16, and G19 hydrogen bond with Vai 1009, Tyr450, Arg447/Ile448, and Thr404, respectively.
  • SpCas9 recognizes the guide:target heteroduplex in a sequence-independent manner.
  • the backbone phosphate groups of the target DNA (nucleotides 1, 9-11, 13, and 20) interact with the REC 1 (Asn497, Trp659, Arg661, and Gln695), RuvC (Gln926), and PI (Glul 108) domains.
  • the C2’ atoms of the target DNA form van der Waals interactions with the RECI domain (Leul69, Tyr450, Met495, Met694, and His698) and the RuvC domain (Ala728).
  • the terminal base pair of the guide:target heteroduplex (Gl :C20’) is recognized by the RuvC domain via end-capping interactions; the sgRNA G1 and target DNA C20’ nucleobases interact with the Tyrl013 and Vai 1015 side chains, respectively, whereas the 20-hydroxyl and phosphate groups of sgRNA G1 interact with Vai 1009 and Gln926, respectively.
  • nucleobases of G21 and U50 in the G21 :U50 wobble pair stack with the terminal C20:G10 pair in the guide:target heteroduplex and Tyr72 on the bridge helix, respectively, with the U50 04 atom hydrogen bonded with Arg75.
  • A51 adopts the syn conformation and is oriented in the direction opposite to U50.
  • the nucleobase of A51 is sandwiched between Phel 105 and U63, with its Nl, N6, and N7 atoms hydrogen bonded with G62, Glyl l03, and Phel 105, respectively.
  • Stem-loop recognition' Stem loop 1 is primarily recognized by the REC lobe, together with the PI domain.
  • the backbone phosphate groups of stem loop 1 (nucleotides 52, 53, and 59- 61) interact with the RECI domain (Leu455, Ser460, Arg467, Thr472, and Ile473), the PI domain (Lysl 123 and Lysl 124), and the bridge helix (Arg70 and Arg74), with the 20-hydroxyl group of G58 hydrogen bonded with Leu455.
  • A52 interacts with Phel 105 through a face-to-edge p-p stacking interaction, and the flipped U59 nucleobase hydrogen bonds with Asn77.
  • the single-stranded linker and stem loops 2 and 3 are primarily recognized by the NUC lobe.
  • the backbone phosphate groups of the linker (nucleotides 63-65 and 67) interact with the RuvC domain (Glu57, Lys742, and Lysl097), the PI domain (Thrl l02), and the bridge helix (Arg69), with the 20-hydroxyl groups of U64 and A65 hydrogen bonded with Glu57 and His721, respectively.
  • the C67 nucleobase forms two hydrogen bonds with Vail 100.
  • Stem loop 2 is recognized by Cas9 via the interactions between the NUC lobe and the non-Watson-Crick A68:G81 pair, which is formed by direct (between the A68 N6 and G81 06 atoms) and water-mediated (between the A68 Nl and G81 Nl atoms) hydrogen-bonding interactions.
  • the A68 and G81 nucleobases contact Serl351 and Tyrl356, respectively, whereas the A68:G81 pair interacts with Thrl358 via a water-mediated hydrogen bond.
  • the 20-hydroxyl group of A68 hydrogen bonds with Hisl349, whereas the G81 nucleobase hydrogen bonds with Lys33.
  • Stem loop 3 interacts with the NUC lobe more extensively, as compared to stem loop 2.
  • the backbone phosphate group of G92 interacts with the RuvC domain (Arg40 and Lys44), whereas the G89 and U90 nucleobases hydrogen bond with Glnl272 and Glul225/Alal227, respectively.
  • the A88 and C91 nucleobases are recognized by Asn46 via multiple hydrogenbonding interactions.
  • Cas9 proteins smaller than SpCas9 allow more efficient packaging of nucleic acids encoding CRISPR systems, e.g., Cas9 and sgRNA into one rAAV (“all-in-one- AAV”) particle.
  • efficient packaging of CRISPR systems can be achieved in other viral vector systems (i.e., lentiviral, integration deficient lentiviral, hd-AAV, etc.) and non viral vector systems (i.e., lipid nanoparticle).
  • Small Cas9 proteins can be advantageous for multidomain-Cas-nuclease-based systems for prime editing.
  • Cas9 proteins include Staphylococcus aureus (SauCas9, 1053 amino acid residues) and Campylobacter jejuni (CjCas9, 984 amino residues).
  • SerCas9 Staphylococcus aureus
  • CjCas9 Campylobacter jejuni
  • Staphylococcus lugdunensis (Siu) Cas9 as having genome-editing activity and provided homology mapping to SpCas9 and SauCas9 to facilitate generation of nickases and inactive (“dead”) enzymes (Schmidt et al., 2021, Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases. Nat Commun 12, 4219. doi.org/10.1038/s41467-021-24454-5) and engineered nucleases with higher cleavage activity by fragmenting and shuffling Cas9 DNAs.
  • the small Cas9s and nickases are useful in the instant disclosure.
  • 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 (SEQ ID NO: 18).
  • 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.
  • prime editors utilized herein comprise CRISPR-Cas system enzymes other than type II enzymes.
  • 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.
  • prime editors comprise a nickase or a dead CRISPR with nuclease function comprised in a different component.
  • the nucleic acid programmable DNA binding proteins utilized herein include, without limitation, Cas9 (e.g., dCas9 and nCas9), Casl2a (Cpfl), Casl2bl (C2cl), Casl2b2, Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), C2c4, C2c5, C2c8, C2c9, C2cl0, Cast 3a (C2c2), Cast 3b (C2c6), Cast 3c (C2c7), Cast 3d, and Argonaute.
  • Cas9 e.g., dCas9 and nCas9
  • Casl2a Casl2a
  • Casl2a Casl2a
  • Casl2bl Casl2b2cl
  • Casl2c3 Casl2d
  • CasY Casl2e
  • CasX
  • 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.l. 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, Casl2a (Cpfl)).
  • Casl2a (Cpfl) 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 Casl2a (Cpfl) mediates robust DNA interference with features distinct from Cas9.
  • Casl2a (Cpfl) is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T- rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpfl cleaves DNA via a staggered DNA double-stranded break.
  • Cpfl proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpfl in complex with guide RNA and target DNA.” Cell (165) 2016, p.949-962; the entire contents of which is hereby incorporated by reference.
  • prime editors used herein comprise the type V CRISPR family includes Francisella novicida U112 Cpfl (FnCpfl) also known as FnCasl2a.
  • FnCpfl 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 (RECI 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 FnCasl2a 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 FnCpfl residues in the WED and RECI domains.
  • FnCasl2a-crRNA complex further reveals that the bases of the seed sequence are solvent exposed and poised for hybridization with target DNA.
  • Structural aspects of FnCpfl are described by Swarts et al., Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Casl2a, Molecular Cell 66, 221-233, April 20, 2017.
  • the crRNA-target DNA strand heteroduplex is enclosed in the central cavity formed by the REC and NUC lobes and interacts extensively with the RECI 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 RECI domains.
  • the 5’-TTN-3’ PAM is recognized in FnCasl2a by a mechanism combining the shapespecific 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-7t stacking interaction with the dA0-dT0* base pair.
  • the phosphate group linking target strand residues dT(-l) and dTO is coordinated by hydrogen-bonding interactions with the side chain of Lys823 and the backbone amide of Gly826.
  • Target strand residue dT(-l) bends away from residue TO, allowing the target strand to interact with the seed sequence of the crRNA.
  • the non-target strand nucleotides dTl*-dT5* interact with the Arg692- Ser702 loop in FnCasl2a 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 FnCasl2a 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.
  • FnCpfl 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 Cpfl/Casl2a enzymes.
  • Another type V CRISPR is AsCpfl from Acidaminococcus sp BV3L6 (Yamano et al., Crystal structure of Cpfl in complex with guide RNA and target DNA, Cell 165, 949-962, May 5, 2016)
  • the nuclease comprises a Casl2f effector.
  • Small CRISPR- associated effector proteins belonging to the type V-F subtype have been identified through the mining of sequence databases and members classified into Casl2fl (Casl4a and type V-U3), Casl2f2 (Casl4b) and Casl2f3 (Casl4c, type V-U2 and U4).
  • Casl2fl Casl4a and type V-U3
  • Casl2f2 Casl4b
  • Casl2f3 Casl4c, type V-U2 and U4
  • protospacer adjacent sequence or “protospacer adjacent motif’ or “PAM” refers to an approximately 2-6 base pair DNA sequence (or a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-long nucleotide sequence) that is an important targeting component of a Cas9 nuclease.
  • PAM sequence is on either strand, and is downstream in the 5' to 3' direction of Cas9 cut site.
  • the 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) DI 135 V, R1335Q, and T1337R “the VQR variant”, which alters the PAM specificity to NGAN or NGNG, (b) D1135E, R1335Q, and T1337R “the EQR variant”, which alters the PAM specificity to NGAG, and (c) DI 135V, G1218R, R1335E, and T1337R “the VRER variant”, which alters the PAM specificity to NGCG.
  • the DI 135E variant of canonical SpCas9 still recognizes NGG, but it is more selective compared to the wild type SpCas9 protein.
  • Cas9 enzymes from different bacterial species can have varying PAM specificities and in some embodiments are therefore chosen based on the desired PAM recognition.
  • Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN.
  • Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT.
  • Cas9 from Streptococcus thermophilis (StCas9) recognizes NNAGAAW.
  • Cas9 from Treponema denticola (TdCas) recognizes NAAAAC. These examples are not meant to be limiting.
  • 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.
  • non-SpCas9s may have other characteristics that make them more useful than SpCas9.
  • 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 editor fusion protein describes a protein that is used in prime editing.
  • Prime editing uses CRISPR enzyme that nicks or cuts only single strand of double stranded DNA, i.e., a nickase; and a nickase can occur either naturally or by mutation or modification of a nuclease that makes double stranded cuts.
  • Such an enzyme can be a catalytically-impaired Cas9 endonuclease (a nickase).
  • a nickase can be a Casl2a/b, MAD7, or variant thereof.
  • the nickase is fused to an engineered reverse transcriptase (RT).
  • the nickase is programmed (directed) with a primeediting guide RNA (pegRNA).
  • pegRNA primeediting guide RNA
  • the pegRNA both specifies the target site and encodes the desired edit.
  • the nickase is a catalytically-impaired Cas9 endonuclease, a Cas9 nickase, that is fused to the reverse transcriptase.
  • the Cas9 nickase part of the protein is guided to the DNA target site by the pegRNA, whereby a nick or single stranded cut occurs.
  • the reverse transcriptase domain then uses the pegRNA to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand.
  • the edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand.
  • the prime editor guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process (typically achieved with a nickase gRNA).
  • PEI refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a wild type MMLV RT having the following N-terminus to C-terminus structure: [NLS]-[Cas9(H840A)]- [linker]-[MMLV_RT(wt)] + a desired PEgRNA.
  • the prime editors disclosed herein is comprised of PEI.
  • PE2 refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a variant MMLV RT having the following N-terminus to C-terminus structure: [NLS]-[Cas9(H840A)]- [linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] + a desired atgRNA (PEgRNA).
  • the prime editors disclosed herein are comprised of PE2.
  • the prime editors disclosed herein are comprised of PE2 and co-expression of MMR protein MLHldn, 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 are comprised of PE3.
  • the prime editors disclosed herein is comprised of PE3 and co-expression of MMR protein MLHldn, 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.
  • a prime editing complex consists of a type II CRISPR PE protein containing an RNA-guided DNA-nicking domain fused to a reverse transcriptase (RT) domain and complexed with a pegRNA.
  • the pegRNA comprises (5’ to 3’) a spacer that is complementary to the target sequence of a genomic DNA, a nickase (e.g. Cas9) binding site, a reverse transcriptase template including editing positions, and primer binding site (PBS).
  • the PE-pegRNA complex binds the target DNA and the CRISPR protein nicks the PAM- containing strand.
  • the resulting 3' end of the nicked target hybridizes to the primer-binding site (PBS) of the pegRNA, then primes reverse transcription of new DNA containing the desired edit using the RT template of the pegRNA.
  • PBS primer-binding site
  • the overall structure of the pegRNA is like that of a typical type II sgRNA with a reverse transcriptase template/primer binding site appended to the 3’ end. The structure leaves the PBS at the 3’ end of the pegRNA free to bind to the nicked strand complementary to the target which forms the primer for reverse transcription.
  • Guide RNAs of CRISPRs differ in overall structure. For example, while the spacer of a type II gRNA is located at the 5’ end, the spacer of a type V gRNA is located towards the 3’ end, with the CRISPR protein (e.g. Casl2a) binding region located toward the 5’ end. Accordingly, the regions of a type V pegRNA are rearranged compared to a type II pegRNA.
  • the overall structure of the pegRNA is like that of a typical type II sgRNA with a reverse transcriptase template/primer binding site appended to the 3’ end.
  • the pegRNA comprises (5’ to 3’) a CRISPR protein-binding region, a spacer which is complementary to the target sequence of a genomic DNA, a reverse transcriptase template including editing positions, and primer binding site (PBS).
  • PBS primer binding site
  • the term “attachment site-containing guide RNA” refers 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).
  • cognate integration recognition site or “integration cognate” or “cognate pair” refers to a first integration recognition site (e.g., any of the integration recognition sites described herein) and a second integration recognition site (e.g., any of the integration recognition sites described herein) that can be recombined. Recombination between a first integration recognition site (e.g., any of the integration recognition sites described herein) and a second recognition site (e.g., any of the integration recognition sites described herein) is mediated by functional symmetry between the two integration recognition sites and the central dinucleotide of each of the two integration recognition sites.
  • a first integration recognition site e.g., any of the integration recognition sites described herein
  • a second integration recognition site e.g., any of the integration recognition sites described herein
  • a non-limiting example of a cognate pair include an attB site and an attP site, whereby a serine integrase mediates recombination between the attB site and the attP site.
  • FIGs. 10A-10E show optimization of the integration recognition site.
  • an atgRNA comprises a reverse transcriptase template that encodes, partially or in its entirety, an integration recognition site (also referred to as an integration target recognition site) or a recombinase recognition site (also referred to as a recombinase target recognition site).
  • the integration target recognition site which is to be placed at a desired location in the genome or intracellular nucleic acid, is referred to as a “beacon,” a “beacon” site or an “attachment site” or a “landing pad” or “landing site.”
  • An integration target recognition site or recombinase target recognition site incorporated into the pegRNA is referred to as an attachment site containing guide RNA (atgRNA).
  • the primer binding site allows the 3’ end of the nicked DNA strand to hybridize to the atgRNA, while the RT template serves as a template for the synthesis of edited genetic information.
  • the atgRNA is capable for instance, without limitation, of (i) identifying the target nucleotide sequence to be edited and (ii) encoding new genetic information that replaces (or in some cases adds) the targeted sequence.
  • the atgRNA is capable of (i) identifying the target nucleotide sequence to be edited and (ii) encoding an integration site that replaces (or inserts/ deletes within) the targeted sequences.
  • the 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, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the first atgRNA’ s reverse transcriptase template encodes for a first single-stranded DNA sequence (i.e., a first DNA flap) that contains a complementary region to a second single-stranded DNA sequence (i.e., a second DNA flap) encoded by a second atgRNA comprising a second reverse transcriptase template.
  • the complementary region between the first and second single-stranded DNA sequences is comprised of more than 5 consecutive bases of an integrase target recognition site.
  • the complementary region between the first and second single-stranded DNA sequences is comprised of more than 10 consecutive bases of an integrase target recognition site.
  • the complementary region between the first and second single-stranded DNA sequences is comprised of more than 20 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 30 consecutive bases of an integrase target recognition site.
  • Use of two guide RNAs that are (or encode DNA that is) partially complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site-containing guide RNAs (atgRNAs).
  • RNAs that are (or encode DNA that is) full complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment sitecontaining guide RNAs (atgRNAs).
  • the first atgRNA upon introducing the nucleic acid construct into a cell, incorporates the first integration recognition site into the cell’s genome at the target sequence.
  • Table 9 includes atgRNAs, sgRNAs and nicking guides that can be used herein. Spacers are labeled in capital font (SPACER), RT regions in bold capital (RT REGION), AttB sites in bold lower case (attb site), and PBS in capital italics (PBS). Unless otherwise denoted, the AttB is for Bxbl. Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9 Table 9
  • a gene editor polypeptide described herein contains an integrase or recombinase.
  • the integrase is delivered as a protein or the integrase is encoded in a delivered polynucleotide.
  • the integration enzyme is selected from the group consisting of Dre, Vika, Bxbl, ⁇ pC31, RDF, cpBTl, Rl, R2, R3, R4, R5, TP901-1, Al 18, cpFCl, cpCl, MR11, TGI, cp370.1, Wp, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, Concept!, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, cpRV, retrotransposases encoded by a Tcl/mariner family member including but not limited to retrotransposases encoded by LI, Tol2, Tel, Tc3, Himar 1 (isolated from the horn fly, Hae
  • Xu et al describes methods for evaluating integrase activity in E. coll and mammalian cells and confirmed at least R4, cpC31, (pBTl, Bxbl, SPBc, TP901-1 and WP integrases to be active on substrates integrated into the genome of HT1080 cells (Xu et al., 2013, Accuracy and efficiency define Bxbl 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.
  • LSRs serine recombinases
  • embodiments can include any serine recombinase such as BceINT, SSCINT, SACINT, and INT10 (see lonnidi et al., 2021; Drag-and-drop genome insertion without DNA cleavage with CRISPR directed integrases.
  • the integration site can be selected from an attB site, an attP site, an attL site, an attR site, a lox71 site a Vox site, or a FRT site.
  • integrases, transposases and the like can depend on nuclear localization.
  • prokaryotic enzymes are adapted to modulate nuclear localization.
  • eukaryotic or vertebrate enzymes are adapted to modulate nuclear localization.
  • the invention provides fusion or hybrid proteins. Such modulation can comprise addition or removal of one or more nuclear localization signal (NLS) and/or addition or removal of one or more nuclear export signal (NES).
  • NLS nuclear localization signal
  • NES nuclear export signal
  • nuclear export signal (NES) of transposases affects the transposition activity of mariner-like elements Ppmarl and Ppmar2 of moso bamboo. Mob DNA. 2019 Aug 19; 10:35. doi: 10.1186/sl3100-019-0179-y).
  • the methods and constructs are used to modulate nuclear localization of system components of the invention.
  • the integrase used herein is selected from below (Table 10).
  • FIGs. 10A-10E show analysis of effect of variant AttP sites on integration efficiency.
  • This disclosure features a gene editor polypeptide useful for site-specifically integrating an integration recognition site into a target nucleic acid sequence (i.e., beacon placement), for example by using a polypeptide comprising a DNA binding nuclease linked to a reverse transcriptase, and integrating an exogenous nucleic acid sequence at the integrated integration recognition site (i.e., PASTE) by introducing into the cell the exogenous nucleic acid and an integration enzyme.
  • a target nucleic acid sequence i.e., beacon placement
  • the gene editor polypeptide includes a polypeptide comprised of a (i) DNA binding nickase (optionally with nickase activity), (ii) a reverse transcriptase, and (iii) an integration enzyme (e.g., an integrase or a recombinase), wherein at least any two elements of (i), (ii), or (iii) are linked via at least a first C-terminal linker.
  • the gene editor polypeptide includes a second linker (see FIG. 1 and FIG. 2).
  • a gene editor polypeptide includes a first and a second linker
  • the first linker links a first domain (e.g., a DNA binding) to a second domain (e.g., a RT) and the second linker links the second domain to a third domain (e.g., an integration enzyme).
  • a first domain e.g., a DNA binding
  • a second domain e.g., a RT
  • a third domain e.g., an integration enzyme
  • the orientation of the DNA binding nuclease, the RT, and the integration enzyme are configured for example from N-terminus to C- terminus: a DNA binding nuclease domain, a RT, and an integration enzyme; a DNA binding nuclease, an integration enzyme, and a RT; an integration enzyme, a DNA binding nuclease, and a RT; an integration enzyme, a RT, and a DNA binding nuclease; a RT, a DNA binding nuclease, and an integration enzyme; and a RT, an integration enzyme, and a DNA binding nuclease, where each of the domains are linked via a linker (see, FIG. 3 and Table 3).
  • the C- terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C- terminus of the reverse transcriptase is linked to the integration enzyme. In one embodiment, the C-terminus of the integration enzyme is linked to the DNA binding nuclease, wherein the C- terminus of the DNA binding nuclease is linked to a reverse transcriptase.
  • the gene editor polypeptide is comprised of at least two of any one of (i) a DNA binding nickase, (ii) a reverse transcriptase, and (iii) an integration enzyme wherein at least any two elements of (i), (ii), or (iii) are linked via at least a first C-terminal linker.
  • the gene editor polypeptide can be comprised of (i) a DNA binding nickase, (ii) a reverse transcriptase, and (iii) an integration enzyme (e.g., an integrase or a recombinase), wherein at least any two elements of (i), (ii), or (iii) may be linked via two or more C-terminal linkers at the same linking position.
  • a gene editor polypeptide includes a DNA binding nickase linked via two linkers to (a reverse transcriptase, and the reverse transcriptase domain fused or linked to an integration enzyme.
  • a linker (e.g., the first linker, the second linker, or both) is selected from a flexible linker, a semi-flexible linker, a rigid linker, a flexible-rigid linker.
  • the gene editor polypeptide includes at least one flexible linker.
  • flexible linkers include LI (SEQ ID NO: 15), L2 (SEQ ID NO: 551), L5 (SEQ ID NO: 19), L6 (SEQ ID NO: 18), and LI 3 (SEQ ID NO: 558).
  • the gene editor polypeptide includes at least one flexible linker selected from: LI, L2, L5, L6, and LI 3.
  • the gene editor polypeptide includes at least a LI linker (SEQ ID NO: 15).
  • the gene editor polypeptide includes at least a L2 linker (SEQ ID NO: 551).
  • the gene editor polypeptide includes at least a L5 linker (SEQ ID NO: 19) linker. In some embodiments, the gene editor polypeptide includes at least a L6 linker (SEQ ID NO: 18). In some embodiments, the gene editor polypeptide includes at least a L13 linker (SEQ ID NO: 558).
  • the gene editor polypeptide includes at least one semi-flexible linker.
  • semi-flexible linkers include L7 (SEQ ID NO: 552) and L8 (SEQ ID NO: 553).
  • the gene editor polypeptide includes at least a L7 linker (SEQ ID NO: 552).
  • the gene editor polypeptide includes at least a L7 linker (SEQ ID NO: 553).
  • the gene editor polypeptide includes at least one rigid linker.
  • rigid linkers include L3 (SEQ ID NO: 16), L4 (SEQ ID NO: 17), and L9 (SEQ ID NO: 554).
  • the gene editor polypeptide includes at least a L3 linker (SEQ ID NO: 16).
  • the gene editor polypeptide includes at least a L4 linker (SEQ ID NO: 17).
  • the gene editor polypeptide includes at least a L9 linker (SEQ ID NO: 554).
  • the gene editor polypeptide includes at least one flexible-rigid linker.
  • flexible-rigid linkers include Li l (SEQ ID NO: 557) and L12 (SEQ ID NO: 558).
  • the gene editor polypeptide includes at least a Li l linker (SEQ ID NO: 557).
  • the gene editor polypeptide includes at least a L12 linker (SEQ ID NO: 558).
  • the gene editor polypeptide includes a linker (e.g., a first linker, a second linker, or both) where the length of the linker is selected from short, medium, and long.
