WO2021138286A1 - Self-complementary aav delivery system for crispr/cas9 - Google Patents

Self-complementary aav delivery system for crispr/cas9 Download PDF

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WO2021138286A1
WO2021138286A1 PCT/US2020/067252 US2020067252W WO2021138286A1 WO 2021138286 A1 WO2021138286 A1 WO 2021138286A1 US 2020067252 W US2020067252 W US 2020067252W WO 2021138286 A1 WO2021138286 A1 WO 2021138286A1
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aav
promoter
vector
sequence
expression cassette
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French (fr)
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Yu Zhang
Eric Olson
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The Board Of Regents Of The University Of Texas System
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/51—Physical structure in polymeric form, e.g. multimers, concatemers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure relates to the fields of molecular biology, medicine and genetics. More particularly, the disclosure relates to compositions and uses thereof for genome editing to correct mutations in vivo.
  • CRISPR/Cas systems can be used to induce double-strand DNA breaks at a specific locus in the genome, which are resolved by endogenous DNA repair mechanisms.
  • a delivery system should (1) target specific tissues or cells, (2) effectively enter cells, (3) avoid activation of the immune system, and (4) prevent off-target events. There remains a need in the art for safe and effective systems to deliver gene editing machinery.
  • AAV delivery vectors and compositions comprising the same, for delivery of gene editing machinery to cells in vitro and in vivo.
  • the instant disclosure provides self-complimentary adeno-associated virus (AAV) delivery systems comprising sequences encoding a guide RNA and a CRISPR nuclease, e.g. , a Cas9.
  • AAV self-complimentary adeno-associated virus
  • the AAV delivery systems disclosed herein may be used, for example, for disrupting a dystrophin splice acceptor site and inducing skipping and/or refraining of an exon of a DMD gene, thereby modifying a DMD gene in a cell or a subject.
  • Compositions and methods according to the disclosure may be used to treat and/or prevent muscular dystrophy.
  • the disclosure provides an AAV expression cassette comprising, from 5’ to 3’, a first promoter, a sequence encoding a first sgRNA comprising a first spacer region and a first scaffold region, a second promoter, a sequence encoding a second sgRNA comprising a second spacer region and a second scaffold region, a third promoter, and a sequence encoding a third gRNA comprising a third sgRNA targeting region and third scaffold region, wherein the expression cassette is flanked by a first inverted terminal repeat (ITR) and a second ITR, wherein the first ITR has the sequence of SEQ ID NO: 2584, wherein the second ITR has the sequence of SEQ ID NO: 2583, and wherein the AAV expression cassette is self-complimentary ( i.e ., a scAAV).
  • ITR inverted terminal repeat
  • the first promoter is a U6 promoter.
  • the U6 promoter may have the sequence of SEQ ID NO: 2589, or a sequence at least 95% identical thereto.
  • the second promoter is the HI promoter.
  • the HI promoter may have the sequence of SEQ ID NO: 2586, or a sequence at least 95% identical thereto.
  • the third promoter is the 7SK promoter.
  • the 7SK promoter may have the sequence of SEQ ID NO: 2587, or a sequence at least 95% identical thereto.
  • each of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are the same. In some embodiments, the at least two of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are different. In some embodiments, each of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are different. [0012] In some embodiments, at least one of the first spacer region, the second spacer region, and the third spacer region targets the human dystrophin gene.
  • the sequences of first spacer region, the second spacer region, and the third spacer region are each independently selected from any one of SEQ ID NO: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036-1043, 1052-1063, 1494-1499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668.
  • the sequences encoding the first, the second, and the third spacer regions are the same, and the sequences are each SEQ ID NO: 2668.
  • the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are the same. In some embodiments, at least two of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are different. In some embodiments, each of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are different. In some embodiments, at least one of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region has the sequence of SEQ ID NO: 2672, or a sequence at least 95% identical thereto.
  • the expression cassette further comprises a fourth sgRNA comprising a fourth spacer region and a fourth scaffold region. In some embodiments, the expression cassette further comprises a fifth sgRNA comprising a fifth spacer region and a fifth scaffold region.
  • the expression cassette has a length less than about 2 kb, less than about 1.8 kb, less than about 1.6 kb, or less than about 1.4kb.
  • an AAV expression cassette comprising, from 5’ to 3’, a first ITR, a promoter, a sequence encoding a Cas9 nuclease, and a second ITR.
  • the first ITR has the sequence of SEQ ID NO: 2585
  • the second ITR has the sequence of SEQ ID NO: 2679
  • the AAV expression cassette is not selfcomplimentary.
  • the promoter is a CK8e promoter.
  • the promoter has the sequence of SEQ ID NO: 2590, or a sequence at least 95% identical thereto.
  • the sequence encoding the Cas9 nuclease is derived from S.
  • the sequence encoding the Cas9 nuclease comprises SEQ ID NO: 2591, or a sequence at least 95% identical thereto.
  • the expression cassette further comprises a sequence encoding a PolyA tail.
  • the PolyA tail comprises a sequence of SEQ ID NO: 2593, or a sequence at least 95% identical thereto.
  • composition comprising an AAV expression cassette of the disclosure.
  • the vector comprising the AAV expression cassette of the disclosure.
  • the vector is a non- viral vector, such as a plasmid.
  • the vector is a viral vector, such as an AAV vector.
  • the AAV vector is a self-complimentary AAV (scAAV).
  • the AAV vector is a recombinant AAV (rAAV).
  • the AAV vector comprises a capsid protein isolated or derived from an AAV vector of serotype 9 (AAV9).