  • linker e.g., a first linker, a second linker, or both
  • short linkers include: LI (SEQ ID NO: 15), L3 (SEQ ID NO: 16), L7 (SEQ ID NO: 552), and L8 (SEQ ID NO: 553).
  • Non-limiting examples of a medium linker include: L2 (SEQ ID NO: 551), L4 (SEQ ID NO: 17), L5 (SEQ ID NO: 19), L6 (SEQ ID NO: 18), L10 (SEQ ID NO: 555), LI 1 (SEQ ID NO: 556), L12 (SEQ ID NO: 557), and L13 (SEQ ID NO: 558).
  • a non-limiting example of a long linker is L9 (SEQ ID NO: 554).
  • the gene editor polypeptide includes a linker (e.g., the first linker, the second linker, or both) that is a non-cleavable linker.
  • linker e.g., the first linker, the second linker, or both
  • Non-limiting examples of non-cleavable linkers include: LI (SEQ ID NO: 15), L2 (SEQ ID NO: 551), L3 (SEQ ID NO: 16), L4 (SEQ ID NO: 17), L5 (SEQ ID NO: 19), L6 (SEQ ID NO: 18), L7 (SEQ ID NO: 552), L8 (SEQ ID NO: 553), L9 (SEQ ID NO: 554), LIO (SEQ ID NO: (555), LI 1 (SEQ ID NO: 556), L12 (SEQ ID NO: 557), and L13 (SEQ ID NO: 558).
  • the gene editor polypeptide include a linker (e.g., the first linker, the second linker, or both) that is a cleavable linker.
  • Cleavable linkers can facilitate the coexpression of multiple proteins within a single open reading frame, effectively separating them post-translationally.
  • Non-limiting examples of self-cleaving peptides include 2A peptides (derived from the foot-and-mouth disease virus), such as F2A, T2A, E2A, and P2A; the “Inteins” that facilitate protein splicing; and the tobacco etch virus (TEV) protease recognition site.
  • the cleavable linker is 2A peptide.
  • the cleavable linker is a P2A peptide.
  • the gene editor polypeptide includes at least a first linker and a second linker where: the at least first linker is a non-cleavable linker and the second linker is a cleavable linker; the at least first linker is a cleavable linker and the second linker is a non- cleavable linker; the at least first linker is a cleavable linker and the second linker is a cleavable linker; or the at least first linker is a non-cleavable linker and the second linker is a non-cleavable linker.
  • the gene editor polypeptide includes the DNA binding nuclease, the RT, and the integration enzyme, where the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, and where any one of L7 (SEQ ID NO: 552), LIO (SEQ ID NO: 555), or L12 (SEQ ID NO: 557) links the DNA binding nuclease to the reverse transcriptase.
  • L7 SEQ ID NO: 552
  • LIO SEQ ID NO: 555
  • L12 SEQ ID NO: 557
  • the gene editor polypeptide includes the DNA binding nuclease, the RT, and the integration enzyme, where the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L8 (SEQ ID NO: 553), L9 (SEQ ID NO: 554), L10 (SEQ ID NO: 555), L12 (SEQ ID NO: 557), or L13 (SEQ ID NO: 558) links the reverse transcriptase to the integration enzyme.
  • the gene editor polypeptide includes the DNA binding nuclease, the RT, and the integration enzyme, where the C-terminus of the integration enzyme is linked to the DNA binding nuclease by L13 (SEQ NO: 558), and where the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by L13 (SEQ ID NO: 558).
  • the gene editor polypeptide is not comprised of aN-mostN-terminal reverse transcriptase.
  • the gene editor polypeptide includes the DNA binding nuclease, the RT, and the integration enzyme, where the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by LI 3 (SEQ ID NO: 558), and where the C-terminus of the reverse transcriptase is linked to the integration enzyme by a cleavable linker (e.g., a P2A (SEQ ID NO: 13).
  • a cleavable linker e.g., a P2A (SEQ ID NO: 13).
  • the gene editor polypeptide includes a nuclear localization signal (NLS). In some embodiments, the gene editor polypeptide comprises a mitochondrial localization signal.
  • the gene editor polypeptide comprises: from N-terminus to C- terminus: a DNA binding nickase domain; a P2A linker (SEQ ID NO: 13), a reverse transcriptase; a mXTEN linker, and an integration enzyme.
  • the gene editor polypeptide comprises: from N-terminus to C- terminus: an integration enzyme, a mXTEN linker (SEQ ID NO: 558), a DNA binding nuclease domain; a mXTEN linker (SEQ ID NO: 558), and a reverse transcriptase.
  • This disclosure also features systems capable of site-specifically integrating at least a first integration recognition site into the genome of a cell where the system includes any of the gene editor polypeptides described herein.
  • This disclosure also features systems capable of site-specifically integrating an exogenous nucleic acid sequence into the genome of a cell where the system includes any of the gene editor polypeptides described herein.
  • the gene editor polypeptide comprises at least one linker that is non-cleavable, and where the at least one linker is selected from a group consisting of LI, L2, L3, L4, L5, L6, L7, L8, L9, L10, LI 1, L12, and L13.
  • LI, L2, L3, L4, L5, L6, L7, L8, L9, L10, LI 1, L12, and L13 are illustrated in FIG. 3 and Table 3. 6.10.
  • a polynucleotide encodes the gene editor polypeptide.
  • the polynucleotide is an RNA or DNA.
  • the polynucleotide is a mRNA.
  • an expression vector comprises the polynucleotide encoding the gene editor polypeptide.
  • the expression vector further comprises any one or more of a one or more of a promoter sequence, atgRNA, nicking gRNA (ngRNA), donor polynucleotide template, or donor polynucleotide template complement.
  • ngRNA nicking gRNA
  • two or more expression vectors are delivered.
  • the expression vector is recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, annelovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, or lipid nanoparticle.
  • the polypeptides described herein are used for programmable polynucleotide (e.g., genes or gene fragments) insertion.
  • Programmable polynucleotide/gene insertion includes site-specifically integrating an exogenous nucleic acid sequence (e.g., a donor polynucleotide template) into a genome of a cell.
  • the method of site- specifically integrating an exogenous nucleic acid sequence into a target nucleic acid comprises incorporating an integration recognition site into a cell by delivering: (i) a polynucleotide encoding a gene editor polypeptide (e.g., any of the gene editor polypeptides described herein), (ii) at least a first target specific atgRNAs comprising a first integration recognition site, and (iii) optionally, a nicking guide RNA.
  • a gene editor polypeptide e.g., any of the gene editor polypeptides described herein
  • at least a first target specific atgRNAs comprising a first integration recognition site
  • optionally, a nicking guide RNA optionally, a nicking guide RNA.
  • the first atgRNA comprises: (i) a domain that is capable of guiding the prime editor system to a target sequence; and (ii) a reverse transcriptase (RT) template that comprises at least a portion of an at least first integration recognition site, whereby the at least portion of the at least first integration recognition site is integrated into the genome of the cell at the target sequence.
  • RT reverse transcriptase
  • the method of site-specifically integrating an exogenous nucleic acid sequence into a target nucleic acid comprises incorporating an integration recognition site into a cell by delivering: (i) a polynucleotide encoding a gene editor polypeptide (e.g., any of the gene editor polypeptides described herein), (ii) a first target specific atgRNA and a second target specific atgRNA.
  • a gene editor polypeptide e.g., any of the gene editor polypeptides described herein
  • 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 an at least first integration recognition site;
  • the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is integrated into the genome of the cell at the target sequence.
  • the method includes integrating the polynucleotide in the cellular genome by delivering a donor polynucleotide template, wherein the donor polynucleotide template is comprised of a second integration recognition site that is a cognate pair of the first integration recognition site, wherein the donor polynucleotide is integrated into the cellular genome at the first integration target recognition site by the gene editor polynucleotide.
  • the method of site-specifically integrating an exogenous nucleic acid sequence into a genome of a cell comprises incorporating an integration target site into a cell by delivering: an expression vector of comprising a polynucleotide encoding a gene editor polypeptide (e.g., any of the gene editor polypeptides described herein), further comprising a promoter, one or more cell genome target specific atgRNA comprising an integration target recognition site, a donor polynucleotide comprising an integration target site, and optionally, a ngRNA.
  • a gene editor polypeptide e.g., any of the gene editor polypeptides described herein
  • the method of polynucleotide integration into a cell genome comprises incorporating an integration target site into a cell by delivering: an expression vector of comprising a polynucleotide encoding a gene editor polypeptide (e.g., any of the gene editor polypeptides described herein), further comprising a promoter, two cell genome target specific atgRNA where the two atgRNA collectively encode the entirety of the an integration recognition site, and a donor polynucleotide comprising an integration target site.
  • a gene editor polypeptide e.g., any of the gene editor polypeptides described herein
  • the method of site-specifically integrating an exogenous nucleic acid sequence into a genome of a cell includes incorporating an integration recognition site into a cell by delivering: any of the polynucleotides described herein; at least a first genome target specific atgRNA comprising a first integration recognition site; optionally, a nicking gRNA; and integrating an exogenous nucleic acid sequence into the cellular genome by delivering into the cell: a donor polynucleotide template, wherein the donor polynucleotide template is comprised of the exogenous nucleic acid sequence positioned relative to a second integration recognition site that is a cognate pair of the first integration recognition site such that the exogenous nucleic acid sequence is integrated into the cellular genome at the first integration target recognition site when contacted with the integration enzyme and the first integration recognition site.
  • the polynucleotides and the at least first atgRNA are delivered concurrently with the donor polynucleotide template
  • the method of site-specifically integrating an exogenous nucleic acid sequence into a target nucleic acid includes: incorporating an integration recognition site into a cell by delivering: any of the expression vectors described herein; and integrating an exogenous nucleic acid sequence into the cellular genome by integrating a donor polynucleotide template, wherein the donor polynucleotide template is comprised of the exogenous nucleic acid sequence positioned relative to a second integration recognition site that is a cognate pair of the first integration recognition site such that the exogenous nucleic acid sequence is integrated into the cellular genome at the first integration target recognition site when contacted with the integration enzyme and the first integration recognition site.
  • the expression vector and the donor polynucleotide template are delivered to the cell concurrently.
  • compositions, systems and methods for correcting or replacing genes or gene fragments (including introns or exons) or inserting genes in new locations comprises recombination or integration into a safe harbor site (SHS).
  • SHS safe harbor site
  • a frequently used human SHS is the AAVS1 site on chromosome 19q, initially identified as a site for recurrent adeno-associated virus insertion.
  • Another locus comprises the human homolog of the murine Rosa26 locus.
  • Yet another SHS comprises the human Hl 1 locus on chromosome 22.
  • a complete gene may be prohibitively large and replacement of an entire gene impractical.
  • a method of the invention 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 invention are adapted to target organoids, including patient derived organoids.
  • methods and compositions of the invention are adapted to treat muscle cells, not limited to cardiomyocytes for Duchene Muscular Dystrophy (DMD).
  • the dystrophin gene is the largest gene in the human genome, spanning ⁇ 2.3 Mb of DNA. DMD is composed of 79 exons resulting in a 14-kb full-length mRNA. Common mutations include mutations that disrupt the reading frame of generate a premature stop codon.
  • An aspect of DMD that lends it to gene editing as a therapeutic approach is the modular structure of the dystrophin protein. Redundancy in the central rod domain permits the deletion of internal segments of the gene that may harbor loss-of-function mutations, thereby restoring the open reading frame (ORFs).
  • the methods and systems described herein are used to treat DMD by site- specifically integrating in the genome a polynucleotide template that repairs or replaces all or a portion of the defective DMD gene.
  • ANCA Anti-Neutrophil Cytoplasmic Antibody
  • SLE Systemic Lupus Erythematosus
  • LN Lupus Nephritis
  • MN Membranous Nephropathy
  • HCU Homocystinuria
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the most common cystic fibrosis (CF) mutation F508del removes a single amino acid.
  • recombining human CFTR into an SHS of a cell that expresses CFTR F508del is a corrective treatment path.
  • the methods and systems described herein are used to CF by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing CF. Proposed validation is detection of persistent CFTR mRNA and protein expression in transduced cells.
  • Sickle cell disease is caused by mutation of a specific amino acid — valine to glutamic acid at amino acid position 6.
  • SCD is corrected by recombination of the HBB gene into a safe harbor site (SHS) and by demonstrating correction in a proportion of target cells that is high enough to produce a substantial benefit.
  • the methods and systems described herein are used to sickle cell disease by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the disease.
  • validation is detection of persistent HBB mRNA and protein expression in transduced cells.
  • DMD Duchenne Muscular Dystrophy.
  • the dystrophin gene is the largest gene in the human genome, spanning ⁇ 2.3 Mb of DNA. DMD is composed of 79 exons resulting in a 14- kb full-length mRNA. Common mutations include mutations that disrupt the reading frame of generate a premature stop codon.
  • An aspect of DMD that lends it to gene editing as a therapeutic approach is the modular structure of the dystrophin protein. Redundancy in the central rod domain permits the deletion of internal segments of the gene that may harbor loss-of-function mutations, thereby restoring the open reading frame (ORFs).
  • recombination will be into safe harbor sites (SHS).
  • SHS safe harbor sites
  • a frequently used human SHS is the A4F5 site on chromosome 19q, initially identified as a site for recurrent adeno-associated virus insertion.
  • the site is the human homolog of the e murine Rosa26 locus (pubmed. ncbi.nlm.nih.gov/18037879).
  • the site is the human Hl 1 locus on chromosome 22.
  • Proposed target cells for recombination include stem cells for example induced pluripotent stem cells (iPSCs) and cells at various stages of differentiation. In some cases, a complete gene may be prohibitively large and replacement of an entire gene impractical. In such instances, rescuing mutants by recombining in corrected gene fragments with the methods and systems described herein is a corrective option.
  • iPSCs induced pluripotent stem cells
  • correcting mutations in exon 44 (or 51) by recombining in a corrective coding sequence downstream of exon 43 (or 50), using the methods and systems described herein is a corrective option.
  • Proposed validation is detection of persistent DMD mRNA and protein expression in transduced cells.
  • F8 Factor VIII
  • F8 Factor VIII
  • F8 A large proportion of severe hemophilia A patients harbor one of two types of chromosomal inversions in the FVIII gene.
  • the recombinase technology and methods described herein are well suited to correcting such inversions (and other mutations) by recombining of the FVIII gene into a SHS.
  • correcting factor VIII deficiency by recombining the FVIII gene into an SHS is a corrective path.
  • the methods and systems described herein are used to correct factor VIII deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the FIX deficiency. Proposed validation is detection of persistent FVIII mRNA and protein expression in transduced cells.
  • Factor 9 Hemophilia B, also called factor IX (FIX) deficiency is a genetic disorder caused by missing or defective factor IX, a clotting protein.
  • the methods and systems described herein are used to correct factor IX deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the FIX deficiency.
  • Proposed validation is detection of persistent FiX mRNA and protein expression in transduced cells.
  • Ornithine transcarbamylase deficiency is a rare genetic condition that causes ammonia to build up in the blood.
  • the condition - more commonly called OTC deficiency - is more common in boys than girls and tends to be more severe when symptoms emerge shortly after birth.
  • the methods and systems described herein are used to correct OTC deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the OTC deficiency or integrates a polynucleotide encoding a functional ornithine transcarbamylase enzyme.
  • Proposed validation is detection of persistent OTC mRNA and protein expression in transduced cells.
  • Phenylketonuria also called PKU, is a rare inherited disorder that causes an amino acid called phenylalanine to build up in the body. PKU is caused by a change in the phenylalanine hydroxylase (PAH) gene. This gene helps create the enzyme needed to break down phenylalanine.
  • PKU phenylalanine hydroxylase
  • the methods and systems described herein are used to correct PKU by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the PKU deficiency or integrates a polynucleotide encoding a functional phenylalanine hydroxylase (PAH) gene.
  • Proposed validation is detection of persistent PAH mRNA and protein expression in transduced cells.
  • Homocystinuria is elevation of the amino acid, homocysteine (protein building block coming from our diet) in the urine or blood.
  • Common causes of HCU include: problems with the enzyme cystathionine beta synthase (CBS), which converts homocysteine to the amino acid cystathionine (which then becomes cysteine) and needs the vitamin B6 (pyridoxine); and roblems with converting homocysteine to the amino acid methionine.
  • CBS cystathionine beta synthase
  • pyridoxine pyridoxine
  • the methods and systems described herein are used to correct HCU by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the HCU or integrates a polynucleotide encoding a functional copy of a gene (e.g., CBS) able to reduce or prevent buildup of homocysteine in the urine.
  • Proposed validation is detection of persistent CBS mRNA and protein expression in transduced cells.
  • IgA Nephropathy (Berger’s disease). IgA nephropathy, also known as Berger's disease, is a kidney/autoimmune disease that occurs when an antibody called immunoglobulin A (IgA) builds up in the kidneys.
  • the methods and systems described herein are used to treat Berger’s disease by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of Berger’s disease.
  • ANCA vasculitis is an autoimmune disease affecting small blood vessels in the body. It is caused by autoantibodies called ANCAs, or Anti-Neutrophilic Cytoplasmic Autoantibodies. ANCAs target and attack a certain kind of white blood cells called neutrophils.
  • the methods and systems described herein are used to treat ANCA vasculitis by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of ANCA vasculitis.
  • Lupus is an autoimmune — a disorder in which the body’s immune system attacks the body’s own cells and organs.
  • the methods and systems described herein are used to treat SLE/LN by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of SLE/LN.
  • MN Membranous Nephropathy
  • the methods and systems described herein are used to treat MN by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of MN.
  • C3 glomerulonephritis C3GN.
  • C3 glomerulopathy is a group of related conditions that cause the kidneys to malfunction.
  • the major features of C3 glomerulopathy include high levels of protein in the urine (proteinuria), blood in the urine (hematuria), reduced amounts of urine, low levels of protein in the blood, and swelling in many areas of the body.
  • Affected individuals may have particularly low levels of a protein called complement component 3 (or C3) in the blood.
  • the methods and systems described herein are used to treat C3 glomerulopathy by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of C3 glomerulopathy.
  • 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.
  • the polynucleotide encoding a gene editor polypeptide are delivered by a vector, wherein the vector is selected from the group consisting of recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, annelovirus, retrovirus, DoggyboneTM DNA (dbDNA), minicircle, plasmid, miniDNA, exosome, fusosome, lipid nanoparticle, or nanoplasmid.
  • 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 polynucleotide encoding a gene editor polypeptide or the gene editor polypeptide described herein include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
  • the gene editor polypeptide or polynucleotide encoding the gene editor polypeptide is packaged in a LNP and administered intravenously. In some embodiments, the gene editor polypeptide or polynucleotide encoding the gene editor polypeptide is packaged in a LNP and administered intrathecally. In some embodiments, the gene editor polypeptide or polynucleotide encoding the gene editor polypeptide is packaged in a LNP and administered by intracerebral ventricular injection. In some embodiments, the gene editor polypeptide or polynucleotide encoding the gene editor polypeptide is packaged in a LNP and administered by intraci sternal magna administration. In some embodiments, the gene editor polypeptide or polynucleotide encoding the gene editor polypeptide is packaged in a LNP and administered by intravitreal injection.
  • lipidmucleic acid complexes including targeted liposomes such as immunolipid complexes
  • Boese et al. Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
  • 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.
  • the charge of the LNP must be taken into consideration. As cationic lipids combined with negatively charged lipids to induce nonbilayer structures that facilitate intracellular delivery. Because charged LNPs are rapidly cleared from circulation following intravenous injection, ionizable cationic lipids with pKa values below 7 were developed (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011). Negatively charged polymers such as RNA may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge. However, at physiological pH values, the LNPs exhibit a low surface charge compatible with longer circulation times.
  • pH 4 e.g., pH 4
  • ionizable cationic lipids Four species of ionizable cationic lipids have been focused upon, namely l,2-dilineoyl-3 -dimethylammonium -propane (DLinDAP), 1,2- dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), l,2-dilinoleyloxy-keto-N,N-dimethyl- 3 -aminopropane (DLinKDMA), and l,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DlinKC2-DMA).
  • DLinDAP 1,2- dilinoleyloxy-3-N,N-dimethylaminopropane
  • DLinKDMA 1,2- dilinoleyloxy-keto-N,N-dimethyl- 3 -aminopropane
  • LNP siRNA systems containing these lipids exhibit remarkably different gene silencing properties in hepatocytes in vivo, with potencies varying according to the series DlinKC2-DMA>DLinKDMA>DLinDMA»DLinDAP employing a Factor VII gene silencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011).
  • a dosage of 1 pg/ml of LNP in or associated with the LNP may be contemplated, especially for a formulation containing DlinKC2-DMA.
  • the LNP composition comprises one or more one or more ionizable lipids.
  • ionizable lipid has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties.
  • an ionizable lipid may be positively charged or negatively charged.
  • the one or more ionizable lipids are selected from the group consisting of 3-(didodecylamino)- N1 ,N1 ,4-tridodecyl- 1 -piperazineethanamine (KL 10), N 1 -[2-(didodecylamino)ethyl]-N 1 ,N4,N4- tridodecyl-l,4-piperazinediethanami- ne (KL22), 14, 25-ditridecyl-15, 18,21, 24-tetraaza- octatriacontane (KL25), l,2-dilinoleyloxy-N,N-dimethylaminopropane (Dlin-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (Dlin-K-DMA), heptatriaconta-6,9,28,31-
  • the lipid nanoparticle may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cationic and/or ionizable lipids.
  • cationic and/or ionizable lipids include, but are not limited to, 3-(didodecylamino)-Nl,Nl,4-tridodecyl-l-piperazineethanamine (KL10), Nl-[2- (didodecylamino)ethyl]-Nl,N4,N4-tridodecyl-l,4-piperazinediethanami- ne (KL22), 14,25- ditridecyl- 15,18,21 ,24-tetraaza-octatriacontane (KL25), 1 ,2-dilinoleyloxy-N,N- dimethylaminopropane (Dlin-DMA), 2, 2-dilinoleyl-4-dimethyla
  • the LNP composition comprises one or more amino lipids.
  • amino lipid and “cationic lipid” are used interchangeably herein to include those lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino head group (e.g., an alkylamino or dialkylamino head group).
  • a pH-titratable amino head group e.g., an alkylamino or dialkylamino head group.
  • the cationic lipid is typically protonated (i.e., positively charged) at a pH below the pKa of the cationic lipid and is substantially neutral at a pH above the pKa.
  • the cationic lipids can also be termed titratable cationic lipids.
  • the one or more cationic lipids include: a protonatable tertiary amine (e.g., pH-titratable) head group; alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and alkyl chains.
  • 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 include, but are not limited to, DSDMA, DODMA, DOTMA, DLinDMA, DLenDMA, . gamma.
  • 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-012, 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, lysylphosphatidyl glycerol, and other anionic modifying groups joined to neutral lipids.
  • Neutral lipids suitable for use in lipid nanoparticles include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, sterols (e.g., cholesterol) and cerebrosides.
  • the lipid nanoparticle comprises cholesterol.
  • Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains and cyclic regions can be used.
  • the neutral lipids used in the disclosure are DOPE, DSPC, DPPC, POPC, or any related phosphatidylcholine.
  • the neutral lipid may be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol.
  • amphipathic lipids are included in nanoparticles. Exemplary amphipathic lipids suitable for use in nanoparticles include, but are not limited to, sphingolipids, phospholipids, fatty acids, and amino lipids.
  • the lipid composition of the pharmaceutical composition may comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof.
  • phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
  • a fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Particular amphipathic lipids can facilitate fusion to a membrane.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • elements e.g., a
  • Non-natural amphipathic lipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond.
  • an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide.
  • 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).
  • 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.