  • the AAV vector comprises a wild type AAV9 capsid protein.
  • composition comprising one or more vectors of the disclosure.
  • the composition comprises a pharmaceutically acceptable carrier or excipient.
  • the disclosure additionally provides a recombinant AAV comprising a capsid protein, and an AAV expression cassette of the disclosure encapsidated by the capsid protein.
  • the capsid protein is isolated or derived from a wild type AAV capsid of one or more of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.
  • the capsid protein is isolated or derived from an AAV9 capsid.
  • AAV vector comprising contacting a vector comprising the AAV expression cassette of the disclosure with an AAV host cell.
  • the AAV host cell may be a mammalian cell, such as a HEK293 cell or modified HEK293 cell.
  • the AAV host cell is an insect cell, such as a Sf9 cell or modified Sf9 cell.
  • the disclosure additionally provides a method of correcting a gene defect in a cell, the method comprising contacting the cell with an AAV vector comprising an AAV expression cassette of the disclosure, wherein the expression cassette comprises a sequence encoding a least one sgRNA comprising a first spacer region and a first scaffold region.
  • the cell is a human cell.
  • the gene defect is a gene defect in the dystrophin gene.
  • the method also comprises contacting the cell with an AAV vector comprising an expression cassette for a Cas9 nuclease.
  • the expression cassette for the Cas9 nuclease is not self-complimentary.
  • the expression cassette for the Cas9 nuclease is an AAV expression cassette of the disclosure.
  • DMD Duchenne Muscular Dystrophy
  • a method of treating a subject in need thereof comprising administering to the subject a first AAV vector comprising an AAV expression cassette comprising a sequence encoding at least one sgRNA comprising a first spacer region and a first scaffold region.
  • the subject is a human.
  • the subject suffers from Duchenne Muscular Dystrophy (DMD).
  • the method also comprises administering to the subject a second AAV vector comprising an expression cassette for a Cas9 nuclease.
  • the expression cassette for the Cas9 nuclease is not self-complimentary.
  • dystrophin expression is at least partially restored in skeletal muscle in the patient.
  • dystrophin expression is at least partially restored in heart muscle in the patient.
  • the dosage of the first AAV required to at least partially restore dystrophin expression is at least about 20-fold lower than the dosage that would be required to achieve the same level of dystrophin expression if the expression cassette of the first AAV was not self-complimentary.
  • FIG. 1A-C Strategies for CRISPR/Cas9-mediated genome editing in Dmd exon 45.
  • FIG. 1A An out-of-frame deletion of Dmd exon 44 results in splicing of exon 43 to 45, generating a premature stop codon in exon 45.
  • a CRISPR/Cas9-mediated “single-cut” strategy was designed to restore the open reading frame (ORF) of the Dmd gene.
  • exon 45 will be reframed with adjacent exon 43 and 46. If the INDEL is large enough to delete the 5 ’-AG-3’ splice acceptor sequence, exon 45 will be skipped, resulting in splicing of exon 43 to 46.
  • FIG. IB Illustration of sgRNA targeting Dmd exon 45.
  • FIG. IB provides SEQ ID NOs: 2733 and 2734.
  • FIG. 1C Illustration of AAV vectors used to deliver the sgRNA expression cassette. Three copies of the same sgRNA are driven by three RNA polymerase III promoters, U6, HI, and 7SK. The top vector produces ssAAV. A 2.3kb staffer sequence was cloned into the ssAAV vector for optimal packaging. The bottom vector produces double-stranded scAAV.
  • FIG. 2 Alkaline denaturing gel electrophoresis confirms integrity of AAV vectors.
  • the viral genomes of the Cas9 vector and sgRNA vectors were analyzed by gel electrophoresis under alkaline denaturing conditions.
  • the size of ssAAV-sgRNA and ssAAV-Cas9 is 3.9 and 5.1 kilobases, respectively, and remains unchanged after alkaline gel electrophoresis.
  • the size of scAAV-sgRNA is 1.4 kilobases and is doubled to 2.8 kilobases under denaturing conditions, indicating its double-stranded viral genome.
  • M marker; knt, kilo-nucleotides.
  • FIG. 3A-3B Primers used throughout experiments and probes used for titration.
  • FIG. 3A provides SEQ ID NOs: 2648 to 2671
  • FIG. 3B provides SEQ ID NOs: 2681 to 2684.
  • FIG. 5 Systemic AAV delivery of CRISPR/Cas9 genome editing components to DEc44 mice rescues dystrophin expression.
  • Immunohistochemistry shows restoration of dystrophin in tibialis anterior (TA), triceps, diaphragm, and heart of DEc44 mice 4 weeks after systemic delivery of ssAAV-packaged SpCas9 and scAAV-packaged sgRNA.
  • SpCas9 vector was kept at constant dose of 8 x 10 13 vg/kg. The dose of sgRNA vector was shown in the figure.
  • FIG. 6 Systemic delivery of CRISPR/Cas9 genome editing components by single- stranded AAV vector to DEc44 mice rescues dystrophin expression.
  • Immunohistochemistry shows restoration of dystrophin in tibialis anterior (TA), triceps, diaphragm, and heart of DEc44 mice 4 weeks after systemic delivery of ssAAV-packaged SpCas9 and ssAAV- packaged sgRNA.
  • SpCas9 vector was kept at constant dose of 8 x 10 13 vg/kg. The dose of sgRNA vector is shown in the figure.