  • 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), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-di-O- octadecenyl-sn-glycero-3 -phosphocholine (POPC
  • phosphorus-lacking compounds such as sphingolipids, glycosphingolipid families, diacylglycerols, and .beta. -acyloxyacids, may also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.
  • the LNP composition comprises one or more helper lipids.
  • helper lipid refers to lipids that enhance transfection (e.g., transfection of an LNP comprising an mRNA that encodes a site-directed endonuclease, such as a SpCas9 polypeptide).
  • the mechanism by which the helper lipid enhances transfection includes enhancing particle stability.
  • the helper lipid enhances membrane fusogenicity.
  • helper lipid of the LNP compositions disclosure herein can be any helper lipid known in the art.
  • helper lipids suitable for the compositions and methods include steroids, sterols, and alkyl resorcinols.
  • helper lipids suitable for use in the present disclosure include, but are not limited to, saturated phosphatidylcholine (PC) such as distearoyl-PC (DSPC) and dipalymitoyl-PC (DPPC), di oleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn- glycero-3 -phosphocholine (DLPC), cholesterol, 5-heptadecylresorcinol, and cholesterol hemi succinate.
  • PC saturated phosphatidylcholine
  • DSPC distearoyl-PC
  • DPPC dipalymitoyl-PC
  • DOPE di oleoylphosphatidylethanolamine
  • DLPC 1,2-dilinoleoyl-sn- glycero-3 -phosphocholine
  • cholesterol 5-heptadecylresorcinol
  • cholesterol hemi succinate hemi succinate.
  • 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 e.g., a lipid nanoparticle
  • a targeting moiety that is specific to a cell type and/or tissue type.
  • a nanoparticle may be targeted to a particular cell, tissue, and/or organ using a targeting moiety.
  • a nanoparticle comprises a targeting moiety.
  • targeting moieties include ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and antibodies (e.g., full-length antibodies, antibody fragments (e.g., Fv fragments, single chain Fv (scFv) fragments, Fab’ fragments, or F(ab’)2 fragments), single domain antibodies, cam elid antibodies and fragments thereof, human antibodies and fragments thereof, monoclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies)).
  • the targeting moiety may be a polypeptide.
  • the targeting moiety may include the entire polypeptide (e.g., peptide or protein) or fragments thereof.
  • a targeting moiety is typically positioned on the outer surface of the nanoparticle in such a manner that the targeting moiety is available for interaction with the target, for example, a cell surface receptor.
  • a variety of different targeting moieties and methods are known and available in the art, including those described, e.g., in Sapra et al., Prog. Lipid Res. 42(5):439-62, 2003 and Abra et al., J. Liposome Res. 12: 1-3, 2002.
  • a lipid nanoparticle may include a surface coating of hydrophilic polymer chains, such as polyethylene glycol (PEG) chains (see, e.g., Allen et al., Biochimica et Biophysica Acta 1237: 99-108, 1995; DeFrees et al., Journal of the American Chemistry Society 118: 6101-6104, 1996; Blume et al., Biochimica etBiophysica Acta 1149: ISO- 184, 1993; Klibanov et al., Journal of Liposome Research 2: 321-334, 1992; U.S. Pat. No.
  • PEG polyethylene glycol
  • a targeting moiety for targeting the lipid nanoparticle is linked to the polar head group of lipids forming the nanoparticle.
  • the targeting moiety is attached to the distal ends of the PEG chains forming the hydrophilic polymer coating (see, e.g., Klibanov et al., Journal of Liposome Research 2: 321-334, 1992; Kirpotin et al., FEBS Letters 388: 115-118, 1996).
  • Standard methods for coupling the targeting moiety or moi eties may be used.
  • phosphatidylethanolamine which can be activated for attachment of targeting moieties
  • derivatized lipophilic compounds such as lipid-derivatized bleomycin
  • Antibody- targeted liposomes can be constructed using, for instance, liposomes that incorporate protein A (see, e.g., Renneisen et al., J. Bio. Chem., 265: 16337-16342, 1990 and Leonetti et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451, 1990).
  • Other examples of antibody conjugation are disclosed in U.S. Pat. No.
  • targeting moieties can also include other polypeptides that are specific to cellular components, including antigens associated with neoplasms or tumors.
  • Polypeptides used as targeting moieties can be attached to the liposomes via covalent bonds (see, for example Heath, Covalent Attachment of Proteins to Liposomes, 149 Methods in Enzymology 111-119 (Academic Press, Inc. 1987)).
  • Other targeting methods include the biotin-avidin system.
  • a lipid nanoparticle includes a targeting moiety that targets the lipid nanoparticle to a cell including, but not limited to, hepatocytes, colon cells, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells (including primary tumor cells and metastatic tumor cells).
  • the targeting moiety targets the lipid nanoparticle to a hepatocyte.
  • the lipid nanoparticles described herein may be lipidoid-based.
  • the synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of polynucleotides (see Mahon et al., Bioconjug Chem. 2010 21 : 1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat. Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011 108: 12996-3001).
  • lipidoid formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse through the extracellular matrix into the blood stream. While a particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see e.g., Akinc et al., Mol Ther. 2009 17:872-879), use of lipidoid oligonucleotides to deliver the formulation to other cells types including, but not limited to, endothelial cells, myeloid cells, and muscle cells may not be similarly size-limited.
  • lipidoid formulations may have a similar component molar ratio.
  • Different ratios of lipidoids and other components including, but not limited to, a neutral lipid (e.g., diacylphosphatidylcholine), cholesterol, a PEGylated lipid (e.g., PEG-DMPE), and a fatty acid (e.g., an omega-3 fatty acid) may be used to optimize the formulation of the mRNA or system for delivery to different cell types including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc.
  • a neutral lipid e.g., diacylphosphatidylcholine
  • cholesterol e.g., a PEGylated lipid
  • PEG-DMPE PEGylated lipid
  • a fatty acid e.g., an omega-3 fatty acid
  • Exemplary lipidoids include, but are not limited to, Dlin-DMA, Dlin-K-DMA, Dlin-KC2-DMA, 98N12-5, C12-200 (including variants and derivatives), Dlin-MC3-DMA and analogs thereof.
  • the use of lipidoid formulations for the localized delivery of nucleic acids to cells (such as, but not limited to, adipose cells and muscle cells) via either subcutaneous or intramuscular delivery may also not require all of the formulation components which may be required for systemic delivery, and as such may comprise the lipidoid and the mRNA or system.
  • a system described herein may be formulated by mixing the mRNA or system, or individual components of the system, with the lipidoid at a set ratio prior to addition to cells.
  • In vivo formulations may require the addition of extra ingredients to facilitate circulation throughout the body.
  • a system or individual components of a system is added and allowed to integrate with the complex. The encapsulation efficiency is determined using a standard dye exclusion assays.
  • In vivo delivery of systems may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle PEGylation, degree of loading, oligonucleotide to lipid ratio, and biophysical parameters such as particle size (Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety).
  • particle size Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety.
  • small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids may result in significant effects on in vivo efficacy.
  • Formulations with the different lipidoids including, but not limited to penta[3-(l- laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401 :61 (2010)), C12-200 (including derivatives and variants), MD1, Dlin-DMA, Dlin-K-DMA, Dlin-KC2-DMA and Dlin-MC3-DMA can be tested for in vivo activity.
  • the lipidoid referred to herein as “98N12-5” is disclosed by Akinc et al., Mol Ther. 2009 17:872-879).
  • 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, doublestranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector wherein virally- derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g.
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.” Vectors for and that result in expression in a eukaryotic cell can be referred to herein as “eukaryotic expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • Vector delivery e.g., plasmid, viral delivery:
  • the CRISPR enzyme for instance a Type V protein such as C2cl or C2c3, and/or any of the present RNAs, for instance a guide RNA
  • Effector proteins and one or more guide RNAs can be packaged into one or more vectors, e.g., plasmid or viral vectors.
  • the vector e.g., plasmid or viral vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the delivery is via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery may be either via a single dose, or multiple doses.
  • the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choice, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc.
  • Such a dosage may further contain, for example, a carrier (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, a pharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), a pharmaceutically-acceptable excipient, and/or other compounds known in the art.
  • a carrier water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.
  • a pharmaceutically-acceptable carrier e.g., phosphate-buffered saline
  • a pharmaceutically-acceptable excipient e.g., phosphate-buffered saline
  • the dosage may further contain one or more pharmaceutically acceptable salts such as, for example, a mineral acid salt such as a hydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and the salts of organic acids such as acetates, propionates, malonates, benzoates, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, gels or gelling materials, flavorings, colorants, microspheres, polymers, suspension agents, etc. may also be present herein.
  • Suitable exemplary ingredients include microcrystalline cellulose, carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin and a combination thereof.
  • REMINGTON’S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which is incorporated by reference herein.
  • the delivery is via an adenovirus, which may be at a single booster dose containing at least 1 x 10 5 particles (also referred to as particle units, pu) of adenoviral vector.
  • the dose preferably is at least about 1 x 10 6 particles (for example, about 1 x 10 6 - 1 x 10 11 particles), more preferably at least about 1 x 10 7 particles, more preferably at least about 1 x 10 8 particles (e.g., about 1 x 10 8 -l x 10 11 particles or about 1 x 10 9 -l x 10 12 particles), and most preferably at least about 1 x IO 10 particles (e.g., about 1 x 10 9 - 1 x IO 10 particles or about 1 x 10 9 -l x 10 12 particles), or even at least about 1 x IO 10 particles (e.g., about 1 x 10 10 -l x 10 12 particles) of the adenoviral vector.
  • the dose comprises no more than about 1 x 10 14 particles, preferably no more than about 1 x 10 13 particles, even more preferably no more than about 1 x 10 12 particles, even more preferably no more than about 1 x 10 11 particles, and most preferably no more than about 1 x IO 10 particles (e.g., no more than about 1 x 10 9 particles).
  • the dose may contain a single dose of adenoviral vector with, for example, about 1 x 10 6 particle units (pu), about 2 x 10 6 pu, about 4 x 10 6 pu, about 1 x 10 7 pu, about 2 x 10 7 pu, about 4 x 10 7 pu, about 1 x 10 8 pu, about 2 x 10 8 pu, about 4 x 10 8 pu, about 1 x 10 9 pu, about 2 x 10 9 pu, about 4 x 10 9 pu, about 1 x IO 10 pu, about 2 x IO 10 pu, about 4 x IO 10 pu, about 1 x 10 11 pu, about 2 x 10 11 pu, about 4 x 10 11 pu, about 1 x 10 12 pu, about 2 x 10 12 pu, or about 4 x 10 12 pu of adenoviral vector.
  • adenoviral vector with, for example, about 1 x 10 6 particle units (pu), about 2 x 10 6 pu, about 4 x 10 6 pu, about 1 x 10 7 pu, about 2 x
  • the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel, et. al., granted on Jun. 4, 2013; incorporated by reference herein, and the dosages at col 29, lines 36-58 thereof.
  • the adenovirus is delivered via multiple doses.
  • the delivery is via an AAV.
  • a therapeutically effective dosage for in vivo delivery of the AAV to a human is believed to be in the range of from about 20 to about 50 ml of saline solution containing from about 1 x 10 10 to about 1 x 10 10 functional AAV/ml solution. The dosage may be adjusted to balance the therapeutic benefit against any side effects.
  • the AAV dose is generally in the range of concentrations of from about 1 x 10 5 to 1 x 10 50 genomes AAV, from about 1 x 10 8 to 1 x IO 20 genomes AAV, from about 1 x 10 10 to about 1 x 10 16 genomes, or about 1 x 10 11 to about 1 x 10 16 genomes AAV.
  • a human dosage may be about 1 x 10 13 genomes AAV. Such concentrations may be delivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of a carrier solution. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. See, for example, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar. 26, 2013, at col. 27, lines 45-60.
  • the promoter used to drive nucleic acid-targeting effector protein coding nucleic acid molecule expression can include: AAV ITR can serve as a promoter: this is advantageous for eliminating the need for an additional promoter element (which can take up space in the vector). The additional space freed up can be used to drive the expression of additional elements (gRNA, etc.). Also, ITR activity is relatively weaker, so can be used to reduce potential toxicity due to over expression of nucleic acid-targeting effector protein. For ubiquitous expression, can use promoters: CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc.
  • promoters For brain or other CNS expression, can use promoters: SynapsinI for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc.
  • For liver expression can use Albumin promoter.
  • For lung expression can use SP-B.
  • For endothelial cells can use ICAM.
  • For hematopoietic cells can use IFNbeta or CD45.
  • Osteoblasts can use OG-2.
  • the promoter used to drive guide RNA can include: Pol III promoters such as U6 or Hl Use of Pol II promoter and intronic cassettes to express guide RNA Adeno Associated Virus (AAV)
  • Pol III promoters such as U6 or Hl Use of Pol II promoter and intronic cassettes to express guide RNA Adeno Associated Virus (AAV)
  • Nucleic acid-targeting effector protein and one or more guide RNA can be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus), U.S. Pat. No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No. 5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus.
  • AAV adeno associated virus
  • lentivirus lentivirus
  • adenovirus or other plasmid or viral vector types in particular, using formulations and doses from, for example, U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus), U.S.
  • the route of administration, formulation and dose can be as in U.S. Pat. No. 8,454,972 and as in clinical trials involving AAV.
  • the route of administration, formulation and dose can be as in U.S. Pat. No. 8,404,658 and as in clinical trials involving adenovirus.
  • the route of administration, formulation and dose can be as in U.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.
  • Doses may be based on or extrapolated to an average 70 kg individual (e.g., a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species.
  • Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed.
  • the viral vectors can be injected into the tissue of interest.
  • the expression of nucleic acid-targeting effector can be driven by a cell-type specific promoter.
  • liver-specific expression might use the Albumin promoter and neuron-specific expression (e.g., for targeting CNS disorders) might use the Synapsin I promoter.
  • AAV is advantageous over other viral vectors for a couple of reasons: Low toxicity (this may be due to the purification method not requiring ultra centrifugation of cell particles that can activate the immune response) and Low probability of causing insertional mutagenesis because it doesn’t integrate into the host genome.
  • AAV has a packaging limit of 4.5 or 4.75 Kb.
  • nucleic acid-targeting effector protein such as a Type V protein such as C2cl or C2c3
  • a promoter and transcription terminator have to be all fit into the same viral vector. Therefore embodiments of the invention include utilizing homologs of nucleic acid-targeting effector protein (such as a Type V protein such as C2cl or C2c3) that are shorter.
  • the AAV can be AAV1, AAV2, AAV5 or any combination thereof.
  • AAV8 is useful for delivery to the liver. The herein promoters and vectors are preferred individually.
  • Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and psi2 cells or PA317 cells, which package retrovirus.
  • Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line may also be infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.
  • Millington-Ward et al. (Molecular Therapy, vol. 19 no. 4, 642-649 April 2011) describes adeno-associated virus (AAV) vectors to deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site.
  • RNAi RNA interference
  • An injection of either 6.0 x 10 8 vp or 1.8 x IO 10 vp AAV were subretinally injected into the eyes by Millington-Ward et al.
  • the AAV vectors of Millington-Ward et al. may be applied to the system of the present invention, contemplating a dose of about 2 x 10 11 to about 6 x 10 11 vp administered to a human.
  • Dalkara et al. also relates to in vivo directed evolution to fashion an AAV vector that delivers wild-type versions of defective genes throughout the retina after noninjurious injection into the eyes’ vitreous humor.
  • Dalkara describes a 7 mer peptide display library and an AAV library constructed by DNA shuffling of cap genes from AAV1, 2, 4, 5, 6, 8, and 9.
  • the rcAAV libraries and rAAV vectors expressing GFP under a CAG or Rho promoter were packaged and deoxyribonuclease-resistant genomic titers were obtained through quantitative PCR.
  • the libraries were pooled, and two rounds of evolution were performed, each consisting of initial library diversification followed by three in vivo selection steps.
  • P30 rho-GFP mice were intravitreally injected with 2 ml of iodixanol-purified, phosphate- buffered saline (PBS)-dialyzed library with a genomic titer of about 1. times.10. sup.12 vg/ml.
  • PBS phosphate- buffered saline
  • the AAV vectors of Dalkara et al. may be applied to the nucleic acid-targeting system of the present invention, contemplating a dose of about 1 x 10 15 to about 1 x 10 16 vg/ml administered to a human.
  • Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SW), human immune deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66 :2731-2739 (1992) ; Johann et al., J. Virol. 66 : 1635-1640 (1992) ; Sommnerfelt et al., Virol. 176 :58-59 (1990) ; Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol.
  • adenoviral based systems may be used.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
  • Adeno-associated virus (“AAV”) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No.
  • Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and yr2 cells or PA317 cells, which package retrovirus.
  • Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line may also be infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.
  • a host cell is transiently or non-transiently transfected with one or more vectors described herein.
  • a cell is transfected as it naturally occurs in a subject.
  • a cell that is transfected is taken from a subject.
  • Cells taken from a subject include, but are not limited to, hepatocytes or cells isolated from muscle, the CNS, eye or lung.
  • Immunological cells are also contemplated, such as but not limited to T cells, HSCs, B-cells and NK cells.
  • mRNA delivery methods and compositions that may be utilized in the present disclosure including, for example, PCT/US2014/028330, US8822663B2, NZ700688A, ES2740248T3, EP2755693A4, EP2755986A4, WO2014152940A1, EP3450553B1, BRI 12016030852A2, and EP3362461A1.
  • Expression of CRISPR systems in particular is described by W02020014577.
  • Each of these publications are incorporated herein by reference in their entireties. Additional disclosure hereby incorporated by reference can be found in Kowalski et al., “Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery,” Mol Therap., 2019; 27(4): 710-728.
  • the cell is derived from cells taken from a subject, such as a cell line.
  • a cell line A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHL231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep
  • a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences.
  • one or more vectors described herein are used to produce a nonhuman transgenic animal or transgenic plant.
  • the transgenic animal is a mammal, such as a mouse, rat, or rabbit.
  • the organism or subject is a plant.
  • the organism or subject or plant is algae. Methods for producing transgenic plants and animals are known in the art, and generally begin with a method of cell transfection, such as described herein.
  • the invention provides for methods of modifying a target polynucleotide in a prokaryotic or eukaryotic cell, which may be in vivo, ex vivo or in vitro.
  • the method comprises sampling a cell or population of cells from a human or non-human animal or plant (including micro-algae) and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant (including micro-algae).
  • pathogens are often host-specific.
  • Fusariumn oxysporum f. sp. Lycopersici causes tomato wilt but attacks only tomato
  • Plants have existing and induced defenses to resist most pathogens. Mutations and recombination events across plant generations lead to genetic variability that gives rise to susceptibility, especially as pathogens reproduce with more frequency than plants.
  • there can be non-host resistance e.g., the host and pathogen are incompatible.
  • Horizontal Resistance e.g., partial resistance against all races of a pathogen, typically controlled by many genes
  • Vertical Resistance e.g., complete resistance to some races of a pathogen but not to other races, typically controlled by a few genes.
  • Plant and pathogens evolve together, and the genetic changes in one balance changes in other. Accordingly, using Natural Variability, breeders combine most useful genes for Yield. Quality, Uniformity, Hardiness, Resistance.
  • the sources of resistance genes include native or foreign Varieties, Heirloom Varieties, Wild Plant Relatives, and Induced Mutations, e.g., treating plant material with mutagenic agents.
  • 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. The transcribed or translated products may be known or unknown and may be at a normal or abnormal level.
  • Embodiment 1 A polypeptide (e.g., gene editor polypeptide), comprising: a DNA binding nuclease, optionally comprising a nickase activity; a reverse transcriptase; an integration enzyme; and wherein at least any two of elements (i), (ii), or (iii) are linked via at least one C-terminal linker.
  • a polypeptide e.g., gene editor polypeptide
  • Embodiment 2 The polypeptide (e.g., gene editor polypeptide) of Embodiment 1, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C- terminus of the reverse transcriptase is linked to the integration enzyme.
  • the polypeptide e.g., gene editor polypeptide
  • Embodiment 3 The polypeptide (e.g., gene editor polypeptide) of Embodiment 1, wherein the C-terminus of the integration enzyme is linked to the DNA binding nuclease, wherein the C- terminus of the DNA binding nuclease is linked to a reverse transcriptase.
  • the polypeptide e.g., gene editor polypeptide
  • Embodiment 4 The polypeptide (e.g., gene editor polypeptide) of any one of Embodiments 1-3, wherein at least one linker is a peptide linker.
  • Embodiment 5 The polypeptide (e.g., gene editor polypeptide) of Embodiment 4, wherein at least one linker is non-cleavable.
  • Embodiment 6 The polypeptide (e.g., gene editor polypeptide) of Embodiment 4, wherein at least one linker is cleavable.
  • Embodiment 7. The polypeptide (e.g., gene editor polypeptide) of Embodiment 5, wherein the at least one linker is selected from a group consisting of LI, L2, L3, L4, L5, L6, L7, L8, L9, LIO, L11, L12, and L13.
  • Embodiment 8 The polypeptide (e.g., gene editor polypeptide) of Embodiment 6, wherein the at least one cleavable linker is selected from a group consisting of P2A, E2A, F2A, and T2A.
  • the at least one cleavable linker is selected from a group consisting of P2A, E2A, F2A, and T2A.
  • Embodiment 9 The polypeptide (e.g., gene editor polypeptide) of any one of Embodiments 1-8, wherein at least one linker is non-cleavable and at least one linker is cleavable.
  • Embodiment 10 The polypeptide (e.g., gene editor polypeptide) of Embodiment 7, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L7, L10, or L12 links the DNA binding nuclease to the reverse transcriptase.
  • the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase
  • the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L7, L10, or L12 links the DNA binding nuclease to the reverse transcriptase.
  • Embodiment 11 The polypeptide (e.g., gene editor polypeptide) of Embodiment 7, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L8, L9, L10, L12, or L13 links the reverse transcriptase to the integration enzyme.
  • the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L8, L9, L10, L12, or L13 links the reverse transcriptase to the integration enzyme.
  • Embodiment 12 The polypeptide (e.g., gene editor polypeptide) of Embodiment 7, wherein the C-terminus of the integration enzyme is linked to the DNA binding nuclease by L13, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by L13.
  • the C-terminus of the integration enzyme is linked to the DNA binding nuclease by L13
  • the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by L13.
  • Embodiment 13 The polypeptide (e.g., gene editor polypeptide) of Embodiment 7, wherein the polypeptide is not comprised of a N-most N-terminal reverse transcriptase.
  • Embodiment 14 The polypeptide (e.g., gene editor polypeptide) of Embodiment 9, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by LI 3, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme by a cleavable linker.
  • the polypeptide e.g., gene editor polypeptide
  • Embodiment 15 The polypeptide (e.g., gene editor polypeptide) of any one of Embodiments 1-14, further comprised of at least one nuclear localization signal.
  • Embodiment 16 A polynucleotide encoding a polypeptide from any one of Embodiments 1-15.
  • Embodiment 17 An expression vector comprising a polynucleotide of Embodiment 16.
  • Embodiment 18 The expression vector of Embodiment 17, further comprising any one or more of a promoter sequence, atgRNA, nicking gRNA (ngRNA), donor polynucleotide template, or donor polynucleotide template complement.
  • a promoter sequence atgRNA, nicking gRNA (ngRNA), donor polynucleotide template, or donor polynucleotide template complement.
  • ngRNA nicking gRNA
  • donor polynucleotide template or donor polynucleotide template complement.
  • Embodiment 19 The expression vector of Embodiment 18, wherein the expression vector is recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, annelovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, or lipid nanoparticle.