  • FIG. 7A-B Whole muscle scanning of immunohistochemistry of TA, triceps, diaphragm, and heart from CRISPR/Cas9-corrected DEc44 mice. Both panels show whole muscle scanning of TA, triceps, diaphragm, and heart from DEc44 mice 4 weeks after systemic delivery of ssAAV-packaged SpCas9 and scAAV-packaged sgRNA (FIG. 7A) or ssAAV- packaged sgRNA (FIG. 7B).
  • SpCas9 vector was kept at constant dose of 8 x 10 13 vg/kg. The dose of sgRNA vector was shown in the figure.
  • FIG. 8A-D Western blot analysis of skeletal muscles and heart from DEc44 mice receiving systemic AAV delivery of CRISPR/Cas9 genome editing components.
  • Western blot analysis shows restoration of dystrophin expression in the TA (FIG. 8A), triceps (FIG. 8B), diaphragm (FIG. 8C), and heart (FIG. 8D) of DEc44 mice 4 weeks after systemic delivery of ssAAV-packaged SpCas9 and scAAV-packaged sgRNA.
  • FIG. 9A-B Quantitative western blot analysis of skeletal muscles and heart from DEc44 mice receiving systemic AAV delivery of CRISPR/Cas9 genome editing components.
  • FIG. 9B Quantification of Cas9 expression in TA, triceps, diaphragm, and heart.
  • FIG. 10A-D Western blot analysis of skeletal muscles and heart from DEc44 mice treated with ssAAV-packaged CRISPR/Cas9 genome editing components.
  • Western blot analysis shows restoration of dystrophin expression in the TA (FIG. 10A), triceps (FIG. 10B), diaphragm (FIG. IOC), and heart (FIG. 10D) of DEc44 mice 4 weeks after systemic delivery of ssAAV-packaged SpCas9 and ssAAV-packaged sgRNA.
  • FIG. 13 Muscle histology of AEx44 mice after systemic delivery of ssAAV expressing CRISPR/Cas9 genome editing components.
  • FIG. 13 shows H&E staining of TA, triceps, diaphragm, and heart of DEc44 mice 4 weeks after systemic delivery but for ssAAV-packaged SpCas9 and ssAAV-packaged sgRNA.
  • FIG. 15A-D Rescue of skeletal muscle function after systemic AAV delivery of CRISPR/Cas9 genome editing components.
  • SpCas9 vector was kept at constant dose of 8 x 10 13 vg/kg. The dose of sgRNA vector was shown in the figure. Data are represented as mean ⁇ SEM.
  • FIG. 16 Serum creatine kinase (CK) analysis of CRISPR/Cas9-corrected DEc44 mice.
  • Serum CK was measured in WT, DEc44 mice untreated, and DEc44 mice 4 weeks after treatment with ssAAV-packaged SpCas9 and scAAV or ssAAV-packaged sgRNA.
  • SpCas9 vector was kept at constant dose of 8 x 10 13 vg/kg. The dose of sgRNA vector was shown in the figure.
  • Serum CK was normalized to WT mice and shown as fold expression. Data are represented as mean ⁇ SEM.
  • FIG. 17A-B scAAV vector induces significant INDELs at genomic and cDNA level.
  • Genomic INDEL analysis (FIG. 17A) and Dystrophin cDNA INDEL analysis (FIG. 17B) of TA, triceps, diaphragm, and heart from DEc44 mice 4 weeks after systemic delivery of ssAAV- packaged SpCas9 and scAAV or ssAAV-packaged sgRNA.
  • SpCas9 vector was kept at constant dose of 8 x 10 13 vg/kg. The dose of sgRNA vector was shown in the figure. Data are represented as mean ⁇ SEM.
  • FIG. 18A-B DEc44 mice sustain higher copies of viral genome after systemic delivery of scAAV-packaged sgRNA.
  • sgRNA viral genome copy number FIG. 18 A
  • Cas9 viral genome copy number FIG. 18A
  • FIG. 19A-B DEc44 mice express more sgRNA and Cas9 transcripts after systemic delivery of scAAV-packaged sgRNA.
  • sgRNA cDNA transcripts FIG. 19A
  • Cas9 cDNA transcripts FIG. 19B
  • SpCas9 vector was kept at constant dose of 8 x 10 13 vg/kg. The dose of sgRNA vector was shown in the figure.
  • Both sgRNA (FIG.
  • FIGS. 20A-B Deep sequencing analysis performed to determine INDEL frequency at the genomic and cDNA levels.
  • the present disclosure relates to a self-complementary adeno-associated virus (scAAV) system for delivery of gene editing systems (e.g., CRISPR/Cas9 systems) to a cell or tissue of interest.
  • scAAV self-complementary adeno-associated virus
  • the scAAV delivery systems may be used for treatment or prevention of a genetic disease including, for example, a genetic muscle disease.
  • the scAAV delivery systems may be used for treatment or prevention of Duchenne Muscular Dystrophy (DMD).
  • DMD Duchenne Muscular Dystrophy
  • DMD Duchenne muscular dystrophy
  • steroid supplementation partially alleviates DMD pathological phenotypes but cannot restore dystrophin expression.
  • Morpholino antisense oligomers allow skipping of mutant DMD exons, but less than 1% of normal levels of dystrophin protein can be restored by this treatment.
  • DMD Becker muscular dystrophy
  • nucleotide sequences are listed in the 5’ to 3’ direction, and amino acid sequences are listed in the N-terminal to C-terminal direction, unless indicated otherwise.
  • single guide RNA refers to a short synthetic RNA composed of a “spacer” (or “targeting”) sequence and a “scaffold” sequence.
  • the gRNA may further comprise a polyA tail.
  • the percent identity between two nucleotide or amino acid sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps which need to be introduced for optimal alignment and the length of each gap.
  • Various computer programs and mathematical algorithms are available in the art to determine percentage identities between nucleotide and amino acid sequences such as GCG Wisconsin package and BLAST.
  • CRISPRs (clustered regularly interspaced short palindromic repeats) are DNA loci containing short repetitions of base sequences. Each repetition is followed by short segments of “spacer DNA” from previous exposures to a virus.
  • CRISPRs are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. CRISPRs are often associated with Cas genes that code for proteins related to CRISPRs.
  • the CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. CRISPR spacers recognize and silence these exogenous genetic elements like RNAi in eukaryotic organisms.
  • CRISPR repeats range in size from 24 to 48 base pairs. They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic. Repeats are separated by spacers of similar length. Some CRISPR spacer sequences exactly match sequences from plasmids and phages, although some spacers match the prokaryote’s genome (self-targeting spacers). New spacers can be added rapidly in response to phage infection. Guide RNA (gRNA or sgRNA)
  • Cas9 requires a short RNA to direct the recognition of DNA targets. Though Cas9 preferentially interrogates DNA sequences containing a PAM sequence NGG it can bind without a protospacer target. However, the Cas9-gRNA complex generally requires a close match to the gRNA to create a double strand break. CRISPR sequences in bacteria are expressed in multiple RNAs and then processed to create guide strands for RNA. Because Eukaryotic systems lack some of the proteins required to process CRISPR RNAs the synthetic construct gRNA was created to combine the essential pieces of RNA for Cas9 targeting into a single RNA expressed with the RNA polymerase type III promoter U6. Synthetic gRNAs are slightly over lOObp at the minimum length and contain a portion which is targets the 20 protospacer nucleotides immediately preceding the PAM sequence NGG; gRNAs do not contain a PAM sequence.
  • Single guide RNAs comprise a spacer region and a scaffold region.
  • a sgRNA “targets” a DNA sequence it means that the spacer region of the sgRNA binds to the DNA sequence.
  • the sgRNA targets a site within a wildtype dystrophin gene.
  • An exemplary wildtype dystrophin sequence includes the human sequence (see GenBank Accession NO. NC_000023.11), located on the human X chromosome, which codes for the protein dystrophin (GenBank Accession No. AAA53189; SEQ ID NO: 5), the sequence of which is reproduced below:
  • the sgRNA targets a site within a mutant dystrophin gene. In some embodiments, the sgRNA targets a dystrophin intron. In some embodiments, the sgRNA targets a dystrophin exon. In some embodiments, the sgRNA targets a site in a dystrophin exon that is expressed and is present in one or more of the dystrophin isoforms shown in Table 1. In embodiments, the sgRNA targets a dystrophin splice site. In some embodiments, the sgRNA targets a splice donor site on the dystrophin gene. In embodiments, the sgRNA targets a splice acceptor site on the dystrophin gene. Table 1: Dystrophin isoforms
  • sgRNAs comprise a spacer and a scaffold region.
  • the spacer targets a mutant DMD exon.
  • the spacer targets any one of exons 1-79 of the DMD gene.
  • the spacer targets at least one of exon 23, 44 or 51.
  • the spacer targets at least one of exons 1, 6, 7, 8, 23, 41, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
  • the spacer targets an intron flanking ( i.e ., immediately 5’ or 3’ to) any one of exons 1-79 of the DMD gene.
  • the spacer targets an intron flanking at least one of exon 23, 44 or 51. In some embodiments, the spacer targets an intron flanking at least one of exons 1, 6, 7, 8, 23, 41, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. In some embodiments, the spacer targets a splice donor or splice acceptor site of any one of exons 1-79 of the DMD gene. In preferred embodiments, the spacer is designed to induce skipping and/or refraining of exon 51, exon 44 or exon 23. In embodiments, the spacer is targeted to a splice acceptor site of exon 51, exon 44 or exon 23.
  • Exemplar ⁇ ' spacers for use in various compositions and methods disclosed herein are provided as SEQ ID NOs: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036-1043, 1052- 1063, 1494-1499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668, and in Tables 5-9, 12, and 14-16.
  • the spacer is selected from any one of SEQ ID No: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036-1043, 1052-1063, 14944499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668.
  • the spacers of the disclosure comprise a sequence that is complementary to a target sequence within a coding sequence or a non-coding sequence corresponding to the DMD gene, and, therefore, hybridize to the target sequence.
  • the scaffold sequence is the sequence within the gRNA that is responsible for nuclease (e.g., Cas9) binding.
  • the scaffold sequence does not include the spacer/targeting sequence.
  • the scaffold may be about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, or about 120 to about 130 nucleotides in length.
  • the scaffold may be about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108,
  • the scaffold may be at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, or at least 125 nucleotides in length. In some embodiments, the scaffold may be 60, 61, 62, 63, 64,
  • the scaffold may have a sequence of any one of SEQ ID NO: 2672-2678 (shown in Table 2 below), or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the scaffold may comprise a sequence of GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTA TCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT (SEQ ID NO: 2672), or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.
  • a gRNA spacer + scaffold
  • a spacer of any one of SEQ ID NO: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036-1043, 1052-1063, 1494- 1499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668, and a scaffold of SEQ ID NO: 2672.
  • a nucleic acid may comprise one or more sequences encoding a gRNA.
  • a nucleic acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 sequences encoding a gRNA.
  • all of the sequences encode the same gRNA. In some embodiments, all of the sequences encode different gRNAs. In some embodiments, at least 2 of the sequences encode the same gRNA, for example at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the sequences encode the same gRNA. Nucleases
  • the CRISPR systems disclosed herein may comprise a nuclease or a nucleic acid encoding the same.