  • Embodiment 20 A method of polynucleotide integration into a cellular genome, the method comprising: incorporating an integration target recognition site into a cell by delivering: the polynucleotide of Embodiment 16; one or more cell genome target specific atgRNA comprising an integration target recognition site; optionally, a nicking gRNA; and integrating the polynucleotide into the cellular genome by delivering: a donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target recognition site, wherein the donor polynucleotide is integrated into the cellular genome at a genomic integration target recognition site by a polypeptide encoded by the polynucleotide of Embodiment 16.
  • Embodiment 21 A method of polynucleotide integration into a cellular genome, the method comprising: (a) incorporating an integration target recognition site into a cell by delivering: the expression vector of Embodiment 18, wherein the expression vector is comprised of a promoter, one or more cell genome target specific atgRNA comprising an integration target recognition site, a donor polynucleotide comprising an integration target site, and optionally, a ngRNA; and (b) integrating the polynucleotide into the cellular genome by integrating the donor polynucleotide into the cellular genome at a genomic integration target recognition site by a polypeptide encoded in the expression vector.
  • Linkers that connect Cas9-nickase, reverse transcriptase, and Bxbl integrase were screened.
  • Linker position 1 (Pl) and Linker position 2 (P2) are illustrated in FIG. 1 and FIG. 2.
  • a panel of selected non-cleavable linkers are shown in FIG. 3, where linkers LI -LI 3 demonstrate various length, structure, and flexibility attributes.
  • atgRNAs were use that targeted the ACTB locus (see Table 9).
  • the atgRNAs included an integration recognition site (e.g., AttB) that is a cognate pair with an integration recognition site located on the donor template polynucleotide (e.g., AttP).
  • the gene editor polypeptides Upon transfection of the atgRNA, nicking gRNA, polynucleotide encoding the gene editor polypeptide, and the donor template polynucleotide, the gene editor polypeptides integrated the integration recognition site from the atgRNAs into the ACTB locus, and then integrated the donor template polynucleotide into the ACTB locus using the cognate pair of integration recognition sites.
  • a PASTE readout in HEK293 cells was used to identify functionally active gene editor compositions, wherein select non-cleavable linkers L1-L13 (see FIG. 3) and a cleavable P2A linker were screened.
  • Droplet digital PCR (ddPCR) in a 96-well plate format was applied to verify gene insertion into the ACTB locus.
  • a total of 135 linker combinations were screened and compared to Cas9(nickase)-mXTEN-RT-P2A-Bxbl and Cas9(nickase)-mXTEN-RT-mXTEN- Bxbl.
  • FIGs. 5, 6, and 7 show the percent integration (as verified by ddPCR) across the screened gene editor compositions.
  • FIG. 5 shows percent integration of a Cas9-Pl-RT-P2-BXB1 configuration tested with various combinations of linkers from FIG. 3 (see x-axis FIG. 5).
  • Columns with asterisks represent the gene editor polypeptides that produced the highest percent insertion of donor polynucleotide template in the ACTB locus.
  • a gene editor polypeptide having a Cas9-L6 (i.e., XTEN)-RT-L5(i.e., (GGS)6)-BXB 1 produced the highest percent insertion of donor polynucleotide template.
  • FIG. 6 shows percent integration of a Cas9-Pl-RT-P2-BXB1 configuration tested with various combination of linkers from FIG. 3 (see x-axis FIG. 6). Columns with asterisks represent the gene editor polypeptides that produced the highest percent insertion of donor polynucleotide template in the ACTB locus.
  • gene editor polypeptides having an L8 linker i.e., PAS12; SEQ ID NO: 553; an L9 linker (i.e., A(EAAK)4ALEA(EAAAK)4A; SEQ ID NO: 554), and/or an L10 linker (i.e., Camel; SEQ ID NO: 555) produced the highest percent insertion of donor polynucleotide template in the ACTB locus.
  • L8 linker i.e., PAS12; SEQ ID NO: 553
  • an L9 linker i.e., A(EAAK)4ALEA(EAAAK)4A
  • an L10 linker i.e., Camel; SEQ ID NO: 555
  • FIG. 7 shows percent integration of a Cas9-Pl-RT-P2-BXB1 configuration tested with various combination of linkers from FIG. 3 (see x-axis FIG. 7).
  • Columns with asterisks include gene editor polypeptides that produced the highest percent insertion of donor polynucleotide template in the ACTB locus.
  • gene editor polypeptides that had the highest percent integration were those where the P2 was an L12 linker (i.e., RFR; SEQ ID NO: 557).
  • the following gene editor polypeptides having an L12 at P2 produced the highest percent integration: (P1/P2) L7 (i.e., PAS8; SEQ ID NO: 552) / L12; L8 (i.e., PAS12; SEQ ID NO: 553) / L12; and LIO (i.e., Camel; SEQ ID NO: 555) / L12.
  • P1/P2 L7
  • PAS8 i.e., PAS8; SEQ ID NO: 552
  • L8 i.e., PAS12; SEQ ID NO: 553
  • L12 i.e., Camel; SEQ ID NO: 555
  • gene editor polypeptides that had the highest percent integration were those that included P1/P2 combinations of: L4 (i.e., (EAAAK)3; SEQ ID NO: 17) /L13 (i.e., mXTEN; SEQ ID NO: 558); LIO (i.e., Camel; SEQ ID NO: 555) / L6 (i.e., XTEN; SEQ ID NO: 18); and L12 (i.e., RFR; SEQ ID NO: 557) / L13).
  • L4 i.e., (EAAAK)3; SEQ ID NO: 17
  • L13 i.e., mXTEN; SEQ ID NO: 558
  • LIO i.e., Camel
  • L6 i.e., XTEN; SEQ ID NO: 18
  • L12 i.e., RFR; SEQ ID NO: 557) / L13
  • lOOng total DNA at a plasmid ratio of 1 : 1:4 of Cas9(nickase)-RT-BxBl i.e., the configurations described in FIG. 8) : nicking guide : atgRNA was delivered to 30,000 HEK293 (see, e.g., FIG. 4).
  • FIGs. 9A-9B show the percent integration (as verified by ddPCR) across mXTEN/P2A or P2A/mXTEN linker gene editor compositions.
  • FIG. 9A shows that a configuration of Cas9-mXTEN-RT-P2A-BXBl produced the highest percent integration for the P2A/mXTEN configurations.
  • FIG. 9B shows that a configuration of BXB1- mXTEN-Cas9-mXTEN-RT produced the highest percent integration for the mXTEN/mXTEN configurations.

Abstract

Provided herein are gene editor compositions that allow for site-specifically integrating an integration recognition site into a target nucleic acid sequence (i.e., beacon placement), for example by using Cas-reverse transcriptase activity, and integrating an exogenous nucleic acid sequence at the integrated integration recognition site by introducing into the cell the exogenous nucleic acid and an integration enzyme, thereby meditating programmable gene insertion (PASTE) For example, the gene editor composition includes a gene editor polypeptide comprised of a (i) DNA binding nickase, (ii) a reverse transcriptase, and (iii) an integration enzyme (i.e., an integrase or recombinase), wherein at least any two elements of (i), (ii), or (iii) are linked via at least one C-terminal linker.

Description

PROGRAMMABLE GENE INSERTION COMPOSITIONS
1. CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/332,857, filed April 20, 2022; which is hereby incorporated in its entirety by reference.
2. SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing with 558 sequences, which has been submitted via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on April 19, 2023, is named 52191WO-SequenceListing.xml, and is 757,270 bytes in size.
3. BACKGROUND
[0003] Programmable, efficient, and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences.
[0004] There is a need in the art for techniques and gene editor compositions which address and overcome these shortcomings and enable the programmable genomic integration of a large gene(s).
4. SUMMARY
[0005] Gene editor compositions are described herein that allow for site-specific integration recognition site placement within a genome (i.e., beacon placement), for example by Cas-reverse transcriptase activity, and subsequent genomic integration at the integrated integration recognition site via integrase activity of the gene editor composition. The gene editor polypeptide can be comprised of a (i) DNA binding nickase, (ii) a reverse transcriptase, and (iii) an integration enzyme (i.e., an integrase or a recombinase), wherein at least any two elements of (i), (ii), or (iii) are linked via at least one C-terminal linker. In preferred embodiments, the linkers are peptide linkers that may be cleavable (i.e., ribosome skipping site, peptide cleavage site, etc.) or non-cleavable. The gene editor polypeptide may be comprised of one or more cleavable and/or non-cleavable linkers at any given linkage position connecting at least any two elements of (i), (ii) or (iii). In certain embodiments, a unique N-terminus to C-terminus configuration of elements (i), (ii), and (iii) is provided herein. The polypeptide compositions described herein can be expressed from and/or delivered as a polynucleotide encoding the polypeptide. Further, an expression vector comprised of the polynucleotide encoding the polypeptides is described herein.
[0006] The present disclosure provides nucleic acid compositions, methods, and an overall platform for site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE) (see lonnidi et al.,' doi: 10.1101/2021.11.01.466786; the entirety of which is incorporated herein by reference), transposon-mediated gene editing, or other suitable gene editing, or gene incorporation technology. Non-limiting examples of PASTE are also described in U.S. Pat. 11,572,556, which is hereby incorporated by reference in its entirety.
[0007] In one aspect, this disclosure features a gene editor polypeptide, comprising: a DNA binding nuclease, optionally comprising a nickase activity; a reverse transcriptase; an integration enzyme; and wherein at least any two of elements (i), (ii), or (iii) are linked via at least a first C- terminal linker.
[0008] In some embodiments, the gene editor polypeptide further comprises a second linker.
[0009] In some embodiments, the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme.
[0010] In some embodiments, the C-terminus of the integration enzyme is linked to the DNA binding nuclease, wherein the C-terminus of the DNA binding nuclease is linked to a reverse transcriptase.
[0011] In some embodiments, the at least first linker, the second linker, or both, is selected from a flexible linker, a semi-flexible linker, a rigid linker, a flexible-rigid linker.
[0012] In some embodiments, the flexible linker is selected from: LI, L2, L5, L6, and L13.
[0013] In some embodiments, the semi-flexible linker is selected from: L7 and L8.
[0014] In some embodiments, the rigid linker is selected from: L3, L4, and L9.
[0015] In some embodiments, the flexible-rigid linker is selected from LI 1 and L12. [0016] In some embodiments, the length of the at least first linker, the length of the second linker, or both, is selected from short, medium, and long.
[0017] In some embodiments, the short linker is selected from: LI, L3, L7, and L8.
[0018] In some embodiments, the medium linker is selected from: L2, L4, L5, L6, L10, LI 1, L12, and L13.
[0019] In some embodiments, the long linker is L9.
[0020] In some embodiments, the at least first linker, the second linker, or both, is a non-cleavable linker.
[0021] In some embodiments, the non-cleavable linker is selected from: LI, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, and L13.
[0022] In some embodiments, the at least first linker, the second linker, or both, is a cleavable linker.
[0023] In some embodiments, the cleavable linker is selected from: a self-cleavable linker, an enzyme cleavable linker, and chemically-induced cleavable linker.
[0024] In some embodiments, the cleavable linker is a self-cleavable linker.
[0025] In some embodiments, the self-cleavable linker is selected from P2A, E2A, F2A, and T2A.
[0026] In some embodiments, the at least first linker is a non-cleavable linker and the second linker is a cleavable linker; the at least first linker is a cleavable linker and the second linker is a non-cleavable linker; the at least first linker is a cleavable linker and the second linker is a cleavable linker; or the at least first linker is a non-cleavable linker and the second linker is a non- cleavable linker.
[0027] In some embodiments, the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L7, L10, or L12 links the DNA binding nuclease to the reverse transcriptase.
[0028] In some embodiments, the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L8, L9, LIO, LI 2, or L13 links the reverse transcriptase to the integration enzyme.
[0029] In some embodiments, the C-terminus of the integration enzyme is linked to the DNA binding nuclease by L13, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by LI 3.
[0030] In some embodiments, the gene editor polypeptide is not comprised of aN-mostN-terminal reverse transcriptase.
[0031] In some embodiments, the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by L13, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme by a cleavable linker.
[0032] In some embodiments, the gene editor polypeptide further comprises of at least one nuclear localization signal.
[0033] In another aspect, this disclosure features a gene editor polypeptide comprising: from N- terminus to C-terminus: a DNA binding nuclease; a P2A linker, a reverse transcriptase; a mXTEN linker, and an integration enzyme.
[0034] In another aspect, this disclosure features a gene editor polypeptide comprising:
[0035] from terminus to C-terminus: an integration enzyme, a mXTEN linker, a DNA binding nuclease; a mXTEN linker, and a reverse transcriptase.
[0036] In another aspect, this disclosure features a polynucleotide encoding any of the gene editor polypeptides described herein.
[0037] In another aspect, this disclosure features an expression vector comprising any of the polynucleotides described herein.
[0038] In some embodiments, the expression vector comprises any of the polynucleotides described herein operably linked to an expression control element. In some embodiments, the expression control element is a promoter.
[0039] In some embodiments, any of the polynucleotides described herein further comprise one or more sequences encoding an atgRNA, nicking gRNA (ngRNA), donor polynucleotide template, or donor polynucleotide template complement. [0040] In some embodiments, any of the expression vectors described herein further comprise one or more sequences encoding an atgRNA, nicking gRNA (ngRNA), donor polynucleotide template, or donor polynucleotide template complement.
[0041] In some embodiments, the expression vector is recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, annelovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, or lipid nanoparticle.
[0042] In another aspect, this disclosure a system capable of site-specifically integrating an exogenous nucleic acid sequence into a genome of a cell, the system comprising: any the gene editor polypeptides described herein or any of the polynucleotides described herein.
[0043] In another aspect, this disclosure features a method of site-specifically integrating an exogenous nucleic acid sequence into a genome of a cell, the method comprising: incorporating an integration recognition site into a cell by delivering: any of the polynucleotides described herein; at least a first atgRNA comprising a first integration recognition site; optionally, a nicking gRNA; and integrating an exogenous nucleic acid sequence into the cellular genome by delivering into the cell concurrently with step (a): a donor polynucleotide template, wherein the donor polynucleotide template is comprised of the exogenous nucleic acid sequence positioned relative to a second integration recognition site that is a cognate pair of the first integration recognition site such that the exogenous nucleic acid sequence is integrated into the cellular genome at the first integration target recognition site when contacted with the integration enzyme and the first integration recognition site.
[0044] In another aspect, this disclosure features a method of site-specifically integrating an exogenous nucleic acid sequence into a target nucleic acid, the method comprising: incorporating an integration recognition site into a cell by delivering: any of the expression vectors described herein; and integrating an exogenous nucleic acid sequence into the cellular genome by integrating a donor polynucleotide template, wherein the donor polynucleotide template is comprised of the exogenous nucleic acid sequence positioned relative to a second integration recognition site that is a cognate pair of the first integration recognition site such that the exogenous nucleic acid sequence is integrated into the cellular genome at the first integration target recognition site when contacted with the integration enzyme and the first integration recognition site. 5. BRIEF DESCRIPTION OF THE DRAWINGS
[0045] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:
[0046] FIG. 1 illustrates an exemplary gene editor composition expression construct with identified linker positions. Linker 1 = position 1 (Pl). Linker 2 = position 2 (P2).
[0047] FIG. 2 illustrates an exemplary gene editor composition with identified linker positions. Linker 1 = position 1 (Pl). Linker 2 = position 2 (P2).
[0048] FIG. 3 illustrates non-cleavable peptide linkers used in the linker screen.
[0049] FIG. 4 illustrates linker screen experimental conditions and setup.
[0050] FIG. 5 illustrates a single plate linker screen with programmable gene insertion (i.e., precent of donor polynucleotide template encoding GFP inserted into ACTB locus) as a functional readout. Linkers tested were selected from FIG. 3. Each column indicates the combination of linkers (e.g., Pl position/P2 position). Dotted line indicates precent integration for a gene editor polypeptide having an mXTEN (i.e., L13) / mXTEN (L13) linker combination (e.g., Cas9- mXTEN-RT-mXTEN-Bxbl). * indicates significant increase compared to dotted line.
[0051] FIG. 6 illustrates a single plate linker screen with programmable gene insertion (i.e., precent of donor polynucleotide template encoding GFP inserted into ACTB locus) as a functional readout. Linkers tested were selected from FIG. 3. Each column indicates the combination of linkers (e.g., Pl position/P2 position). Dotted line indicates precent integration for a gene editor polypeptide having an mXTEN (i.e., L13) / mXTEN (L13) linker combination (e.g., Cas9- mXTEN-RT-mXTEN-Bxbl). * indicates significant increase compared to dotted line.
[0052] FIG. 7 illustrates a single plate linker screen with programmable gene insertion (i.e., precent of donor polynucleotide template encoding GFP inserted into ACTB locus) as a functional readout. Linkers tested were selected from FIG. 3. Each column indicates the combination of linkers (e.g., Pl position/P2 position). Dotted line indicates precent integration for a gene editor polypeptide having an mXTEN (i.e., L13) / mXTEN (L13) linker combination (e.g., Cas9- mXTEN-RT-mXTEN-Bxbl). * indicates significant increase compared to dotted line.
[0053] FIG. 8 illustrates non-limiting examples of protein domain configurations that include a DNA binding nuclease (e.g., Cas9), a reverse transcriptase (RT), and an integration enzyme (e.g., Bxbl). mXTEN refers to a L13 linker (SEQ ID NO: 558) (see FIG. 3). P2A refers to a porcine teschovirus-1 2A peptide (SEQ ID NO: 13).
[0054] FIGs. 9A-9B show percent integration (i.e., percent PASTE) for various gene editor polypeptide configurations where the DNA binding nuclease, the RT, and the integration enzyme are linked via the indicated cleavable and non-cleavable linkers. FIG. 9A shows percent integration as a functional indicator of programmable gene insertion (i.e., precent of donor polynucleotide template encoding GFP inserted into ACTB locus). FIG. 9B shows percent integration as a functional indicator of programmable gene insertion (i.e., precent of donor polynucleotide template encoding GFP inserted into ACTB locus).
[0055] FIG. 10A-10E shows analysis of AttP variants. FIG. 10A shows a non-limiting schematic of AttP mutations tested for improving integration efficiency (SEQ ID NOS: 394 and 540-542, respectively, in order of appearance). FIG. 10B shows integration efficiencies of wildtype and mutant AttP sites across a panel of AttB lengths. FIG. 10C shows a non-limiting schematic of multiplexed integration of different cargo sets at specific genomic loci. Three fluorescent cargos (GFP, mCherry, and YFP) are inserted orthogonally at three different loci (ACTB, LMNB1, NOLC1) for in-frame gene tagging. FIG. 10D shows orthogonality of top 4 AttB/AttP dinucleotide pairs evaluated for GFP integration with PASTE at the ACTB locus. FIG. 10E shows efficiency of multiplexed PASTE insertion of combinations of fluorophores at ACTB, LMNB1, and NOLC1 loci. Data are mean (n= 3) ± s.e.m.
[0056] FIG. 11 illustrates a schematic of single atgRNA and dual atgRNA approaches for step 1 : beacon placement (“integration recognition site”) and step 2: gene insertion, thereby illustrating PASTE.
6. DETAILED DESCRIPTION OF THE INVENTION
[0057] Described herein are gene editor polypeptides comprising a DNA binding nuclease domain (e.g., comprising nickase activity), a reverse transcriptase (RT), and an integration enzyme used for programmable gene insertion/PASTE. In typical embodiments of the gene editor polypeptide, the DNA binding nuclease, the RT, and the integration enzyme are linked (e.g., fused) via at least a first C-terminal linker. In typical embodiments, the gene editor polynucleotide also includes a second C-terminal linker. A linker in a gene editor polypeptide can serve as a spatial buffer that helps maintain the structural and functional integrity of each of the DNA binding nuclease domain, the RT, and the integration enzyme. As described herein, the linker enabled each of these domains to perform their respective roles without hindrance, thereby preserving their biological activities. In some embodiments, the gene editor polypeptide includes linkers designed to be cleavable, allowing for the selective separation of the fused proteins if desired. Overall, this disclosure is based in part on the finding that gene editor polypeptides configured in various orientations and comprising different linkers and different combinations of linkers (e.g., a gene editor polypeptide that includes a first linker and a second linker where the first and second linkers are different types of linkers or is a linker with the same amino acid sequence) performed with varying efficiencies (e.g., varying percent integration of donor polynucleotide templates). In particular, this disclosure identified preferred orientations and preferred linkers that when combined resulted in improved integration efficiency compared to the other orientation and linker combinations tested..
6.1. Terminology
[0058] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.
[0059] “Gene editor” as used herein, is a protein that that can be used to perform gene editing, gene modification, gene insertion, gene deletion, or gene inversion. As used herein, the terms “gene editor polynucleotide” refers to polynucleotide sequence encoding the gene editor protein. Such an enzyme or enzyme fusion may contain DNA or RNA targetable nuclease protein (i.e., Cas protein, ADAR, or AD AT), wherein target specificity is mediated by a complexed nucleic acid (i.e., guide RNA). Such an enzyme or enzyme fusion may be a DNA/RNA targetable protein, wherein target specificity is mediated by internal, conjugated, fused, or linked amino acids, such as within TALENs, ZFNs, or meganucleases. The skilled person in the art would appreciate that the gene editor can demonstrate targeted nuclease activity, targeted binding with no nuclease activity, or targeted nickase activity (or cleaves 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 a prime editor fusion protein or a gene writer fusion protein.
[0060] “Prime editor fusion protein” as used herein, describes a protein that is used in prime editing. “Prime editor system” as used herein describes the components used in prime editing. Prime editing uses CRISPR enzyme 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. The nickase is programmed (directed) with a prime-editing guide RNA (pegRNA). The skilled person in the art would appreciate that the pegRNA both specifies the target site and encodes the desired edit. Described herein are attachment site containing guide RNA (atgRNA) that both specifies the target and encodes for the desired integrase target recognition site. The nickase may be programmed (directed) with an atgRNA. Advantageously the nickase is a catalytically-impaired Cas9 endonuclease, a Cas9 nickase, that is fused to the reverse transcriptase. During genetic editing, the Cas9 nickase part of the protein is guided to the DNA target site by the atgRNA (pegRNA), whereby a nick or single stranded cut occurs. The reverse transcriptase domain then uses the atgRNA (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. Afterward, optionally, the prime editor (PE) guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process (typically achieved with a nickase gRNA). Other enzymes that can be used to nick or cut only a single strand of double stranded DNA includes a cleavase (e.g., cleavase I enzyme).
[0061] In some embodiments, an additional agent or agents may be added that improve the efficiency and outcome purity of the prime edit. In some embodiments, 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). In typical embodiments, the agent is a MMR-inhibiting protein. In certain embodiments, the MMR-inhibiting protein is dominant negative MMR protein. In certain embodiments, the dominant negative MMR protein is MLHldn. In particular embodiments, the MMR-inhibiting agent is incorporated into the single nucleic acid construct design described herein that encodes any of the gene editor polypeptides described herein. In some embodiments, the MMR-inhibiting agent is linked or fused to the prime 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 Writer™ protein, which may or may not have a linked or fused integrase.