  • the nuclease is a Type II, Type V-A, Type V-B, Type V-C, Type V-U, Type VI-B nuclease.
  • the nuclease is a transcription activator-like effector nuclease (TALEN), a meganuclease, or a zinc-finger nuclease.
  • TALEN transcription activator-like effector nuclease
  • the nuclease is a Cas9, Casl2a (Cpfl), Casl2b, Casl2c, Tnp- B like, Casl3a (C2c2), Casl3b, or Casl4 nuclease.
  • the nuclease is a Cas9 nuclease or a Cpfl nuclease.
  • the nuclease is a modified form or variant of a Cas9, Casl2a (Cpfl), Casl2b, Casl2c, Tnp-B like, Casl3a (C2c2), Casl3b, or Casl4 nuclease.
  • the nuclease is a modified form or variant of a TAL nuclease, a meganuclease, or a zinc-finger nuclease
  • a “modified” or “variant” nuclease is one that is, for example, truncated, fused to another protein (such as another nuclease), catalytically inactivated, etc.
  • the nuclease may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a naturally occurring Cas9, Casl2a (Cpfl), Casl2b, Casl2c, Tnp-B like, Casl3a (C2c2), Casl3b, Casl4 nuclease, or a TALEN, meganuclease, or zinc-finger nuclease.
  • the nuclease is a Cas9 nuclease derived from S. pyogenes (SpCas9).
  • SpCas9 S. pyogenes
  • An exemplary SpCas9 sequence is provided in SEQ ID NO: 2591.
  • the nuclease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2591.
  • the nuclease is a Cas9 derived from S. aureus (SaCas9).
  • An exemplar ⁇ ' SaCas9 sequence is provided in SEQ ID NO: 873.
  • the nuclease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 873.
  • the Cpfl is a Cpfl enzyme from Acidaminococcus (species BV3L6, UniProt Accession No. U2UMQ6).
  • An exemplary Acidaminococcus Cpfl sequence is provided in SEQ ID NO: 870.
  • the nuclease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 870.
  • the Cpfl is a Cpfl enzyme from Lachnospiraceae (species ND2006, UniProt Accession No. A0A182DWE3).
  • An exemplary Lachnospiraceae Cpfl sequence is provided in SEQ ID NO: 871.
  • the nuclease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 871.
  • a sequence encoding the nuclease is codon optimized for expression in mammalian cells. In some embodiments, the sequence encoding the nuclease is codon optimized for expression in human cells or mouse cells.
  • CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays.
  • Cas protein families As of 2013, more than forty different Cas protein families had been described. Of these protein families, Casl appears to be ubiquitous among different CRISPR/Cas systems. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat- associated mysterious proteins (RAMPs). More than one CRISPR subtype may occur in a single genome. The sporadic distribution of the CRISPR/Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.
  • Exogenous DNA is apparently processed by proteins encoded by Cas genes into small elements (-30 base pairs in length), which are then somehow inserted into the CRISPR locus near the leader sequence.
  • RNAs from the CRISPR loci are constitutively expressed and are processed by Cas proteins to small RNAs composed of individual, exogenously-derived sequence elements with a flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level.
  • Evidence suggests functional diversity among CRISPR subtypes.
  • the Cse (Cas subtype Ecoli ) proteins (called CasA-E in E. coli ) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer- repeat units that Cascade retains.
  • Cas6 processes the CRISPR transcripts.
  • CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Casl and Cas2.
  • the Cmr (Cas RAMP module) proteins found in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs.
  • RNA-guided CRISPR enzymes are classified as type V restriction enzymes.
  • Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix.
  • tracrRNA and spacer RNA can be combined into a "single-guide RNA" molecule that, mixed with Cas9, can find and cut the correct DNA targets and such synthetic guide RNAs are used for gene editing.
  • Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR/Cas- mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during bacterial interaction with eukaryotic hosts.
  • Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence. Delivery of Cas9 DNA sequences also is contemplated.
  • compositions of the disclosure include a small version of a Cas9 from the bacterium Staphylococcus aureus (UniProt Accession No. J7RUA5).
  • the small version of the Cas9 provides advantages over wildtype or full length Cas9.
  • the Cas9 is a SpCas9.
  • Cpfl Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 or CRISPR/Cpfl is a DNA-editing technology which shares some similarities with the CRISPR/Cas9 system.
  • Cpfl is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. It prevents genetic damage from viruses.
  • Cpfl genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA.
  • Cpfl is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations.
  • Cpfl appears in many bacterial species.
  • the ultimate Cpfl endonuclease that was developed into a tool for genome editing was taken from one of the first 16 species known to harbor it.
  • the Cpfl is a Cpfl enzyme from Acidaminococcus (species BV3L6, UniProt Accession No. U2UMQ6; SEQ ID NO: 870), having the sequence set forth below: 1 MIQFEGFZNL YQVSKTLRFE LIPQGKTLKH IQEQGFIEED KARKDHYKEL KPIIDRIYKT
  • the Cpfl is a Cpfl enzyme from Lachnospiraceae (species ND2006, UniProt Accession No. A0A182DWE3; SEQ ID NO: 871), having the sequence set forth below:
  • the Cpfl is codon optimized for expression in mammalian cells. In some embodiments, the Cpfl is codon optimized for expression in human cells or mouse cells.
  • the Cpfl locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain.
  • the Cpfl protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cpfl does not have a HNH endonuclease domain, and the N-terminal of Cpfl does not have the alpha-helical recognition lobe of Cas9.