[0062] The prime editor or gene editor system can be used to achieve DNA deletion and replacement. In some embodiments, the DNA deletion replacement is induced using a pair of atgRNA 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 a/.BioRxiv, November 2, 2021; Anzalone et al. is incorporated herein by reference). In some embodiments described herein, the DNA deletion is induced using a single atgRNA. In some embodiments, the DNA deletion and replacement is induced using a wild type Cas9 prime editor (PE-Cas9) system (i.e., PED AR by Jiang et al. Nat. Biotechnology, October 14, 2021; Jiang et al. is incorporated herein by reference in its entirety) In some embodiments, the DNA replacement is an integrase target recognition site or recombinase target recognition site. In certain embodiments, the constructs and methods described herein may be utilized to incorporate the pair of pegRNAs used in PrimeDel, TwinPE (WO2021226558 incorporated by reference herein in its entirety), or PED AR, the prime 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 single nucleic acid construct described herein. The integrase may be directly linked, for example by a peptide linker, to the prime editor fusion or gene writer protein.
[0063] In some embodiments, 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. In some embodiments, 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. In some embodiments 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. Such a complex is called PEI. In some embodiments, the CRISPR enzyme nickase, e.g., Cas9(H840A), i.e., a Cas9nickase, can be linked to a non-M-MLV reverse transcriptase such as an AMV-RT or XRT (Cas9(H840A)-AMV-RT or XRT). In some embodiments, instead of the CRISPR enzyme nickase 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(D10A). A CRISPR enzyme, such as a CRISPR enzyme nickase, such as Cas9 (wild type), Cas9(H840A), Cas9(D10A) 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. In some embodiments, the M-MLV RT comprise one or more of the mutations Y8H, P51L, S56A, S67R, E69K, V129P, L139P, T197A, 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.
Figure imgf000013_0001
[0064] In some embodiments, 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). In some embodiments, the reverse transcriptase can be a fusion of MMuLV to the Sto7d DNA binding domain (see lonnidi et al.; https://doi.org/10.1101/2021. l l.01.466786). The fusion of MMuLV to the Sto7d DNA binding domain sequence is given in Table 2.
Figure imgf000013_0002
[0065] PE3, PE3b, PE4, PE5, and/or PEmax, which a skilled person can incorporate into a single nucleic acid construct described herein, involves nicking the non-edited strand, potentially causing the cell to remake that strand using the edited strand as the template to induce HR. The nicking of the non-edited strand can involve the use of a nicking guide RNA (ngRNA).
[0066] The skilled person can readily incorporate into a gene editor composition described herein a prime editing or CRISPR system. Examples of prime editors or gene editors can be found in the following: W02020/191153, W02020/191171, WO2020/191233,
WO2020/191234, WO2020/191239, W02020/191241, WO2020/191242, WO2020/191243, WO2020/191245, WO2020/191246, WO2020/191248, WO2020/191249, each of which is incorporated by reference herein in its entirety. In addition, mention is made, and can be used herein, of CRISPR Patent Applications and Patents of the Zhang laboratory and/or Broad
Institute, Inc. and Massachusetts Institute of Technology and/or Broad Institute, Inc., Massachusetts Institute of Technology and President and Fellows of Harvard College and/or Editas Medicine, Inc. Broad Institute, Inc., The University of Iowa Research Foundation and Massachusetts Institute of Technology, including those claiming priority to US Application
61/736,527, filed December 12, 2012, including US Patents 11,104,937, 11,091,798,
11,060,115, 11,041,173, 11,021,740, 11,008,588, 11,001,829, 10,968,257, 10,954,514,
10,946,108, 10,930,367, 10,876,100, 10,851,357, 10,781,444, 10,711,285, 10,689,691,
10,648,020, 10,640,788, 10,577,630, 10,550,372, 10,494,621, 10,377,998, 10,266,887,
10,266,886, 10,190,137, 9,840,713, 9,822,372, 9,790,490, 8,999,641, 8,993,233, 8,945,839,
8,932,814, 8,906,616, 8,895,308, 8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965,
8,771,945, and 8,697,359; CRISPRPatent Applications and Patents of the Doudna laboratory and/or of Regents of the University of California, the University of Vienna and Emmanuelle
Charpentier, including those claiming priority to US application 61/652,086, filed May 25, 2012, and/or 61/716,256, filed October 19, 2012, and/or 61/757,640, filed January 28, 2013, and/or 61/765,576, filed February 15, 2013 and/or 13/842,859, including US Patents
11,028,412, 11,008,590, 11,008,589, 11,001,863, 10,988,782, 10,988,780, 10,982,231,
10,982,230, 10,900,054, 10,793,878, 10,774,344, 10,752,920, 10,676,759, 10,669,560,
10,640,791, 10,626,419, 10,612,045, 10,597,680, 10,577,631, 10,570,419, 10,563,227,
10,550,407, 10,533,190, 10,526,619, 10,519,467, 10,513,712, 10,487,341, 10,443,076,
10,428,352, 10,421,980, 10,415,061, 10,407,697, 10,400,253, 10,385,360, 10,358,659,
10,358,658, 10,351,878, 10,337,029, 10,308,961, 10,301,651, 10,266,850, 10,227,611,
10,113,167, and 10,000,772; CRISPR Patent Applications and Patents of Vilnius University and/or the Siksnys laboratory, including those claiming priority to US application 62/046384 and/or 61/625,420 and/or 61/613,373 and/or PCT/IB2015/056756, including US Patent 10,385,336; CRISPR Patent Applications and Patents of the President and Fellows of Harvard College, including those of George Church’s laboratory and/or claiming priority to US application 61/738,355, filed December 17, 2012, including 11,111,521, 11,085,072, 11,064,684, 10,959,413, 10,925,263, 10,851,369, 10,787,684, 10,767,194, 10,717,990, 10,683,490, 10,640,789, 10,563,225, 10,435,708, 10,435,679, 10,375,938, 10,329,587,
10,273,501, 10,100,291, 9,970,024, 9,914,939, 9,777,262, 9,587,252, 9,267,135, 9,260,723, 9,074,199, 9,023,649; CRISPR Patent Applications and Patents of the President and Fellows of Harvard College, including those of David Liu’s laboratory, including 11,111,472, 11,104,967, 11,078,469, 11,071,790, 11,053,481, 11,046,948, 10,954,548, 10,947,530,
10,912,833, 10,858,639, 10,745,677, 10,704,062, 10,682,410, 10,612,011, 10,597,679,
10,508,298, 10,465,176, 10,323,236, 10,227,581, 10,167,457, 10,113,163, 10,077,453,
9,999,671, 9,840,699, 9,737,604, 9,526,784, 9,388,430, 9,359,599, 9,340,800, 9,340,799, 9,322,037, 9,322,006, 9,228,207, 9,163,284, and 9,068,179; and CRISPR Patent Applications and Patents of Toolgen Incorporated and/or the Kim laboratory and/or claiming priority to US application 61/717,324, filed October 23, 2012 and/or 61/803,599, filed March 20, 2013 and/or 61/837,481, filed June 20, 2013 and/or 62/033,852, filed August 6, 2014 and/or PCT/KR2013/009488 and/or PCT/KR2015/008269, including US Patent 10,851,380, and 10,519,454; and CRISPR Patent Applications and Patents of Sigma and/or Millipore and/or the Chen laboratory and/or claiming priority to US application 61/734,256, filed December 6, 2012 and/or 61/758,624, filed January 30, 2013 and/or 61/761,046, filed February 5, 2013 and/or 61/794,422, filed March 15, 2013, including US Patent 10,731,181, each of which is hereby incorporated herein by reference, and from the disclosures of the foregoing, the skilled person can readily make and use a prime editing or CRISPR system, and can especially appreciate impaired endonucleases, such as a mutated Cas9 that only nicks a single strand of DNA and is hence a nickase, or a CRISPR enzyme that only makes a single-stranded cut that can be employed in a PASTE system of the invention. Further, from the disclosures of the foregoing, the skilled person can incorporate the selected CRISPR enzyme, as part of the gene editor, gene editor fusion, prime editor, or prime editor fusion compositions described herein.
[0067] Prior to RT-mediated edit incorporation, 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. However, in some embodiments 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. In addition, to the extent the “targeting rules” of other napDNAbp are known or are newly determined, it becomes possible to use new napDNAbp, beyond Cas9, to site specifically target and modify genomic sites of interest.
[0068] Similar to a prime editor protein, 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 Writer™ proteins and related systems can be found in US20200109398, which is incorporated by reference herein in its entirety.
[0069] In some embodiments, the prime editor or Gene Writer protein fusion or prime editor protein linked or fused to an integrase is expressed as a split construct. In typical embodiments, the split construct in reconstituted in a cell. In some embodiments, the split construct can be fused or ligated via intein protein splicing. In some embodiments, the split construct can be reconstituted via protein-protein inter-molecular bonding and/or interactions. In some embodiments, the split construct can be reconstituted via chemical, biological, or environmental induced oligomerization. In some embodiments, the split construct can be reconstituted via nanobody binding ALFA-tagged proteins. In certain embodiments, the split construct can be adapted into one or more single nucleic acid polynucleotides.
[0070] In some embodiments, 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. Suitable linkers, for example between die Cas9, RT, and integrase, may be selected from Table 3:
Figure imgf000016_0001
Figure imgf000017_0001
[0071] In some embodiments, the prime editor or Gene Writer protein fusion or prime editor protein linked or fused to an integrase is expressed as a split construct. In typical embodiments, the split construct in reconstituted in a cell. In some embodiments, the split construct can be fused or ligated via intein protein splicing. In some embodiments, the split construct can be reconstituted via protein-protein inter-molecular bonding and/or interactions. In some embodiments, the split construct can be reconstituted via chemical, biological, or environmental induced oligomerization. In certain embodiments, the split construct can be adapted into one or more nucleic acid constructs described herein.
6.2. Type II CRISPR proteins
[0072] The skilled person can incorporate a selected CRISPR enzyme, described below, as part of the prime editor fusion, as a component of the gene editor polypeptide described herein. Streptococcus pyogenes Cas9 (SpCas9), the most common enzyme used in genome-editing applications, is a large nuclease of 1368 amino acid residues. Advantages of SpCas9 include its short, 5'-NGG-3' PAM and very high average editing efficiency. SpCas9 consists of two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe can be divided into three regions, a long a helix referred to as the bridge helix (residues 60-93), the RECI (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. In the NUC lobe, 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.
[0073] REC lobe'. The REC lobe includes the RECI 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. Further, 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. In striking contrast, the deletion of either the repeat-interacting region (D97-150) or the anti -repeat-interacting region (D312-409) of the RECI domain abolished the DNA cleavage activity, indicating that the recognition of the repeatanti -repeat duplex by the RECI domain is critical for the Cas9 function.
[0074] PAM-interacting domain'. The NUC lobe contains the PAM-interacting (PI) domain that is positioned to recognize the PAM sequence on the noncomplementary DNA strand. The PI domain of SpCas9 is required for the recognition of 5’-NGG-3’ PAM, and deletion of the PI domain (A1099-1368) abolished the cleavage activity, indicating that the PI domain is critical for SpCas9 function and a major determinant for the PAM specificity.
[0075] RuvC domain-. The RuvC nucleases of SpCas9 have an RNase H fold and four catalytic residues, AsplO (Ala), Glu762, His983, and Asp986, that are critical for the two-metal cleavage of the noncomplementary strand of the target DNA. In addition to the conserved RNase H fold, the Cas9 RuvC domain has other structural elements involved in interactions with the guide :target heteroduplex (an end-capping loop between a42 and a43) and the PI domain/stem loop 3 (P hairpin formed by P3 and [34).
[0076] 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.
[0077] sgRNA:DNA recognition'. The sgRNA guide region is primarily recognized by the REC lobe. The backbone phosphate groups of the guide region (nucleotides 2, 4-6, and 13-20) interact with the RECI domain (Argl65, Glyl66, Arg403, Asn407, Lys510, Tyr515, and Arg661) and the bridge helix (Arg63, Arg66, Arg70, Arg71, Arg74, and Arg78). The 20-hydroxyl groups of Gl, Cl 5, U16, and G19 hydrogen bond with Vai 1009, Tyr450, Arg447/Ile448, and Thr404, respectively.
[0078] A mutational analysis demonstrated that the R66A, R70A, and R74A mutations on the bridge helix markedly reduced the DNA cleavage activities, highlighting the functional significance of the recognition of the sgRNA “seed” region by the bridge helix. Although Arg78 and Argl65 also interact with the “seed” region, the R78A and R165A mutants showed only moderately decreased activities. These results are consistent with the fact that Arg66, Arg70, and Arg74 form multiple salt bridges with the sgRNA backbone, whereas Arg78 and Argl65 form a single salt bridge with the sgRNA backbone. Moreover, the alanine mutations of the repeatantirepeat duplex-interacting residues (Arg75 and Lysl63) and the stemloop- 1 -interacting residue (Arg69) resulted in decreased DNA cleavage activity, confirming the functional importance of the recognition of the repeat: anti -repeat duplex and stem loop 1 by Cas9.
[0079] RNA-guided DNA targeting'. SpCas9 recognizes the guide:target heteroduplex in a sequence-independent manner. The backbone phosphate groups of the target DNA (nucleotides 1, 9-11, 13, and 20) interact with the REC 1 (Asn497, Trp659, Arg661, and Gln695), RuvC (Gln926), and PI (Glul 108) domains. The C2’ atoms of the target DNA (nucleotides 5, 7, 8, 11, 19, and 20) form van der Waals interactions with the RECI domain (Leul69, Tyr450, Met495, Met694, and His698) and the RuvC domain (Ala728). The terminal base pair of the guide:target heteroduplex (Gl :C20’) is recognized by the RuvC domain via end-capping interactions; the sgRNA G1 and target DNA C20’ nucleobases interact with the Tyrl013 and Vai 1015 side chains, respectively, whereas the 20-hydroxyl and phosphate groups of sgRNA G1 interact with Vai 1009 and Gln926, respectively.
[0080] Repeat: Anti-Repeat duplex recognition'. The nucleobases of U23/A49 and A42/G43 hydrogen bond with the side chain of Argl l22 and the main-chain carbonyl group of Phe351, 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.
[0081] 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 04 atom hydrogen bonded with Arg75. Notably, A51 adopts the syn conformation and is oriented in the direction opposite to U50. The nucleobase of A51 is sandwiched between Phel 105 and U63, with its Nl, N6, and N7 atoms hydrogen bonded with G62, Glyl l03, and Phel 105, respectively.
[0082] Stem-loop recognition'. Stem loop 1 is primarily recognized by the REC lobe, together with the PI domain. The backbone phosphate groups of stem loop 1 (nucleotides 52, 53, and 59- 61) interact with the RECI domain (Leu455, Ser460, Arg467, Thr472, and Ile473), the PI domain (Lysl 123 and Lysl 124), and the bridge helix (Arg70 and Arg74), with the 20-hydroxyl group of G58 hydrogen bonded with Leu455. A52 interacts with Phel 105 through a face-to-edge p-p stacking interaction, and the flipped U59 nucleobase hydrogen bonds with Asn77.
[0083] The single-stranded linker and stem loops 2 and 3 are primarily recognized by the NUC lobe. The backbone phosphate groups of the linker (nucleotides 63-65 and 67) interact with the RuvC domain (Glu57, Lys742, and Lysl097), the PI domain (Thrl l02), and the bridge helix (Arg69), with the 20-hydroxyl groups of U64 and A65 hydrogen bonded with Glu57 and His721, respectively. The C67 nucleobase forms two hydrogen bonds with Vail 100.
[0084] Stem loop 2 is recognized by Cas9 via the interactions between the NUC lobe and the non-Watson-Crick A68:G81 pair, which is formed by direct (between the A68 N6 and G81 06 atoms) and water-mediated (between the A68 Nl and G81 Nl atoms) hydrogen-bonding interactions. The A68 and G81 nucleobases contact Serl351 and Tyrl356, respectively, whereas the A68:G81 pair interacts with Thrl358 via a water-mediated hydrogen bond. The 20-hydroxyl group of A68 hydrogen bonds with Hisl349, whereas the G81 nucleobase hydrogen bonds with Lys33.
[0085] Stem loop 3 interacts with the NUC lobe more extensively, as compared to stem loop 2. The backbone phosphate group of G92 interacts with the RuvC domain (Arg40 and Lys44), whereas the G89 and U90 nucleobases hydrogen bond with Glnl272 and Glul225/Alal227, respectively. The A88 and C91 nucleobases are recognized by Asn46 via multiple hydrogenbonding interactions.
[0086] 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. In addition, efficient packaging of CRISPR systems can be achieved in other viral vector systems (i.e., lentiviral, integration deficient lentiviral, hd-AAV, etc.) and non viral vector systems (i.e., lipid nanoparticle). Small Cas9 proteins can be advantageous for multidomain-Cas-nuclease-based systems for prime editing. Well characterized smaller Cas9 proteins include Staphylococcus aureus (SauCas9, 1053 amino acid residues) and Campylobacter jejuni (CjCas9, 984 amino residues). However, both recognize longer PAMs, 5'-NNGRRT-3' for SauCas9 (R = A or G) and 5'-NNNNRYAC-3' for CjCas9 (Y = C or T), which reduces the number of uniquely addressable target sites in the genome, in comparison to the NGG SpCas9 PAM. Among smaller Cas9s, Schmidt et al. identified Staphylococcus lugdunensis (Siu) Cas9 as having genome-editing activity and provided homology mapping to SpCas9 and SauCas9 to facilitate generation of nickases and inactive (“dead”) enzymes (Schmidt et al., 2021, Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases. Nat Commun 12, 4219. doi.org/10.1038/s41467-021-24454-5) and engineered nucleases with higher cleavage activity by fragmenting and shuffling Cas9 DNAs. The small Cas9s and nickases are useful in the instant disclosure.
[0087] Besides dead Cas9 and Cas9 nickase variants, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 (SEQ ID NO: 18).
[0088] In some embodiments, the disclosure also may utilize Cas9 fragments that retain their functionality and that are fragments of any herein disclosed Cas9 protein. In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, 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.
[0089] In various embodiments, 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.
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
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Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
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Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
[0090] In some embodiments, prime editors utilized herein comprise CRISPR-Cas system enzymes other than type II enzymes. In certain embodiments, 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.
[0091] In various embodiments, the nucleic acid programmable DNA binding proteins utilized herein include, without limitation, Cas9 (e.g., dCas9 and nCas9), Casl2a (Cpfl), Casl2bl (C2cl), Casl2b2, Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), C2c4, C2c5, C2c8, C2c9, C2cl0, Cast 3a (C2c2), Cast 3b (C2c6), Cast 3c (C2c7), Cast 3d, and Argonaute. 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.l. 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, Casl2a (Cpfl)). Similar to Cas9, Casl2a (Cpfl) 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 Casl2a (Cpfl) mediates robust DNA interference with features distinct from Cas9. Casl2a (Cpfl) is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T- rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpfl cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpfl -family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpfl proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpfl 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
[0092] In some embodiments, prime editors used herein comprise the type V CRISPR family includes Francisella novicida U112 Cpfl (FnCpfl) also known as FnCasl2a. FnCpfl 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 (RECI 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. Notably, nucleotides 1-5 of the crRNA are ordered in the central cavity of FnCasl2a 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 FnCpfl residues in the WED and RECI domains. These include residues Thrl6, Lys595, His804, and His881 from the WED domain and residues Tyr47, Lys51, Phel82, and Argl86 from the RECI domain. The structure of the FnCasl2a-crRNA complex further reveals that the bases of the seed sequence are solvent exposed and poised for hybridization with target DNA. Structural aspects of FnCpfl are described by Swarts et al., Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Casl2a, Molecular Cell 66, 221-233, April 20, 2017.
[0093] Pre-crRNA processing'. Essential residues for crRNA processing include His843, Lys852, and Lys869. Structural observations are consistent with an acid-base catalytic mechanism in which Lys869 acts as the general base catalyst to deprotonate the attacking 2’ -hydroxyl group of U(-19), while His843 acts as a general acid to protonate the 5’-oxygen leaving group of A(-18). In turn, the side chain of Lys852 is involved in charge stabilization of the transition state. Collectively, these interactions facilitate the intra-molecular attack of the 20-hydroxyl group of U(-19) on the scissile phosphate and promote the formation of the 2’, 3 ’-cyclic phosphate product.
[0094] 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 RECI 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 RECI domains. The 5’-TTN-3’ PAM is recognized in FnCasl2a by a mechanism combining the shapespecific recognition of a narrowed minor groove, with base-specific recognition of the PAM bases by two invariant residues, Lys671 and Lys613. Directly downstream of the PAM, 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-7t stacking interaction with the dA0-dT0* base pair. The phosphate group linking target strand residues dT(-l) and dTO is coordinated by hydrogen-bonding interactions with the side chain of Lys823 and the backbone amide of Gly826. Target strand residue dT(-l) bends away from residue TO, allowing the target strand to interact with the seed sequence of the crRNA. The non-target strand nucleotides dTl*-dT5* interact with the Arg692- Ser702 loop in FnCasl2a 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 FnCasl2a 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.
[0095] In the FnCasl2a R-loop complex, 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.
[0096] Target DNA cleavage'. FnCpfl 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 Cpfl/Casl2a enzymes.
[0097] Another type V CRISPR is AsCpfl from Acidaminococcus sp BV3L6 (Yamano et al., Crystal structure of Cpfl in complex with guide RNA and target DNA, Cell 165, 949-962, May 5, 2016)
[0098] In certain embodiments, the nuclease comprises a Casl2f effector. Small CRISPR- associated effector proteins belonging to the type V-F subtype have been identified through the mining of sequence databases and members classified into Casl2fl (Casl4a and type V-U3), Casl2f2 (Casl4b) and Casl2f3 (Casl4c, type V-U2 and U4). (See, e.g., Karvelis et al., PAM recognition by miniature CRISPR-Casl2f nucleases triggers programmable double-stranded DNA target cleavage. Nucleic Acids Research, 21 May 2020, 48(9), 5016-23 doi.org/10.1093/nar/gkaa208). Xu et al. described development of a 529 amino acid Casl2f-based system for mammalian genome engineering through multiple rounds of iterative protein engineering and screening. (Xu, X. et al., Engineered Miniature CRISPR-Cas System for Mammalian Genome Regulation and Editing. Molecular Cell, October 21, 2021, 81(20): 4333-45, doi.org/10.1016/j.molcel.2021.08.008). [0099] Exemplary CRISPR-Cas proteins and enzymes used in the Prime Editors herein include the following without limitation.
Figure imgf000068_0001
Figure imgf000069_0001
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Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
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Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0002
Figure imgf000093_0001
Table 6 - Casl2b (C2cl) orthologs
Figure imgf000094_0001
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Figure imgf000098_0001
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Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000108_0002
6.4. Protospacer Adjacent Motif
[0100] As used herein, the term “protospacer adjacent sequence” or “protospacer adjacent motif’ or “PAM” refers to an approximately 2-6 base pair DNA sequence (or a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-long nucleotide sequence) that is an important targeting component of a Cas9 nuclease. Typically, 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. Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes alternative PAM sequence. [0101] For example, with reference to the canonical SpCas9 amino acid sequence, the PAM specificity can be modified by introducing one or more mutations, including (a) DI 135 V, R1335Q, and T1337R “the VQR variant”, which alters the PAM specificity to NGAN or NGNG, (b) D1135E, R1335Q, and T1337R “the EQR variant”, which alters the PAM specificity to NGAG, and (c) DI 135V, G1218R, R1335E, and T1337R “the VRER variant”, which alters the PAM specificity to NGCG. In addition, the DI 135E variant of canonical SpCas9 still recognizes NGG, but it is more selective compared to the wild type SpCas9 protein.