  • Cpfl CRISPR-Cas domain architecture shows that Cpfl is functionally unique, being classified as Class 2, type V CRISPR system.
  • the Cpfl loci encode Casl, Cas2 and Cas4 proteins more similar to types I and III than from type II systems.
  • Database searches suggest the abundance of Cpfl -family proteins in many bacterial species.
  • Cpfl does not require a tracrRNA, therefore, only crRNA is required. This benefits genome editing because Cpfl is not only smaller than Cas9, but also it has a smaller sgRNA molecule (approximately half as many nucleotides as Cas9).
  • the Cpfl-crRNA complex cleaves target DNA or RNA by identification of a protospacer adjacent motif 5'-YTN-3' (where "Y” is a pyrimidine and “N” is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cpfl introduces a sticky-end-like DNA double- stranded break of 4 or 5 nucleotides overhang.
  • the CRISPR/Cpfl system consists of a Cpfl enzyme and a guide RNA that finds and positions the complex at the correct spot on the double helix to cleave target DNA.
  • CRISPR/Cpfl systems activity has three stages: 1) Adaptation, during which Casl and Cas2 proteins facilitate the adaptation of small fragments of DNA into the CRISPR array; 2) Formation of crRNAs: processing of pre-cr-RNAs producing of mature crRNAs to guide the Cas protein; and 3) Interference, in which the Cpfl is bound to a crRNA to form a binary complex to identify and cleave a target DNA sequence.
  • the first step in editing the DMD gene using CRISPR/Cpfl or CRISPR/Cas9 is to identify the genomic target sequence.
  • the genomic target for the gRNAs of the disclosure can be any ⁇ 24 nucleotide DNA sequence, provided that the sequence is unique compared to the rest of the genome.
  • the genomic target sequence corresponds to a sequence within exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene.
  • the genomic target sequence is a 5’ or 3’ splice site of exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene.
  • the genomic target sequence corresponds to a sequence within an intron immediately upstream or downstream of exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene.
  • the next step in editing the DMD gene is to identify all Protospacer Adjacent Motif (PAM) sequences within the genetic region to be targeted.
  • the target sequence must be immediately upstream of a PAM. Once all possible PAM sequences and putative target sites have been identified, the next step is to choose which site is likely to result in the most efficient on-target cleavage.
  • the gRNA targeting sequence needs to match the target sequence, and the gRNA targeting sequence must not match additional sites within the genome.
  • the gRNA targeting sequence has perfect homology to the target with no homology elsewhere in the genome. In some embodiments, a given gRNA targeting sequence will have additional sites throughout the genome where partial homology exists.
  • off-targets sites are called “off-targets” and should be considered when designing a gRNA.
  • off-target sites are not cleaved as efficiently when mismatches occur near the PAM sequence, so gRNAs with no homology or those with mismatches close to the PAM sequence will have the highest specificity.
  • on-target activity factors that maximize cleavage of the desired target sequence (“on-target activity”) must be considered. It is known to those of skill in the art that two gRNA targeting sequences, each having 100% homology to the target DNA may not result in equivalent cleavage efficiency. In fact, cleavage efficiency may increase or decrease depending upon the specific nucleotides within the selected target sequence.
  • the next step is to synthesize and clone desired gRNAs.
  • Targeting oligos can be synthesized, annealed, and inserted into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, the exact cloning strategy will depend on the gRNA vector that is chosen.
  • the gRNAs for Cpfl are notably simpler than the gRNAs for Cas9, and only consist of a single crRNA containing direct repeat scaffold sequence followed by -24 nucleotides of guide sequence.
  • Each gRNA should then be validated in one or more target cell lines. For example, after the Cas9 or Cpfl and the gRNA are delivered to the cell, the genomic target region may be amplified using PCR and sequenced according to methods known to those of skill in the art.
  • gene editing may be performed in vitro or ex vivo.
  • cells are contacted in vitro or ex vivo with a Cas9 or a Cpfl and a gRNA that targets a dystrophin splice site.
  • the cells are contacted with one or more nucleic acids encoding the Cas9 or Cpfl and the guide RNA.
  • the one or more nucleic acids are introduced into the cells using, for example, lipofection or electroporation.
  • Gene editing may also be performed in zygotes.
  • zygotes may be injected with one or more nucleic acids encoding Cas9 or Cpfl and a gRNA that targets a dystrophin splice site. The zygotes may subsequently be injected into a host.
  • the Cas9 or Cpfl is provided on a vector.
  • the vector contains a Cas9 derived from S. pyogenes (SpCas9, SEQ ID NO: 872).
  • the vector contains a Cas9 derived from S. aureus (SaCas9, SEQ ID NO: 873).
  • the vector contains a Cpfl sequence derived from a Lachnospiraceae bacterium. See, for example, Uniprot Accession No. A0A182DWE3; SEQ ID NO: 871.
  • the vector contains a Cpfl sequence derived from an Acidaminococcus bacterium. See, for example, Uniprot Accession No.
  • the Cas9 or Cpfl sequence is codon optimized for expression in human cells or mouse cells.
  • the vector further contains a sequence encoding a fluorescent protein, such as GFP, which allows Cas9 or Cpfl -expressing cells to be sorted using fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • the vector is a viral vector such as an adeno-associated viral vector.
  • the gRNA is provided on a vector.
  • the vector is a viral vector such as an adeno-associated viral vector.
  • the Cas9 or Cpfl and the guide RNA are provided on the same vector. In embodiments, the Cas9 or Cpfl and the guide RNA are provided on different vectors.
  • the cells are additionally contacted with a single-stranded DMD oligonucleotide to effect homology directed repair.
  • small INDELs restore the protein reading frame of dystrophin (“reframing” strategy).
  • the cells may be contacted with a single gRNA.
  • a splice donor or splice acceptor site is disrupted, which results in exon skipping and restoration of the protein reading frame (“exon skipping” strategy).
  • exon skipping strategy the cells may be contacted with two or more gRNAs.
  • Efficiency of in vitro or ex vivo Cas9 or Cpfl -mediated DNA cleavage may be assessed using techniques known to those of skill in the art, such as the T7 El assay. Restoration of DMD expression may be confirmed using techniques known to those of skill in the art, such as RT-PCR, western blotting, and immunocytochemistry.
  • in vitro or ex vivo gene editing is performed in a muscle or satellite cell.
  • gene editing is performed in iPSC or iCM cells.
  • the iPSC cells are differentiated after gene editing.
  • the iPSC cells may be differentiated into a muscle cell or a satellite cell after editing.
  • the iPSC cells are differentiated into cardiac muscle cells, skeletal muscle cells, or smooth muscle cells.
  • the iPSC cells are differentiated into cardiomyocytes. iPSC cells may be induced to differentiate according to methods known to those of skill in the art.
  • contacting the cell with the Cas9 or the Cpfl and the gRNA restores dystrophin expression.
  • cells which have been edited in vitro or ex vivo , or cells derived therefrom show levels of dystrophin protein that are comparable to wildtype cells.
  • the edited cells, or cells derived therefrom express dystrophin at a level that is 50%, 60%, 70%, 80%, 90%, 95% or any percentage in between of wildtype dystrophin expression levels.
  • the cells which have been edited in vitro or ex vivo, or cells derived therefrom have a mitochondrial number that is comparable to that of wildtype cells.
  • the edited cells, or cells derived therefrom have 50%, 60%, 70%, 80%, 90%, 95% or any percentage in between as many mitochondria as wildtype cells.
  • the edited cells, or cells derived therefrom show an increase in oxygen consumption rate (OCR) compared to non-edited cells at baseline.
  • OCR oxygen consumption rate
  • expression cassettes are employed to express a transcription factor product, either for subsequent purification and delivery to a cell/subject, or for use directly in a genetic -based delivery approach.
  • expression vectors which contain one or more nucleic acids encoding Cas9 or Cpfl and at least one DMD guide RNA that targets a dystrophin splice site.
  • a nucleic acid encoding Cas9 or Cpf 1 and a nucleic acid encoding at least one guide RNA are provided on the same vector.
  • a nucleic acid encoding Cas9 or Cpfl and a nucleic acid encoding least one guide RNA are provided on separate vectors.
  • Expression requires that appropriate signals be provided in the vectors, and include various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
  • various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in cells.
  • Elements designed to optimize messenger RNA stability and translatability in host cells also are defined.
  • the conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
  • expression cassette is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i.e., is under the control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • An “expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • At least one module in each promoter functions to position the start site for RNA synthesis.
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • RNA polymerase III (also called Pol III) transcribes DNA to synthesize ribosomal 5S rRNA, tRNA and other small RNAs.
  • the genes transcribed by RNA Pol III fall in the category of “housekeeping” genes whose expression is required in all cell types and most environmental conditions. Therefore, the regulation of Pol III transcription is primarily tied to the regulation of cell growth and the cell cycle, thus requiring fewer regulatory proteins than RNA polymerase II. Under stress conditions however, the protein Mail represses Pol III activity.
  • RNA polymerase complex In the process of transcription (by any polymerase) there are three main stages: (i) initiation, requiring construction of the RNA polymerase complex on the gene's promoter; (ii) elongation, the synthesis of the RNA transcript; and (iii) termination, the finishing of RNA transcription and disassembly of the RNA polymerase complex.
  • Promoters under the control of RNA Pol III include those for ribosomal 5S rRNA, tRNA and few other small RNAs such as U6 spliceosomal RNA, RNase P and RNase MRP RNA, 7SL RNA (the RNA component of the signal recognition particles), Vault RNAs, Y RNA, SINEs (short interspersed repetitive elements), 7SK RNA, two microRNAs, several small nucleolar RNAs and several few regulatory antisense RNAs.
  • the Cas9 or Cpfl constructs of the disclosure are expressed by a cell type-specific promoter.
  • the Cas9 or Cpfl constructs of the disclosure are expressed by a muscle cell-specific promoter.
  • This cell type-specific promoter e.g., the muscle-specific promoter
  • Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
  • viral promoters such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma vims long terminal repeat, rat insulin promoter and glyceraldehyde- 3 -phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest.
  • CMV human cytomegalovirus
  • SV40 early promoter the Rous sarcoma vims long terminal repeat
  • rat insulin promoter and glyceraldehyde- 3 -phosphate dehydrogenase
  • glyceraldehyde- 3 -phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest.
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given
  • the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • the promoter and/or enhancer may be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and/or DQ b, b-interferon, interleukin-2, interleukin-2 receptor, MHC class II, MHC class II HLA-Dra, b-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, oc-fetoprotein, t-globin, b-globin, c-fos, c-HA-mv, insulin, neural cell adhesion molecule (NCAM), ai-antitrypain, H2B (TH2B) histone, mouse and/or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), plate
  • inducible elements may be used.
  • the inducible element is, for example, MTII, MMTV (mouse mammary tumor vims), b-interferon, adenovirus 5 E2, collagenase, stromelysin, SV40, murine MX gene, GRP78 gene, oc-2- macroglobulin, vimentin, MHC class I gene H-2Kb, HSP70, proliferin, tumor necrosis factor, and/or thyroid stimulating hormone a gene.