[0102] It will also be appreciated that Cas9 enzymes from different bacterial species (i.e., Cas9 orthologs) can have varying PAM specificities and in some embodiments are therefore chosen based on the desired PAM recognition. For example, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN. In addition, Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT. In another example, Cas9 from Streptococcus thermophilis (StCas9) recognizes NNAGAAW. In still another example, Cas9 from Treponema denticola (TdCas) 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). Further reference may be made to Shah et al., “Protospacer recognition motifs: mixed identities and functional diversity,” RNA Biology, 10(5): 891-899 (which is incorporated herein by reference). Gasiunas used cell-free biochemical screens to identify protospacer adjacent motif (PAM) and guide RNA requirements of 79 Cas9 proteins. (Gasiunas et al., A catalogue of biochemically diverse CRISPR-Cas9 orthologs, Nature Communications 11 :5512 doi.org/10.1038/s41467-020-19344-l) The authors described 7 classes of gRNA and 50 different PAM requirement.
[0103] Oh, Y. et al. describe linking reverse transcriptase to a Francisella novicida Cas9 [FnCas9(H969A)] nickase module. (Oh, Y. et al., Expansion of the prime editing modality with Cas9 from Francisella novicida, bioRxiv 2021.05.25.445577; doi.org/10.1101/2021.05.25.445577). By increasing the distance to the PAM, the FnCas9(H969A) nickase module expands the region of a reverse transcription template (RTT) following the primer binding site. 6.5. Prime Editors
[0104] “Prime editor fusion protein” describes a protein that is used in prime editing. Prime editing uses CRISPR enzyme that nicks or cuts only single strand of double stranded DNA, i.e., a nickase; and a nickase can occur either naturally or by mutation or modification of a nuclease that makes double stranded cuts. Such an enzyme can be a catalytically-impaired Cas9 endonuclease (a nickase). 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 primeediting guide RNA (pegRNA). The skilled person in the art would appreciate that the pegRNA both specifies the target site and encodes the desired edit. Advantageously the nickase is a catalytically-impaired Cas9 endonuclease, a Cas9 nickase, that is fused to the reverse transcriptase. During genetic editing, the Cas9 nickase part of the protein is guided to the DNA target site by the pegRNA, whereby a nick or single stranded cut occurs. The reverse transcriptase domain then uses the pegRNA to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand. The edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand. Afterward, optionally, the prime editor (PE) guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process (typically achieved with a nickase gRNA).
[0105] As used herein, “PEI” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a wild type MMLV RT having the following N-terminus to C-terminus structure: [NLS]-[Cas9(H840A)]- [linker]-[MMLV_RT(wt)] + a desired PEgRNA. In various embodiments, the prime editors disclosed herein is comprised of PEI.
[0106] As used herein, “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 (PEgRNA). In various embodiments, the prime editors disclosed herein are comprised of PE2.
[0107] In various embodiments, the prime editors disclosed herein are comprised of PE2 and co-expression of MMR protein MLHldn, that is PE4.
[0108] As used herein, “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. In various embodiments, the prime editors disclosed herein are comprised of PE3. [0109] In various embodiments, the prime editors disclosed herein is comprised of PE3 and co-expression of MMR protein MLHldn, that is PE5.
[0110] As used herein, “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. Guides for Prime Editing
[oni] Anzalone et al., 2019 (Nature 576:149) describes prime editing and a prime editing complex using a type II CRISPR and can be used herein. 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. 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.
[0112] Guide RNAs of CRISPRs differ in overall structure. For example, while the spacer of a type II gRNA is located at the 5’ end, the spacer of a type V gRNA is located towards the 3’ end, with the CRISPR protein (e.g. Casl2a) binding region located toward the 5’ end. Accordingly, the regions of a type V pegRNA are rearranged compared to a type II pegRNA. The overall structure of the pegRNA is like that of a typical type II sgRNA with a reverse transcriptase template/primer binding site appended to the 3’ end. The pegRNA comprises (5’ to 3’) a CRISPR protein-binding region, a spacer which is complementary to the target sequence of a genomic DNA, a reverse transcriptase template including editing positions, and primer binding site (PBS). 6.7. Attachment Site-Containing Guide RNA (atgRNA)
[0113] As used herein, 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. In some embodiments, the RT template comprises a clamp sequence and an integration recognition site. As referred to herein 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).
[0114] As used herein, the term “cognate integration recognition site” or “integration cognate” or “cognate pair” refers to a first integration recognition site (e.g., any of the integration recognition sites described herein) and a second integration recognition site (e.g., any of the integration recognition sites described herein) that can be recombined. Recombination between a first integration recognition site (e.g., any of the integration recognition sites described herein) and a second recognition site (e.g., any of the integration recognition sites described herein) is mediated by functional symmetry between the two integration recognition sites and the central dinucleotide of each of the two integration recognition sites. In some cases, a first integration recognition site (e.g., any of the integration recognition sites described herein) that can be recombined with a second integration recognition site (e.g., any of the integration recognition sites described herein) are referred to as a “cognate pair.” A non-limiting example of a cognate pair include an attB site and an attP site, whereby a serine integrase mediates recombination between the attB site and the attP site. FIGs. 10A-10E show optimization of the integration recognition site.
[0115] In typical embodiments, an atgRNA comprises a reverse transcriptase template that encodes, partially or in its entirety, an integration recognition site (also referred to as an integration target recognition site) or a recombinase recognition site (also referred to as a recombinase target recognition site). The integration target recognition site, which is to be placed at a desired location in the genome or intracellular nucleic acid, is referred to as a “beacon,” a “beacon” site or an “attachment site” or a “landing pad” or “landing site.” An integration target recognition site or recombinase target recognition site incorporated into the pegRNA is referred to as an attachment site containing guide RNA (atgRNA).
[0116] During genome editing, the primer binding site allows the 3’ end of the nicked DNA strand to hybridize to the atgRNA, while the RT template serves as a template for the synthesis of edited genetic information. The atgRNA is capable for instance, without limitation, of (i) identifying the target nucleotide sequence to be edited and (ii) encoding new genetic information that replaces (or in some cases adds) the targeted sequence. In some embodiments, 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.
[0117] In some embodiments, where the system contains a first atgRNA and a second atgRNA (see FIG. 11), 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; and the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
[0118] In some embodiments, 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. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 5 consecutive bases of an integrase target recognition site. In certain embodiments, 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. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 20 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 30 consecutive bases of an integrase target recognition site. Use of two guide RNAs that are (or encode DNA that is) partially complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site-containing guide RNAs (atgRNAs). In certain embodiments, use of two guide RNAs that are (or encode DNA that is) full complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment sitecontaining guide RNAs (atgRNAs).
I l l [0119] In some embodiments, upon introducing the nucleic acid construct into a cell, the first atgRNA incorporates the first integration recognition site into the cell’s genome at the target sequence.
[0120] Table 9 includes atgRNAs, sgRNAs and nicking guides that can be used herein. Spacers are labeled in capital font (SPACER), RT regions in bold capital (RT REGION), AttB sites in bold lower case (attb site), and PBS in capital italics (PBS). Unless otherwise denoted, the AttB is for Bxbl.
Figure imgf000114_0001
Table 9
Figure imgf000115_0001
Table 9
Figure imgf000116_0001
Table 9
Figure imgf000117_0001
Table 9
Figure imgf000118_0001
Table 9
Figure imgf000119_0001
Table 9
Figure imgf000120_0001
Table 9
Figure imgf000121_0001
Table 9
Figure imgf000122_0001
Table 9
Figure imgf000123_0001
Table 9
Figure imgf000124_0001
Table 9
Figure imgf000125_0001
Table 9
Figure imgf000126_0001
Table 9
Figure imgf000127_0001
Table 9
Figure imgf000128_0001
Table 9
Figure imgf000129_0001
Table 9
Figure imgf000130_0001
Table 9
Figure imgf000131_0001
Table 9
Figure imgf000132_0001
Table 9
Figure imgf000133_0001
Table 9
Figure imgf000134_0001
6.8. Integrases/Recombinases and Integration/Recombination Sites
[0121] In typical embodiments, a gene editor polypeptide described herein contains an integrase or recombinase. In some embodiments, the integrase is delivered as a protein or the integrase is encoded in a delivered polynucleotide. In some embodiments, the integration enzyme is selected from the group consisting of Dre, Vika, Bxbl, <pC31, RDF, cpBTl, Rl, R2, R3, R4, R5, TP901-1, Al 18, cpFCl, cpCl, MR11, TGI, cp370.1, Wp, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, Concept!!, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, cpRV, retrotransposases encoded by a Tcl/mariner family member including but not limited to retrotransposases encoded by LI, Tol2, Tel, Tc3, Himar 1 (isolated from the horn fly, Haematobia irrilans). Mosl (Mosaic element of Drosophila mauritiana), and Minos, and any mutants thereof. As can be used herein, Xu et al describes methods for evaluating integrase activity in E. coll and mammalian cells and confirmed at least R4, cpC31, (pBTl, Bxbl, SPBc, TP901-1 and WP integrases to be active on substrates integrated into the genome of HT1080 cells (Xu et al., 2013, Accuracy and efficiency define Bxbl 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 Bxbl in episomal and chromosomal integration efficiency, LSRs that achieve both efficient and sitespecific 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 lonnidi et al., 2021; Drag-and-drop genome insertion without DNA cleavage with CRISPR directed integrases. bioRxiv 2021.11 .01.466786, doi. org/10.1101/2021.11.01.466786). In some embodiments, the integration site can be selected from an attB site, an attP site, an attL site, an attR site, a lox71 site a Vox site, or a FRT site.
[0122] It will be appreciated that desired activity of integrases, transposases and the like can depend on nuclear localization. In certain embodiments, prokaryotic enzymes are adapted to modulate nuclear localization. In certain embodiments, eukaryotic or vertebrate enzymes are adapted to modulate nuclear localization. In certain embodiments, 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). Xu et al compared derivatives of fourteen serine integrases that either possess or lack a nuclear localization signal (NLS) to conclude that certain integrases benefit from addition of an NLS whereas others are transported efficiently without addition, and a major determinant of activity in yeast and vertebrate cells is avoidance of toxicity. (Xu et al., 2016, Comparison and optimization of ten phage encoded serine integrases for genome engineering in Saccharomyces cerevisiae. BMC Biotechnol. 2016 Feb 9; 16:13. doi: 10.1186/sl2896-016-0241 -5). Ramakrishnan et al. systematically studied the effect of different NES mutants developed from mariner-like elements (MLEs) on transposase localization and activity and concluded that nuclear export provides a means of controlling transposition activity and maintaining genome integrity. (Ramakrishnan et al. Nuclear export signal (NES) of transposases affects the transposition activity of mariner-like elements Ppmarl and Ppmar2 of moso bamboo. Mob DNA. 2019 Aug 19; 10:35. doi: 10.1186/sl3100-019-0179-y). The methods and constructs are used to modulate nuclear localization of system components of the invention.
[0123] In typical embodiments, the integrase used herein is selected from below (Table 10).
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
[0124] Sequences of insertion sites (i.e., recognition target sites) suitable for use in embodiments of the disclosure are presented below (Table 11). FIGs. 10A-10E show analysis of effect of variant AttP sites on integration efficiency.
Figure imgf000143_0002
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
6.9. Gene Editor Polypeptide Compositions
[0125] This disclosure features a gene editor polypeptide useful for site-specifically integrating an integration recognition site into a target nucleic acid sequence (i.e., beacon placement), for example by using a polypeptide comprising a DNA binding nuclease linked to a reverse transcriptase, and integrating an exogenous nucleic acid sequence at the integrated integration recognition site (i.e., PASTE) by introducing into the cell the exogenous nucleic acid and an integration enzyme. The gene editor polypeptide includes a polypeptide comprised of a (i) DNA binding nickase (optionally with nickase activity), (ii) a reverse transcriptase, and (iii) an integration enzyme (e.g., an integrase or a recombinase), wherein at least any two elements of (i), (ii), or (iii) are linked via at least a first C-terminal linker. In some embodiments, the gene editor polypeptide includes a second linker (see FIG. 1 and FIG. 2). For example, where a gene editor polypeptide includes a first and a second linker, the first linker links a first domain (e.g., a DNA binding) to a second domain (e.g., a RT) and the second linker links the second domain to a third domain (e.g., an integration enzyme). In some embodiments, the orientation of the DNA binding nuclease, the RT, and the integration enzyme are configured for example from N-terminus to C- terminus: a DNA binding nuclease domain, a RT, and an integration enzyme; a DNA binding nuclease, an integration enzyme, and a RT; an integration enzyme, a DNA binding nuclease, and a RT; an integration enzyme, a RT, and a DNA binding nuclease; a RT, a DNA binding nuclease, and an integration enzyme; and a RT, an integration enzyme, and a DNA binding nuclease, where each of the domains are linked via a linker (see, FIG. 3 and Table 3). In one embodiment, the C- terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C- terminus of the reverse transcriptase is linked to the integration enzyme. In one embodiment, the C-terminus of the integration enzyme is linked to the DNA binding nuclease, wherein the C- terminus of the DNA binding nuclease is linked to a reverse transcriptase.
[0126] In some embodiments, the gene editor polypeptide is comprised of at least two of any one of (i) a DNA binding nickase, (ii) a reverse transcriptase, and (iii) an integration enzyme wherein at least any two elements of (i), (ii), or (iii) are linked via at least a first C-terminal linker.
[0127] In certain embodiments, the gene editor polypeptide can be comprised of (i) a DNA binding nickase, (ii) a reverse transcriptase, and (iii) an integration enzyme (e.g., an integrase or a recombinase), wherein at least any two elements of (i), (ii), or (iii) may be linked via two or more C-terminal linkers at the same linking position. For example, a gene editor polypeptide includes a DNA binding nickase linked via two linkers to (a reverse transcriptase, and the reverse transcriptase domain fused or linked to an integration enzyme.
[0128] In some embodiments, a linker (e.g., the first linker, the second linker, or both) is selected from a flexible linker, a semi-flexible linker, a rigid linker, a flexible-rigid linker.
[0129] In some embodiments, the gene editor polypeptide includes at least one flexible linker. Non-limiting examples of flexible linkers include LI (SEQ ID NO: 15), L2 (SEQ ID NO: 551), L5 (SEQ ID NO: 19), L6 (SEQ ID NO: 18), and LI 3 (SEQ ID NO: 558). In some embodiments, the gene editor polypeptide includes at least one flexible linker selected from: LI, L2, L5, L6, and LI 3. In some embodiments, the gene editor polypeptide includes at least a LI linker (SEQ ID NO: 15). In some embodiments, the gene editor polypeptide includes at least a L2 linker (SEQ ID NO: 551). In some embodiments, the gene editor polypeptide includes at least a L5 linker (SEQ ID NO: 19) linker. In some embodiments, the gene editor polypeptide includes at least a L6 linker (SEQ ID NO: 18). In some embodiments, the gene editor polypeptide includes at least a L13 linker (SEQ ID NO: 558).
[0130] In some embodiments, the gene editor polypeptide includes at least one semi-flexible linker. Non-limiting examples of semi-flexible linkers include L7 (SEQ ID NO: 552) and L8 (SEQ ID NO: 553). In some embodiments, the gene editor polypeptide includes at least a L7 linker (SEQ ID NO: 552). In some embodiments, the gene editor polypeptide includes at least a L7 linker (SEQ ID NO: 553).
[0131] In some embodiments, the gene editor polypeptide includes at least one rigid linker. Nonlimiting examples of rigid linkers include L3 (SEQ ID NO: 16), L4 (SEQ ID NO: 17), and L9 (SEQ ID NO: 554). In some embodiments, the gene editor polypeptide includes at least a L3 linker (SEQ ID NO: 16). In some embodiments, the gene editor polypeptide includes at least a L4 linker (SEQ ID NO: 17). In some embodiments, the gene editor polypeptide includes at least a L9 linker (SEQ ID NO: 554).
[0132] In some embodiments, the gene editor polypeptide includes at least one flexible-rigid linker. Non-limiting examples of flexible-rigid linkers include Li l (SEQ ID NO: 557) and L12 (SEQ ID NO: 558). In some embodiments, the gene editor polypeptide includes at least a Li l linker (SEQ ID NO: 557). In some embodiments, the gene editor polypeptide includes at least a L12 linker (SEQ ID NO: 558).
[0133] In some embodiments, the gene editor polypeptide includes a linker (e.g., a first linker, a second linker, or both) where the length of the linker is selected from short, medium, and long. Non-limiting examples of short linkers include: LI (SEQ ID NO: 15), L3 (SEQ ID NO: 16), L7 (SEQ ID NO: 552), and L8 (SEQ ID NO: 553). Non-limiting examples of a medium linker include: L2 (SEQ ID NO: 551), L4 (SEQ ID NO: 17), L5 (SEQ ID NO: 19), L6 (SEQ ID NO: 18), L10 (SEQ ID NO: 555), LI 1 (SEQ ID NO: 556), L12 (SEQ ID NO: 557), and L13 (SEQ ID NO: 558). A non-limiting example of a long linker is L9 (SEQ ID NO: 554).
[0134] In some embodiments, the gene editor polypeptide includes a linker (e.g., the first linker, the second linker, or both) that is a non-cleavable linker. Non-limiting examples of non-cleavable linkers include: LI (SEQ ID NO: 15), L2 (SEQ ID NO: 551), L3 (SEQ ID NO: 16), L4 (SEQ ID NO: 17), L5 (SEQ ID NO: 19), L6 (SEQ ID NO: 18), L7 (SEQ ID NO: 552), L8 (SEQ ID NO: 553), L9 (SEQ ID NO: 554), LIO (SEQ ID NO: (555), LI 1 (SEQ ID NO: 556), L12 (SEQ ID NO: 557), and L13 (SEQ ID NO: 558).
[0135] In some embodiments, the gene editor polypeptide include a linker (e.g., the first linker, the second linker, or both) that is a cleavable linker. Cleavable linkers can facilitate the coexpression of multiple proteins within a single open reading frame, effectively separating them post-translationally. Non-limiting examples of self-cleaving peptides include 2A peptides (derived from the foot-and-mouth disease virus), such as F2A, T2A, E2A, and P2A; the “Inteins” that facilitate protein splicing; and the tobacco etch virus (TEV) protease recognition site. In some embodiments, the cleavable linker is 2A peptide. In some embodiments, the cleavable linker is a P2A peptide.
[0136] In some embodiments, the gene editor polypeptide includes at least a first linker and a second linker where: the at least first linker is a non-cleavable linker and the second linker is a cleavable linker; the at least first linker is a cleavable linker and the second linker is a non- cleavable linker; the at least first linker is a cleavable linker and the second linker is a cleavable linker; or the at least first linker is a non-cleavable linker and the second linker is a non-cleavable linker.
[0137] In some embodiments, the gene editor polypeptide includes the DNA binding nuclease, the RT, and the integration enzyme, where the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, and where any one of L7 (SEQ ID NO: 552), LIO (SEQ ID NO: 555), or L12 (SEQ ID NO: 557) links the DNA binding nuclease to the reverse transcriptase.
[0138] In some embodiments, the gene editor polypeptide includes the DNA binding nuclease, the RT, and the integration enzyme, where the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L8 (SEQ ID NO: 553), L9 (SEQ ID NO: 554), L10 (SEQ ID NO: 555), L12 (SEQ ID NO: 557), or L13 (SEQ ID NO: 558) links the reverse transcriptase to the integration enzyme.
[0139] In some embodiments, the gene editor polypeptide includes the DNA binding nuclease, the RT, and the integration enzyme, where the C-terminus of the integration enzyme is linked to the DNA binding nuclease by L13 (SEQ NO: 558), and where the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by L13 (SEQ ID NO: 558). [0140] In some embodiments, the gene editor polypeptide is not comprised of aN-mostN-terminal reverse transcriptase.
[0141] In some embodiments, the gene editor polypeptide includes the DNA binding nuclease, the RT, and the integration enzyme, where the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by LI 3 (SEQ ID NO: 558), and where the C-terminus of the reverse transcriptase is linked to the integration enzyme by a cleavable linker (e.g., a P2A (SEQ ID NO: 13).
[0142] In some embodiments, the gene editor polypeptide includes a nuclear localization signal (NLS). In some embodiments, the gene editor polypeptide comprises a mitochondrial localization signal.
[0143] In one embodiment, the gene editor polypeptide comprises: from N-terminus to C- terminus: a DNA binding nickase domain; a P2A linker (SEQ ID NO: 13), a reverse transcriptase; a mXTEN linker, and an integration enzyme.
[0144] In one embodiment, the gene editor polypeptide comprises: from N-terminus to C- terminus: an integration enzyme, a mXTEN linker (SEQ ID NO: 558), a DNA binding nuclease domain; a mXTEN linker (SEQ ID NO: 558), and a reverse transcriptase.
[0145] This disclosure also features systems capable of site-specifically integrating at least a first integration recognition site into the genome of a cell where the system includes any of the gene editor polypeptides described herein.
[0146] This disclosure also features systems capable of site-specifically integrating an exogenous nucleic acid sequence into the genome of a cell where the system includes any of the gene editor polypeptides described herein.
[0147] In some embodiments, the gene editor polypeptide comprises at least one linker that is non-cleavable, and where the at least one linker is selected from a group consisting of LI, L2, L3, L4, L5, L6, L7, L8, L9, L10, LI 1, L12, and L13. LI, L2, L3, L4, L5, L6, L7, L8, L9, L10, LI 1, L12, and L13 are illustrated in FIG. 3 and Table 3. 6.10. Gene Editor Polynucleotide Compositions
[0148] In certain embodiments, a polynucleotide encodes the gene editor polypeptide. In some embodiments, the polynucleotide is an RNA or DNA. In some embodiments, the polynucleotide is a mRNA.
[0149] In certain embodiments, an expression vector comprises the polynucleotide encoding the gene editor polypeptide. In some embodiments, the expression vector further comprises any one or more of a one or more of a promoter sequence, atgRNA, nicking gRNA (ngRNA), donor polynucleotide template, or donor polynucleotide template complement. In some embodiments, two or more expression vectors are delivered.
[0150] In typical embodiments, the expression vector is recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, annelovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, or lipid nanoparticle.
6.11. Programmable Gene Insertion
[0151] In some embodiments, the polypeptides described herein are used for programmable polynucleotide (e.g., genes or gene fragments) insertion. Programmable polynucleotide/gene insertion includes site-specifically integrating an exogenous nucleic acid sequence (e.g., a donor polynucleotide template) into a genome of a cell. In typical embodiments, the method of site- specifically integrating an exogenous nucleic acid sequence into a target nucleic acid comprises incorporating an integration recognition site into a cell by delivering: (i) a polynucleotide encoding a gene editor polypeptide (e.g., any of the gene editor polypeptides described herein), (ii) at least a first target specific atgRNAs comprising a first integration recognition site, and (iii) optionally, a nicking guide RNA. In some embodiments, the first atgRNA comprises: (i) a domain that is capable of guiding the prime editor system to a target sequence; and (ii) a reverse transcriptase (RT) template that comprises at least a portion of an at least first integration recognition site, whereby the at least portion of the at least first integration recognition site is integrated into the genome of the cell at the target sequence.
[0152] In some embodiments, the method of site-specifically integrating an exogenous nucleic acid sequence into a target nucleic acid, the method comprises incorporating an integration recognition site into a cell by delivering: (i) a polynucleotide encoding a gene editor polypeptide (e.g., any of the gene editor polypeptides described herein), (ii) a first target specific atgRNA and a second target specific atgRNA. In such embodiments, 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 an at least first integration recognition site; the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is integrated into the genome of the cell at the target sequence.
[0153] In some embodiments, the method includes integrating the polynucleotide in the cellular genome by delivering a donor polynucleotide template, wherein the donor polynucleotide template is comprised of a second integration recognition site that is a cognate pair of the first integration recognition site, wherein the donor polynucleotide is integrated into the cellular genome at the first integration target recognition site by the gene editor polynucleotide..
[0154] In some embodiments, the method of site-specifically integrating an exogenous nucleic acid sequence into a genome of a cell comprises incorporating an integration target site into a cell by delivering: an expression vector of comprising a polynucleotide encoding a gene editor polypeptide (e.g., any of the gene editor polypeptides described herein), further comprising a promoter, one or more cell genome target specific atgRNA comprising an integration target recognition site, a donor polynucleotide comprising an integration target site, and optionally, a ngRNA. In some embodiments, the method of polynucleotide integration into a cell genome comprises incorporating an integration target site into a cell by delivering: an expression vector of comprising a polynucleotide encoding a gene editor polypeptide (e.g., any of the gene editor polypeptides described herein), further comprising a promoter, two cell genome target specific atgRNA where the two atgRNA collectively encode the entirety of the an integration recognition site, and a donor polynucleotide comprising an integration target site.
[0155] In some embodiments, the method of site-specifically integrating an exogenous nucleic acid sequence into a genome of a cell includes incorporating an integration recognition site into a cell by delivering: any of the polynucleotides described herein; at least a first genome target specific atgRNA comprising a first integration recognition site; optionally, a nicking gRNA; and integrating an exogenous nucleic acid sequence into the cellular genome by delivering into the cell: a donor polynucleotide template, wherein the donor polynucleotide template is comprised of the exogenous nucleic acid sequence positioned relative to a second integration recognition site that is a cognate pair of the first integration recognition site such that the exogenous nucleic acid sequence is integrated into the cellular genome at the first integration target recognition site when contacted with the integration enzyme and the first integration recognition site. In some embodiments, the polynucleotides and the at least first atgRNA are delivered concurrently with the donor polynucleotide template.
[0156] In some embodiments, the method of site-specifically integrating an exogenous nucleic acid sequence into a target nucleic acid includes: incorporating an integration recognition site into a cell by delivering: any of the expression vectors described herein; and integrating an exogenous nucleic acid sequence into the cellular genome by integrating a donor polynucleotide template, wherein the donor polynucleotide template is comprised of the exogenous nucleic acid sequence positioned relative to a second integration recognition site that is a cognate pair of the first integration recognition site such that the exogenous nucleic acid sequence is integrated into the cellular genome at the first integration target recognition site when contacted with the integration enzyme and the first integration recognition site. In some embodiments, the expression vector and the donor polynucleotide template are delivered to the cell concurrently.
6.12. Genes and Targets
[0157] 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. In certain embodiments, such a method comprises recombination or integration into a safe harbor site (SHS). A frequently used human SHS is the AAVS1 site on chromosome 19q, initially identified as a site for recurrent adeno-associated virus insertion. Another locus comprises the human homolog of the murine Rosa26 locus. Yet another SHS comprises the human Hl 1 locus on chromosome 22. In some cases, a complete gene may be prohibitively large and replacement of an entire gene impractical. In certain embodiments, a method of the invention comprises recombining corrective gene fragments into a defective locus.
[0158] 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. In certain embodiments, methods and compositions of the invention are adapted to target organoids, including patient derived organoids.
[0159] In certain embodiments, methods and compositions of the invention 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). In some embodiments, 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.
[0160] The following are non-limiting diseases that may be treated utilizing the methods and compositions of the present disclosure:
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)
• S YNGAP 1 -ID (S YNGAP 1 )
• 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’s disease)
Anti-Neutrophil Cytoplasmic Antibody (ANCA) Vasculitis
Systemic Lupus Erythematosus (SLE) / Lupus Nephritis (LN) Membranous Nephropathy (MN)
C3 glomerulonephritis (C3GN)
Blood disorders:
• Sickle Cell
• Hemophilia
• Factor VIII or
• Factor IX
• Ornithine transcarbamylase deficiency (OTCD)
• Homocystinuria (HCU)
• Phenylketonuria (PKU)
Cancer
• Prostate cancer
• Renal cell cancer
• Thyroid cancer
[0161] CFTR (cystic fibrosis transmembrane conductance regulator). The most common cystic fibrosis (CF) mutation F508del removes a single amino acid. In some embodiments, recombining human CFTR into an SHS of a cell that expresses CFTR F508del is a corrective treatment path. In some embodiments, 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.
[0162] Sickle cell disease (SCD) is caused by mutation of a specific amino acid — valine to glutamic acid at amino acid position 6. In some embodiments, 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. In some embodiments, 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. In some embodiments, validation is detection of persistent HBB mRNA and protein expression in transduced cells.
[0163] 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).
[0164] In some embodiments, recombination will be into safe harbor sites (SHS). A frequently used human SHS is the A4F5 site on chromosome 19q, initially identified as a site for recurrent adeno-associated virus insertion. In some embodiments, 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 Hl 1 locus on chromosome 22. Proposed target cells for recombination include stem cells for example induced pluripotent stem cells (iPSCs) and cells at various stages of differentiation. In some cases, a complete gene may be prohibitively large and replacement of an entire gene impractical. In such instances, rescuing mutants by recombining in corrected gene fragments with the methods and systems described herein is a corrective option.
[0165] In some embodiments, 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.
[0166] F8 (Factor VIII). A large proportion of severe hemophilia A patients harbor one of two types of chromosomal inversions in the FVIII gene. The recombinase technology and methods described herein are well suited to correcting such inversions (and other mutations) by recombining of the FVIII gene into a SHS.
[0167] In some embodiments, correcting factor VIII deficiency by recombining the FVIII gene into an SHS is a corrective path. In some embodiments, 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.
[0168] 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.
[0169] In some embodiments, 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.
[0170] 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.
[0171] In some embodiments, 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.
[0172] Phenylketonuria, also called PKU, is a rare inherited disorder that causes an amino acid called phenylalanine to build up in the body. PKU is caused by a change in the phenylalanine hydroxylase (PAH) gene. This gene helps create the enzyme needed to break down phenylalanine.
[0173] In some embodiments, 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.
[0174] Homocystinuria (HCU). Homocystinuria is elevation of the amino acid, homocysteine (protein building block coming from our diet) in the urine or blood. Common causes of HCU include: problems with the enzyme cystathionine beta synthase (CBS), which converts homocysteine to the amino acid cystathionine (which then becomes cysteine) and needs the vitamin B6 (pyridoxine); and roblems with converting homocysteine to the amino acid methionine.
[0175] In some embodiments, the methods and systems described herein are used to correct HCU by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the HCU or integrates a polynucleotide encoding a functional copy of a gene (e.g., CBS) able to reduce or prevent buildup of homocysteine in the urine. Proposed validation is detection of persistent CBS mRNA and protein expression in transduced cells. [0176] 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.
[0177] In some embodiments, 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. 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.
[0178] Anti-Neutrophil Cytoplasmic Antibody (ANCA) Vasculitis. 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.
[0179] In some embodiments, 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. 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.
[0180] Systemic Lupus Erythematosus (SLE) / Lupus Nephritis (LN). Lupus is an autoimmune — a disorder in which the body’s immune system attacks the body’s own cells and organs.
[0181] In some embodiments, 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. 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.
[0182] Membranous Nephropathy (MN). 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).
[0183] In some embodiments, 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. 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.
[0184] 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.
[0185] In some embodiments, 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. 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
[0186] 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. In typical embodiments, the polynucleotide encoding a gene editor polypeptide are delivered by a vector, wherein the vector is selected from the group consisting of recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, annelovirus, retrovirus, Doggybone™ DNA (dbDNA), minicircle, plasmid, miniDNA, exosome, fusosome, lipid nanoparticle, or nanoplasmid.
[0187] 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.
[0188] Methods of non-viral delivery of the polynucleotide encoding a gene editor polypeptide or the gene editor polypeptide described herein include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
6.13.1. Lipid Nanoparticle Delivery
[0189] In some embodiments, the gene editor polypeptide or polynucleotide encoding the gene editor polypeptide is packaged in a LNP and administered intravenously. In some embodiments, the gene editor polypeptide or polynucleotide encoding the gene editor polypeptide is packaged in a LNP and administered intrathecally. In some embodiments, the gene editor polypeptide or polynucleotide encoding the gene editor polypeptide is packaged in a LNP and administered by intracerebral ventricular injection. In some embodiments, the gene editor polypeptide or polynucleotide encoding the gene editor polypeptide is packaged in a LNP and administered by intraci sternal magna administration. In some embodiments, the gene editor polypeptide or polynucleotide encoding the gene editor polypeptide is packaged in a LNP and administered by intravitreal injection.
[0190] The preparation of lipidmucleic 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. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
[0191] In another embodiment, 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.
[0192] The charge of the LNP must be taken into consideration. As cationic lipids combined with negatively charged lipids to induce nonbilayer structures that facilitate intracellular delivery. Because charged LNPs are rapidly cleared from circulation following intravenous injection, ionizable cationic lipids with pKa values below 7 were developed (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011). Negatively charged polymers such as RNA may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge. However, at physiological pH values, the LNPs exhibit a low surface charge compatible with longer circulation times. Four species of ionizable cationic lipids have been focused upon, namely l,2-dilineoyl-3 -dimethylammonium -propane (DLinDAP), 1,2- dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), l,2-dilinoleyloxy-keto-N,N-dimethyl- 3 -aminopropane (DLinKDMA), and l,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DlinKC2-DMA). It has been shown that LNP siRNA systems containing these lipids exhibit remarkably different gene silencing properties in hepatocytes in vivo, with potencies varying according to the series DlinKC2-DMA>DLinKDMA>DLinDMA»DLinDAP employing a Factor VII gene silencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011). A dosage of 1 pg/ml of LNP in or associated with the LNP may be contemplated, especially for a formulation containing DlinKC2-DMA.
[0193] In some embodiments, the LNP composition comprises one or more one or more ionizable lipids. As used herein, the term “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. In some embodiments, the one or more ionizable lipids are selected from the group consisting of 3-(didodecylamino)- N1 ,N1 ,4-tridodecyl- 1 -piperazineethanamine (KL 10), N 1 -[2-(didodecylamino)ethyl]-N 1 ,N4,N4- tridodecyl-l,4-piperazinediethanami- ne (KL22), 14, 25-ditridecyl-15, 18,21, 24-tetraaza- octatriacontane (KL25), l,2-dilinoleyloxy-N,N-dimethylaminopropane (Dlin-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (Dlin-K-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate (Dlin-MC3-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-di oxolane (Dlin-KC2-DMA), 1, 2-di oleyloxy -N,N- dimethylaminopropane (DODMA), 2-({8-[(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z,12Z)-octad- eca-9,12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA), (2R)-2-({8-[(3)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z) — octadeca-9, 12-dien-l- yloxy]propan-l -amine (Octyl-CLinDMA (2R)), and (2S)-2-({8-[(3)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z, 12Z) — octadeca-9, 12-dien-l-y loxy]propan-l -amine (Octyl-CLinDMA (2S)). In one embodiment, the ionizable lipid may be selected from, but not limited to, an ionizable lipid described in International Publication Nos. WO2013086354 and WO2013116126.
[0194] In some embodiments, 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)-Nl,Nl,4-tridodecyl-l-piperazineethanamine (KL10), Nl-[2- (didodecylamino)ethyl]-Nl,N4,N4-tridodecyl-l,4-piperazinediethanami- ne (KL22), 14,25- ditridecyl- 15,18,21 ,24-tetraaza-octatriacontane (KL25), 1 ,2-dilinoleyloxy-N,N- dimethylaminopropane (Dlin-DMA), 2, 2-dilinoleyl-4-dimethylaminomethyl-[l,3]-di oxolane (Dlin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (Dlin-MC3- DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (Dlin-KC2-DMA), 2-({8- [(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- -octadeca-9, 12-dien-l- yloxy]propan-l -amine (Octyl-CLinDMA), (2R)-2-({8-[(3.beta.)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z- ,12Z)-octadeca-9,12-dien-l-yl oxy]propan-l -amine (Octyl-CLinDMA (2R)), (2S)-2-({8-[(3Pcholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z- ,12Z)-octadeca-9,12-dien-l-yl oxy]propan-l -amine (Octyl-CLinDMA (2S)).N,N-dioleyl-N,N- dimethylammonium chloride (“DODAC”); N-(2,3-dioleyloxy)propyl-N,N — N- triethylammonium chloride (“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”); 1,2- Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP. Cl”); 3-.beta.-(N — (N’,N’- dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”), N-(l-(2,3-dioleyloxy)propyl)-N-2- (sperminecarboxamido)ethyl)-N,N-dimethyl- -ammonium trifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine (“DOGS”), l,2-dioleoyl-3-dimethylammonium propane (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), and N-(l,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DMRIE”). Additionally, a number of commercial preparations of cationic and/or ionizable lipids can be used, such as, e g., LIPOFECTIN.RTM. (including DOTMA and DOPE, available from GIBCO/BRL), and 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. [0195] In some embodiments, the LNP composition comprises one or more amino lipids. The terms “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). In principle, there are no specific limitations concerning the 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. In some embodiments, 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. 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-012, Dlin-MC2-DMA (also known as MC2), and Dlin-MC3-DMA (also known as MC3).
[0196] 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, lysylphosphatidyl glycerol, and other anionic modifying groups joined to neutral lipids.
[0197] Neutral lipids (including both uncharged and zwitterionic 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. In some embodiments, 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. In some embodiments, the neutral lipids used in the disclosure are DOPE, DSPC, DPPC, POPC, or any related phosphatidylcholine. In some embodiments, the neutral lipid may be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol. [0198] In some embodiments, 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.
[0199] 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. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.
[0200] 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.
[0201] 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.
[0202] Particular amphipathic lipids can facilitate fusion to a membrane. For example, 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.
[0203] Non-natural amphipathic lipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, 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). Under appropriate reaction conditions, 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).
[0204] 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.
[0205] In some embodiments, the LNP composition comprises one or more phospholipids. In some embodiments, 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), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-di-O- octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OchemsPC), l-hexadecyl-sn-glycero-3- phosphocholine (Cl 6 Lyso PC), l,2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn- glycero-3 -phosphoethanolamine (ME 16:0 PE), l,2-distearoyl-sn-glycero-3- phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1,2-dilinolenoyl-sn- glycero-3 -phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3-phosphoethanolaminel,2- didocosahexaenoyl — sn-glycero-3 -phosphoethanolamine, l,2-dioleoyl-sn-glycero-3 -phosphorac^ 1 -glycerol) sodium salt (DOPG), sphingomyelin, and any mixtures thereof.
[0206] Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and .beta. -acyloxyacids, may also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.
[0207] In some embodiments, the LNP composition comprises one or more helper lipids. The term “helper lipid” as used herein 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). In principle, 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. Generally, 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. Particularly helper lipids suitable for use in the present disclosure include, but are not limited to, saturated phosphatidylcholine (PC) such as distearoyl-PC (DSPC) and dipalymitoyl-PC (DPPC), di oleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn- glycero-3 -phosphocholine (DLPC), cholesterol, 5-heptadecylresorcinol, and cholesterol hemi succinate. In some embodiments, the helper lipid of the LNP composition includes cholesterol.
[0208] In some embodiments, the LNP composition comprises one or more structural lipids. As used herein, the term “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. In some embodiments, the structural lipid is a sterol. As defined herein, "sterols" are a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol.
[0209] The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. In some embodiments, the LNP composition disclosed herein comprise one or more polyethylene glycol (PEG) lipid. The term “PEG-lipid” refers to polyethylene glycol (PEG)-modified lipids. Such lipids are also referred to as PEGylated lipids. Non-limiting examples of 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 For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiment, the PEG-lipid is PEG2k-DMG. In some embodiments, the one or more PEG lipids of the LNP composition comprises PEG-DMPE. In some embodiments, the one or more PEG lipids of the LNP composition comprises PEG-DMG.
[0210] In some embodiments, the ratio between the lipid components and the nucleic acid molecules of the LNP composition, e.g., the weight ratio, 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.
[0211] In certain embodiments, it is desirable to target a nanoparticle, e.g., a lipid nanoparticle, using a targeting moiety that is specific to a cell type and/or tissue type. In some embodiments, a nanoparticle may be targeted to a particular cell, tissue, and/or organ using a targeting moiety. In particular embodiments, a nanoparticle comprises a targeting moiety. Exemplary non-limiting targeting moieties include ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and antibodies (e.g., full-length antibodies, antibody fragments (e.g., Fv fragments, single chain Fv (scFv) fragments, Fab’ fragments, or F(ab’)2 fragments), single domain antibodies, cam elid antibodies and fragments thereof, human antibodies and fragments thereof, monoclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies)). In some embodiments, 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.
[0212] In some embodiments, a lipid nanoparticle (e.g., a liposome) 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 etBiophysica Acta 1149: ISO- 184, 1993; Klibanov et al., Journal of Liposome Research 2: 321-334, 1992; U.S. Pat. No. 5,013,556; Zalipsky, Bioconjugate Chemistry 4: 296-299, 1993; Zalipsky, FEBS Letters 353: 71- 74, 1994; Zalipsky, in Stealth Liposomes Chapter 9 (Lasic and Martin, Eds) CRC Press, Boca Raton Fla., 1995). In one approach, a targeting moiety for targeting the lipid nanoparticle is linked to the polar head group of lipids forming the nanoparticle. In another approach, 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).
[0213] Standard methods for coupling the targeting moiety or moi eties may be used. For example, 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.
[0214] In some embodiments, 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). In particular embodiments, the targeting moiety targets the lipid nanoparticle to a hepatocyte.
[0215] The lipid nanoparticles described herein may be lipidoid-based. The synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of polynucleotides (see Mahon et al., Bioconjug Chem. 2010 21 : 1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat. Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011 108: 12996-3001).
[0216] The characteristics of optimized lipidoid formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse through the extracellular matrix into the blood stream. While a particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see e.g., Akinc et al., Mol Ther. 2009 17:872-879), use of lipidoid oligonucleotides to deliver the formulation to other cells types including, but not limited to, endothelial cells, myeloid cells, and muscle cells may not be similarly size-limited.
[0217] In one aspect, effective delivery to myeloid cells, such as monocytes, lipidoid formulations may have a similar component molar ratio. Different ratios of lipidoids and other components including, but not limited to, a neutral lipid (e.g., diacylphosphatidylcholine), cholesterol, a PEGylated lipid (e.g., PEG-DMPE), and a fatty acid (e.g., an omega-3 fatty acid) may be used to optimize the formulation of the mRNA or system for delivery to different cell types including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc. 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. The use of lipidoid formulations for the localized delivery of nucleic acids to cells (such as, but not limited to, adipose cells and muscle cells) via either subcutaneous or intramuscular delivery, 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.
[0218] According to the present disclosure, 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. After formation of the particle, 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.
[0219] 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). As an example, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids may result in significant effects on in vivo efficacy. Formulations with the different lipidoids, including, but not limited to penta[3-(l- laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401 :61 (2010)), C12-200 (including derivatives and variants), MD1, Dlin-DMA, Dlin-K-DMA, Dlin-KC2-DMA and Dlin-MC3-DMA can be tested for in vivo activity. The lipidoid referred to herein as “98N12-5” is disclosed by Akinc et al., Mol Ther. 2009 17:872-879). The lipidoid referred to herein as “C12-200” is disclosed by Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869 and Liu and Huang, Molecular Therapy. 2010 669- 670. [0220] 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.
[0221] In some embodiments, 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. This process and the apparatus for carrying out this process are known in the art. More information in this regard can be found in, for example, U.S. Patent Publication No. 20040142025. The action of continuously introducing lipid and buffer solutions into a mixing environment, such as in a mixing chamber, causes a continuous dilution of the lipid solution with the buffer solution, thereby producing a lipid vesicle substantially instantaneously upon mixing. By mixing the aqueous solution comprising a nucleic acid molecule with the organic lipid solution, the organic lipid solution undergoes a continuous stepwise dilution in the presence of the buffer solution (e.g., aqueous solution) to produce a nucleic acid-lipid particle.
[0222] In some embodiments, 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. In some embodiments, the collection vessel includes one or more elements configured to stir the contents of the collection vessel to facilitate dilution. In some embodiments, the amount of dilution buffer present in the collection vessel is substantially equal to the volume of lipid vesicle solution introduced thereto.
[0223] In some embodiments, 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. In these embodiments, 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. These processes and the apparatuses for carrying out direct dilution and in-line dilution processes are known in the art. More information in this regard can be found in, for example, U.S. Patent Publication No.
20070042031.
6.13.2. Viral Vector Delivery
[0224] In certain aspects 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 used herein, 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. Generally, a vector is capable of replication when associated with the proper control elements. In general, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, doublestranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally- derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)). 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. Moreover, certain 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.
[0225] 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. Within a recombinant expression vector, “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). With regards to recombination and cloning methods, mention is made of U.S. patent application Ser. No. 10/815,730, published Sep. 2, 2004 as US 2004-0171156 Al, the contents of which are herein incorporated by reference in their entirety.
[0226] Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme, for instance a Type V protein such as C2cl or C2c3, and/or any of the present RNAs, for instance a guide RNA, 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. Effector proteins and one or more guide RNAs can be packaged into one or more vectors, e.g., plasmid or viral vectors. In some embodiments, 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. One skilled in the art understands that 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.
[0227] 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. 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. Additionally, 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. In addition, 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. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON’S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which is incorporated by reference herein.
[0228] In an embodiment herein 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 adenoviral vector. In an embodiment herein, the dose preferably is at least about 1 x 106 particles (for example, about 1 x 106- 1 x 1011 particles), more preferably at least about 1 x 107 particles, more preferably at least about 1 x 108 particles (e.g., about 1 x 108-l x 1011 particles or about 1 x 109-l x 1012 particles), and most preferably at least about 1 x IO10 particles (e.g., about 1 x 109- 1 x IO10 particles or about 1 x 109-l x 1012 particles), or even at least about 1 x IO10 particles (e.g., about 1 x 1010-l x 1012 particles) of the adenoviral vector. Alternatively, the dose comprises no more than about 1 x 1014 particles, preferably no more than about 1 x 1013 particles, even more preferably no more than about 1 x 1012 particles, even more preferably no more than about 1 x 1011 particles, and most preferably no more than about 1 x IO10 particles (e.g., no more than about 1 x 109 particles). Thus, the dose may contain a single dose of adenoviral vector with, for example, about 1 x 106 particle units (pu), about 2 x 106 pu, about 4 x 106 pu, about 1 x 107 pu, about 2 x 107 pu, about 4 x 107 pu, about 1 x 108 pu, about 2 x 108 pu, about 4 x 108 pu, about 1 x 109 pu, about 2 x 109 pu, about 4 x 109 pu, about 1 x IO10 pu, about 2 x IO10 pu, about 4 x IO10 pu, about 1 x 1011 pu, about 2 x 1011 pu, about 4 x 1011 pu, about 1 x 1012 pu, about 2 x 1012 pu, or about 4 x 1012 pu of adenoviral vector. See, for example, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel, et. al., granted on Jun. 4, 2013; incorporated by reference herein, and the dosages at col 29, lines 36-58 thereof. In an embodiment herein, the adenovirus is delivered via multiple doses.
[0229] In an embodiment herein, the delivery is via an AAV. A therapeutically effective dosage for in vivo delivery of the AAV to a human is believed to be in the range of from about 20 to about 50 ml of saline solution containing from about 1 x 1010 to about 1 x 1010 functional AAV/ml solution. The dosage may be adjusted to balance the therapeutic benefit against any side effects. In an embodiment herein, the AAV dose is generally in the range of concentrations of from about 1 x 105 to 1 x 1050 genomes AAV, from about 1 x 108 to 1 x IO20 genomes AAV, from about 1 x 1010 to about 1 x 1016 genomes, or about 1 x 1011 to about 1 x 1016 genomes AAV. A human dosage may be about 1 x 1013 genomes AAV. Such concentrations may be delivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of a carrier solution. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. See, for example, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar. 26, 2013, at col. 27, lines 45-60.
[0230] The promoter used to drive nucleic acid-targeting effector protein coding nucleic acid molecule expression can include: AAV ITR can serve as a promoter: this is advantageous for eliminating the need for an additional promoter element (which can take up space in the vector). The additional space freed up can be used to drive the expression of additional elements (gRNA, etc.). Also, ITR activity is relatively weaker, so can be used to reduce potential toxicity due to over expression of nucleic acid-targeting effector protein. For ubiquitous expression, can use promoters: CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc. For brain or other CNS expression, can use promoters: SynapsinI for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. For liver expression, can use Albumin promoter. For lung expression, can use SP-B. For endothelial cells, can use ICAM. For hematopoietic cells can use IFNbeta or CD45. For Osteoblasts can use OG-2.
[0231] The promoter used to drive guide RNA can include: Pol III promoters such as U6 or Hl Use of Pol II promoter and intronic cassettes to express guide RNA Adeno Associated Virus (AAV)
[0232] Nucleic acid-targeting effector protein and one or more guide RNA can be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus), U.S. Pat. No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No. 5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus. For examples, for AAV, the route of administration, formulation and dose can be as in U.S. Pat. No. 8,454,972 and as in clinical trials involving AAV. For Adenovirus, the route of administration, formulation and dose can be as in U.S. Pat. No. 8,404,658 and as in clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation and dose can be as in U.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids. Doses may be based on or extrapolated to an average 70 kg individual (e.g., a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed. The viral vectors can be injected into the tissue of interest. For celltype specific genome modification, the expression of nucleic acid-targeting effector can be driven by a cell-type specific promoter. For example, liver-specific expression might use the Albumin promoter and neuron-specific expression (e.g., for targeting CNS disorders) might use the Synapsin I promoter.
[0233] In terms of in vivo delivery, AAV is advantageous over other viral vectors for a couple of reasons: Low toxicity (this may be due to the purification method not requiring ultra centrifugation of cell particles that can activate the immune response) and Low probability of causing insertional mutagenesis because it doesn’t integrate into the host genome.
[0234] AAV has a packaging limit of 4.5 or 4.75 Kb. This means that nucleic acid-targeting effector protein (such as a Type V protein such as C2cl or C2c3) as well as a promoter and transcription terminator have to be all fit into the same viral vector. Therefore embodiments of the invention include utilizing homologs of nucleic acid-targeting effector protein (such as a Type V protein such as C2cl or C2c3) that are shorter.
[0235] As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof. One can select the AAV of the AAV with regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue. AAV8 is useful for delivery to the liver. The herein promoters and vectors are preferred individually.
[0236] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and psi2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.
[0237] Millington-Ward et al. (Molecular Therapy, vol. 19 no. 4, 642-649 April 2011) describes adeno-associated virus (AAV) vectors to deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site. An injection of either 6.0 x 108 vp or 1.8 x IO10 vp AAV were subretinally injected into the eyes by Millington-Ward et al. The AAV vectors of Millington-Ward et al. may be applied to the system of the present invention, contemplating a dose of about 2 x 1011 to about 6 x 1011 vp administered to a human.
[0238] Dalkara et al. (Sci Transl Med 5, 189ra76 (2013)) also relates to in vivo directed evolution to fashion an AAV vector that delivers wild-type versions of defective genes throughout the retina after noninjurious injection into the eyes’ vitreous humor. Dalkara describes a 7 mer peptide display library and an AAV library constructed by DNA shuffling of cap genes from AAV1, 2, 4, 5, 6, 8, and 9. The rcAAV libraries and rAAV vectors expressing GFP under a CAG or Rho promoter were packaged and deoxyribonuclease-resistant genomic titers were obtained through quantitative PCR. The libraries were pooled, and two rounds of evolution were performed, each consisting of initial library diversification followed by three in vivo selection steps. In each such step, P30 rho-GFP mice were intravitreally injected with 2 ml of iodixanol-purified, phosphate- buffered saline (PBS)-dialyzed library with a genomic titer of about 1. times.10. sup.12 vg/ml. The AAV vectors of Dalkara et al. may be applied to the nucleic acid-targeting system of the present invention, contemplating a dose of about 1 x 1015 to about 1 x 1016 vg/ml administered to a human.
[0239] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SW), human immune deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66 :2731-2739 (1992) ; Johann et al., J. Virol. 66 : 1635-1640 (1992) ; Sommnerfelt et al., Virol. 176 :58-59 (1990) ; Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81 :6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).
[0240] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and yr2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.
[0241] In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, 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.
[0242] Another useful method to deliver proteins, enzymes, and guides comprises transfection of messenger RNA (mRNA). 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, BRI 12016030852A2, and EP3362461A1. Expression of CRISPR systems in particular is described by W02020014577. Each of these publications are incorporated herein by reference in their entireties. Additional disclosure hereby incorporated by reference can be found in Kowalski et al., “Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery,” Mol Therap., 2019; 27(4): 710-728.
[0243] In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHL231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHOIR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr-/-, COR-L23, COR-L23/CPR, COR-L23/5010, COR- L23/R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB- 435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCLH69/CPR, NCI- H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VcaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, 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.
[0244] In some embodiments, one or more vectors described herein are used to produce a nonhuman transgenic animal or transgenic plant. In some embodiments, the transgenic animal is a mammal, such as a mouse, rat, or rabbit. In certain embodiments, the organism or subject is a plant. In certain embodiments, the organism or subject or plant is algae. Methods for producing transgenic plants and animals are known in the art, and generally begin with a method of cell transfection, such as described herein.
[0245] In one aspect, 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. In some embodiments, 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).
[0246] In plants, pathogens are often host-specific. For example, Fusariumn oxysporum f. sp. Lycopersici causes tomato wilt but attacks only tomato, and F. oxysporum f. dianthii Puccinia graminis f. sp. Tritici attacks only wheat. 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. In plants there can be non-host resistance, e.g., the host and pathogen are incompatible. There can also be Horizontal Resistance, e.g., partial resistance against all races of a pathogen, typically controlled by many genes and Vertical Resistance, e.g., complete resistance to some races of a pathogen but not to other races, typically controlled by a few genes. In a Gene-for-Gene level, plants 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. Using the present invention, plant breeders are provided with a new tool to induce mutations. Accordingly, one skilled in the art can analyze the genome of sources of resistance genes, and in Varieties having desired characteristics or traits employ the present invention to induce the rise of resistance genes, with more precision than previous mutagenic agents and hence accelerate and improve plant breeding programs. [0247] Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Examples of 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. The transcribed or translated products may be known or unknown and may be at a normal or abnormal level.
7. ADDITIONAL EMBODIMENTS
[0248] Embodiment 1. A polypeptide (e.g., gene editor polypeptide), comprising: a DNA binding nuclease, optionally comprising a nickase activity; a reverse transcriptase; an integration enzyme; and wherein at least any two of elements (i), (ii), or (iii) are linked via at least one C-terminal linker.
[0249] Embodiment 2. The polypeptide (e.g., gene editor polypeptide) of Embodiment 1, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C- terminus of the reverse transcriptase is linked to the integration enzyme.
[0250] Embodiment 3. The polypeptide (e.g., gene editor polypeptide) of Embodiment 1, wherein the C-terminus of the integration enzyme is linked to the DNA binding nuclease, wherein the C- terminus of the DNA binding nuclease is linked to a reverse transcriptase.
[0251] Embodiment 4. The polypeptide (e.g., gene editor polypeptide) of any one of Embodiments 1-3, wherein at least one linker is a peptide linker.
[0252] Embodiment 5. The polypeptide (e.g., gene editor polypeptide) of Embodiment 4, wherein at least one linker is non-cleavable.
[0253] Embodiment 6. The polypeptide (e.g., gene editor polypeptide) of Embodiment 4, wherein at least one linker is cleavable. [0254] Embodiment 7. The polypeptide (e.g., gene editor polypeptide) of Embodiment 5, wherein the at least one linker is selected from a group consisting of LI, L2, L3, L4, L5, L6, L7, L8, L9, LIO, L11, L12, and L13.
[0255] Embodiment 8. The polypeptide (e.g., gene editor polypeptide) of Embodiment 6, wherein the at least one cleavable linker is selected from a group consisting of P2A, E2A, F2A, and T2A.
[0256] Embodiment 9. The polypeptide (e.g., gene editor polypeptide) of any one of Embodiments 1-8, wherein at least one linker is non-cleavable and at least one linker is cleavable.
[0257] Embodiment 10. The polypeptide (e.g., gene editor polypeptide) of Embodiment 7, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L7, L10, or L12 links the DNA binding nuclease to the reverse transcriptase.
[0258] Embodiment 11. The polypeptide (e.g., gene editor polypeptide) of Embodiment 7, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L8, L9, L10, L12, or L13 links the reverse transcriptase to the integration enzyme.
[0259] Embodiment 12. The polypeptide (e.g., gene editor polypeptide) of Embodiment 7, wherein the C-terminus of the integration enzyme is linked to the DNA binding nuclease by L13, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by L13.
[0260] Embodiment 13. The polypeptide (e.g., gene editor polypeptide) of Embodiment 7, wherein the polypeptide is not comprised of a N-most N-terminal reverse transcriptase.
[0261] Embodiment 14. The polypeptide (e.g., gene editor polypeptide) of Embodiment 9, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by LI 3, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme by a cleavable linker.
[0262] Embodiment 15. The polypeptide (e.g., gene editor polypeptide) of any one of Embodiments 1-14, further comprised of at least one nuclear localization signal.
[0263] Embodiment 16. A polynucleotide encoding a polypeptide from any one of Embodiments 1-15. [0264] Embodiment 17. An expression vector comprising a polynucleotide of Embodiment 16.
[0265] Embodiment 18. The expression vector of Embodiment 17, further comprising any one or more of a promoter sequence, atgRNA, nicking gRNA (ngRNA), donor polynucleotide template, or donor polynucleotide template complement.
[0266] Embodiment 19. The expression vector of Embodiment 18, wherein the expression vector is recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, annelovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, or lipid nanoparticle.
[0267] Embodiment 20. A method of polynucleotide integration into a cellular genome, the method comprising: incorporating an integration target recognition site into a cell by delivering: the polynucleotide of Embodiment 16; one or more cell genome target specific atgRNA comprising an integration target recognition site; optionally, a nicking gRNA; and integrating the polynucleotide into the cellular genome by delivering: a donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target recognition site, wherein the donor polynucleotide is integrated into the cellular genome at a genomic integration target recognition site by a polypeptide encoded by the polynucleotide of Embodiment 16.
[0268] Embodiment 21. A method of polynucleotide integration into a cellular genome, the method comprising: (a) incorporating an integration target recognition site into a cell by delivering: the expression vector of Embodiment 18, wherein the expression vector is comprised of a promoter, one or more cell genome target specific atgRNA comprising an integration target recognition site, a donor polynucleotide comprising an integration target site, and optionally, a ngRNA; and (b) integrating the polynucleotide into the cellular genome by integrating the donor polynucleotide into the cellular genome at a genomic integration target recognition site by a polypeptide encoded in the expression vector.
8. EXAMPLES
8.1. Example 1: PASTE With Selected Gene Editor Compositions
[0269] Linkers that connect Cas9-nickase, reverse transcriptase, and Bxbl integrase were screened. Linker position 1 (Pl) and Linker position 2 (P2) are illustrated in FIG. 1 and FIG. 2. A panel of selected non-cleavable linkers are shown in FIG. 3, where linkers LI -LI 3 demonstrate various length, structure, and flexibility attributes. [0270] For these experiments atgRNAs were use that targeted the ACTB locus (see Table 9). The atgRNAs included an integration recognition site (e.g., AttB) that is a cognate pair with an integration recognition site located on the donor template polynucleotide (e.g., AttP). Upon transfection of the atgRNA, nicking gRNA, polynucleotide encoding the gene editor polypeptide, and the donor template polynucleotide, the gene editor polypeptides integrated the integration recognition site from the atgRNAs into the ACTB locus, and then integrated the donor template polynucleotide into the ACTB locus using the cognate pair of integration recognition sites.
[0271] A PASTE readout in HEK293 cells was used to identify functionally active gene editor compositions, wherein select non-cleavable linkers L1-L13 (see FIG. 3) and a cleavable P2A linker were screened. Droplet digital PCR (ddPCR) in a 96-well plate format was applied to verify gene insertion into the ACTB locus. A total of 135 linker combinations were screened and compared to Cas9(nickase)-mXTEN-RT-P2A-Bxbl and Cas9(nickase)-mXTEN-RT-mXTEN- Bxbl. Using Lipofectamine 3000 transfection reagent, lOOng total DNA at a plasmid ratio of 1 : 1 :4 of Cas9(nickase)-Pl-RT-P2-Bxbl : nicking guide : atgRNA was delivered to 30,000 HEK293 (FIG. 4). FIGs. 5, 6, and 7 show the percent integration (as verified by ddPCR) across the screened gene editor compositions.
[0272] FIG. 5 shows percent integration of a Cas9-Pl-RT-P2-BXB1 configuration tested with various combinations of linkers from FIG. 3 (see x-axis FIG. 5). Columns with asterisks represent the gene editor polypeptides that produced the highest percent insertion of donor polynucleotide template in the ACTB locus. A gene editor polypeptide having a Cas9-L6 (i.e., XTEN)-RT-L5(i.e., (GGS)6)-BXB 1 produced the highest percent insertion of donor polynucleotide template.
[0273] FIG. 6 shows percent integration of a Cas9-Pl-RT-P2-BXB1 configuration tested with various combination of linkers from FIG. 3 (see x-axis FIG. 6). Columns with asterisks represent the gene editor polypeptides that produced the highest percent insertion of donor polynucleotide template in the ACTB locus. In general, gene editor polypeptides having an L8 linker (i.e., PAS12; SEQ ID NO: 553); an L9 linker (i.e., A(EAAK)4ALEA(EAAAK)4A; SEQ ID NO: 554), and/or an L10 linker (i.e., Camel; SEQ ID NO: 555) produced the highest percent insertion of donor polynucleotide template in the ACTB locus.
[0274] FIG. 7 shows percent integration of a Cas9-Pl-RT-P2-BXB1 configuration tested with various combination of linkers from FIG. 3 (see x-axis FIG. 7). Columns with asterisks include gene editor polypeptides that produced the highest percent insertion of donor polynucleotide template in the ACTB locus. In general, gene editor polypeptides that had the highest percent integration were those where the P2 was an L12 linker (i.e., RFR; SEQ ID NO: 557). For example, the following gene editor polypeptides having an L12 at P2 produced the highest percent integration: (P1/P2) L7 (i.e., PAS8; SEQ ID NO: 552) / L12; L8 (i.e., PAS12; SEQ ID NO: 553) / L12; and LIO (i.e., Camel; SEQ ID NO: 555) / L12. FIG. 7 also showed that gene editor polypeptides that had the highest percent integration were those that included P1/P2 combinations of: L4 (i.e., (EAAAK)3; SEQ ID NO: 17) /L13 (i.e., mXTEN; SEQ ID NO: 558); LIO (i.e., Camel; SEQ ID NO: 555) / L6 (i.e., XTEN; SEQ ID NO: 18); and L12 (i.e., RFR; SEQ ID NO: 557) / L13).
8.2. Example 2: PASTE With Configuration Selected Gene Editor Compositions
[0275] In this example, various configurations of Cas9-nickase protein, reverse transcriptase protein, and Bxbl integrase protein were screened (see FIG. 8). Similar to Example 1, atgRNA targeting the ACTB locus and a donor polynucleotide template comprising a sequence encoding GFP were used to test efficiency of the gene editor polypeptides. Also similar to Example 1, a PASTE readout in HEK293 cells was used to identify functionally active gene editor compositions with variable protein domain configuration. Droplet digital PCR (ddPCR) in a 96-well plate format was applied to verify gene insertion into the ACTB locus. Using Lipofectamine 3000 transfection reagent, lOOng total DNA at a plasmid ratio of 1 : 1:4 of Cas9(nickase)-RT-BxBl (i.e., the configurations described in FIG. 8) : nicking guide : atgRNA was delivered to 30,000 HEK293 (see, e.g., FIG. 4).
[0276] A total of 3 configurations were screened wherein either the first linker position (Pl) or the second linker position (P2) were non-cleavable mXTEN (i.e., mXTEN/mXTEN) or cleavable P2A (see FIG. 8). FIGs. 9A-9B show the percent integration (as verified by ddPCR) across mXTEN/P2A or P2A/mXTEN linker gene editor compositions. In particular, FIG. 9A shows that a configuration of Cas9-mXTEN-RT-P2A-BXBl produced the highest percent integration for the P2A/mXTEN configurations. FIG. 9B shows that a configuration of BXB1- mXTEN-Cas9-mXTEN-RT produced the highest percent integration for the mXTEN/mXTEN configurations.
[0277] Overall, this data establishes optimal gene editor polypeptide compositions that depend both on configuration and linker selection. 9. EQUIVALENTS AND INCORPORATION BY REFERENCE
[0278] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GenelD entries), patent application, or patent, was specifically and individually indicated incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and every individual publication, database entry (e.g., Genbank sequences or GenelD entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. §1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.
[0279] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it is understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS:
1. A gene editor polypeptide, comprising:
(i) a DNA binding nuclease, optionally comprising a nickase activity;
(ii) a reverse transcriptase;
(iii) an integration enzyme; and wherein at least any two of elements (i), (ii), or (iii) are linked via at least a first C- terminal linker.
2. The gene editor polypeptide of claim 1, further comprising a second linker.
3. The gene editor polypeptide of claim 2, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme.
4. The gene editor polypeptide of claim 2, wherein the C-terminus of the integration enzyme is linked to the DNA binding nuclease, wherein the C-terminus of the DNA binding nuclease is linked to a reverse transcriptase.
5. The gene editor polypeptide of any one of claims 1-4, wherein the at least first linker, the second linker, or both, is selected from a flexible linker, a semi-flexible linker, a rigid linker, a flexible-rigid linker.
6. The gene editor polypeptide of claim 5, wherein the flexible linker is selected from: LI, L2, L5, L6, and L13.
7. The gene editor polypeptide of claim 5, wherein the semi-flexible linker is selected from: L7 and L8.
8. The gene editor polypeptide of claim 5, wherein the rigid linker is selected from: L3, L4, and L9.
9. The gene editor polypeptide of claim 5, wherein the flexible-rigid linker is selected from Li l and L12. The gene editor polypeptide of any one of claims 1-5, wherein the length of the at least first linker, the length of the second linker, or both, is selected from short, medium, and long. The gene editor polypeptide of claim 10, wherein the short linker is selected from: LI, L3, L7, and L8. The gene editor polypeptide of claim 10, wherein the medium linker is selected from: L2, L4, L5, L6, LIO, Lil, L12, and L13. The gene editor polypeptide of claim 10, wherein the long linker is L9. The gene editor polypeptide of any one of claims 1-5, wherein the at least first linker, the second linker, or both, is a non-cleavable linker. The gene editor polypeptide of claim 14, wherein the non-cleavable linker is selected from: LI , L2, L3, L4, L5, L6, L7, L8, L9, LI O, LI 1, LI 2, and LI 3. The gene editor polypeptide of any one of claims 1-15, wherein the at least first linker, the second linker, or both, is a cleavable linker. The gene editor polypeptide of claim 16, wherein the cleavable linker is selected from: a self-cleavable linker, an enzyme cleavable linker, and a chemically-induced cleavable linker. The gene editor polypeptide of claim 17, wherein the cleavable linker is a self-cleavable linker. The gene editor polypeptide of claim 18, wherein the self-cleavable linker is selected from P2A, E2A, F2A, and T2A. The gene editor polypeptide of any one of claims 1-19, wherein: the at least first linker is a non-cleavable linker and the second linker is a cleavable linker; the at least first linker is a cleavable linker and the second linker is a non-cleavable linker; the at least first linker is a cleavable linker and the second linker is a cleavable linker; or the at least first linker is a non-cleavable linker and the second linker is a non- cleavable linker. The gene editor polypeptide of claim 1, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L7, LIO, or L12 links the DNA binding nuclease to the reverse transcriptase. The gene editor polypeptide of claim 1, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme, wherein any one of L8, L9, LIO, L12, or LI 3 links the reverse transcriptase to the integration enzyme. The gene editor polypeptide of claim 1, wherein the C-terminus of the integration enzyme is linked to the DNA binding nuclease by L13, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by L13. The gene editor polypeptide of claim 1, wherein the gene editor polypeptide is not comprised of a N-most N-terminal reverse transcriptase. The gene editor polypeptide of claim 1, wherein the C-terminus of the DNA binding nuclease is linked to the reverse transcriptase by L13, wherein the C-terminus of the reverse transcriptase is linked to the integration enzyme by a cleavable linker. The gene editor polypeptide of any one of claims 1-25, further comprising at least one nuclear localization signal. A gene editor polypeptide comprising: from N-terminus to C-terminus: a DNA binding nuclease; a P2A linker, a reverse transcriptase; a mXTEN linker, and an integration enzyme. A gene editor polypeptide comprising: from terminus to C-terminus: an integration enzyme, a mXTEN linker, a DNA binding nuclease; a mXTEN linker, and a reverse transcriptase. A polynucleotide encoding a gene editor polypeptide from any one of claims 1-28. An expression vector comprising the polynucleotide of claim 29. The expression vector of claim 30, wherein the polynucleotide is operably linked to an expression control element. The expression vector of claim 31, wherein the expression control element is a promoter. The expression vector of any one of claims 30-32, further comprising one or more sequences encoding an atgRNA, nicking gRNA (ngRNA), donor polynucleotide template, or donor polynucleotide template complement. The expression vector of any one of claims 30-33, wherein the expression vector is recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, annelovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, or lipid nanoparticle. A system capable of site-specifically integrating an exogenous nucleic acid sequence into a genome of a cell, the system comprising: the gene editor polypeptide of any one of claims 1-28, the polynucleotide of claim 29, or the expression vector of any one of claims 30-34. A method of site-specifically integrating an exogenous nucleic acid sequence into a genome of a cell, the method comprising:
(a) incorporating an integration recognition site into a cell by delivering:
(i) the polynucleotide of claim 29;
(ii) at least a first atgRNA comprising a first integration recognition site;
(iii) optionally, a nicking gRNA; and (b) integrating an exogenous nucleic acid sequence into the cellular genome by delivering into the cell concurrently with step (a):
(i) a donor polynucleotide template, wherein the donor polynucleotide template is comprised of the exogenous nucleic acid sequence positioned relative to a second integration recognition site that is a cognate pair of the first integration recognition site such that the exogenous nucleic acid sequence is integrated into the cellular genome at the first integration target recognition site when contacted with the integration enzyme and the first integration recognition site. A method of site-specifically integrating an exogenous nucleic acid sequence into a target nucleic acid, the method comprising:
(a) incorporating an integration recognition site into a cell by delivering:
(i) the expression vector of any one of claims 30-34; and
(b) integrating an exogenous nucleic acid sequence into the cellular genome by integrating
(i) a donor polynucleotide template, wherein the donor polynucleotide template is comprised of the exogenous nucleic acid sequence positioned relative to a second integration recognition site that is a cognate pair of the first integration recognition site such that the exogenous nucleic acid sequence is integrated into the cellular genome at the first integration target recognition site when contacted with the integration enzyme and the first integration recognition site. The method of claim 37, wherein the expression vector and the donor polynucleotide template are delivered to the cell concurrently.
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