  • the inducer is phorbol ester (TFA), heavy metals, glucocorticoids, poly(rI)x, poly(rc), E1A, phorbol ester (TPA), interferon, Newcastle Disease Virus, A23187, IL-6, semm, interferon, SV40 large T antigen, PMA, and/or thyroid hormone.
  • TFA phorbol ester
  • Any of the inducible elements described herein may be used with any of the inducers described herein.
  • muscle specific promoters include the myosin light chain-2 promoter, the a-actin promoter, the troponin 1 promoter; the Na + /Ca 2+ exchanger promoter, the dystrophin promoter, the a7 integrin promoter, the brain natriuretic peptide promoter and the aB-crystallin/small heat shock protein promoter, a-myosin heavy chain promoter and the ANF promoter.
  • the muscle specific promoter is the CK8 promoter.
  • the CK8 promoter has the following sequence (SEQ ID NO: 874):
  • the muscle-cell cell specific promoter is a variant of the CK8 promoter, called CK8e.
  • the CK8e promoter has the following sequence (SEQ ID NO. 2590):
  • a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • Any polyadenylation sequence may be employed such as human growth hormone and SV40 polyadenylation signals.
  • a terminator is also contemplated as an element of the expression cassette. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • the self-cleaving peptide is a 2A peptide.
  • a 2A-like self-cleaving domain from the insect virus Thosea asigna (TaV 2A peptide) (SEQ ID NO: 876, EGRGSLLTCGDVEENPGP) is used. These 2A-like domains have been shown to function across eukaryotes and cause cleavage of amino acids to occur co-translationally within the 2A-like peptide domain. Therefore, inclusion of T aV 2A peptide allows the expression of multiple proteins from a single mRNA transcript.
  • equine rhinitis A virus EAV 2A peptide
  • PTV1 porcine teschovirus-1
  • FMDV foot and mouth disease vims
  • the 2A peptide is used to express a reporter and a Cas9 or a Cpfl simultaneously.
  • the reporter may be, for example, GFP.
  • Other self-cleaving peptides that may be used include, but are not limited to nuclear inclusion protein a (Nia) protease, a PI protease, a 3C protease, an L protease, a 3C-like protease, or modified versions thereof.
  • an AAV expression cassette comprises a sequence encoding a Cas9 nuclease.
  • the AAV expression cassette comprises, from 5’ to 3’, a promoter and a sequence encoding a Cas9 nuclease, wherein the expression cassette is flanked by a first inverted terminal repeat (ITR) and a second ITR, wherein the first ITR has the sequence of SEQ ID NO: 2585, wherein the second ITR has the sequence of SEQ ID NO: 2679.
  • ITR inverted terminal repeat
  • the AAV expression cassette is self-complementary. In some embodiments, the AAV expression cassette is not self-complimentary.
  • the promoter is a CK8e promoter. In some embodiments, the promoter has the sequence of SEQ ID NO: 2590, or a sequence at least 95% identical thereto.
  • the sequence encoding the Cas9 nuclease is derived from S. aureus or S. pyogenes. In some embodiments, the sequence encoding the Cas9 nuclease comprises SEQ ID NO: 2591, or a sequence at least 95% identical thereto.
  • the expression cassette further comprises a sequence encoding a PolyA tail.
  • the PolyA tail comprises a sequence of SEQ ID NO: 2593, or a sequence at least 95% identical thereto.
  • an expression cassette encoding a Cas9 may be packaged into an AAV vector.
  • the AAV vector is a wildtype AAV vector.
  • the AAV vector contains one or more mutations.
  • the AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, A AVI 2, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof.
  • Exemplar ⁇ ' AAV-Cas9 vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the Cas9 sequence.
  • the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, A AVI 2, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof.
  • the ITRs comprise or consist of full-length and/or wildtype sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, deletion, insertion, inversion, or transposition.
  • the ITRs comprise or consist of at least 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs.
  • the ITRs comprise or consist of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs.
  • the ITRs have a length of 110 ⁇ 10 base pairs.
  • the ITRs have a length of 120 ⁇ 10 base pairs. In some embodiments, the ITRs have a length of 130 ⁇ 10 base pairs. In some embodiments, the ITRs have a length of 140 ⁇ 10 base pairs. In some embodiments, the ITRs have a length of 150 ⁇ 10 base pairs. In some embodiments, the ITRs have a length of 115, 145, or 141 base pairs. In some embodiments, the ITRs have a sequence selected from SEQ ID NO: 2584, SEQ ID NO: 2583, SEQ ID NO: 2585, SEQ ID NO: 2679 and SEQ ID. NO: 2582.
  • the AAV-Cas9 vector may contain one or more nuclear localization signals (NLS).
  • the AAV-Cas9 vector contains 1, 2, 3, 4, or 5 nuclear localization signals.
  • Exemplary NLS include the c-myc NLS (SEQ ID NO: 884), the SV40 NLS (SEQ ID NO: 885), the hnRNPAI M9 NLS (SEQ ID NO: 886), the nucleoplasmin NLS (SEQ ID NO: 887), the sequence
  • RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 888) of the IBB domain from importin-alpha
  • the sequence PQPKKKPL (SEQ ID NO: 891) of human p53 the sequence SALIKKKKKMAP (SEQ ID NO: 892) of mouse c-abl IV
  • the sequences DRLRR (SEQ ID NO: 893) and KQKKRK (SEQ ID NO: 894) of the influenza virus NS1
  • nuclear localization signals include bipartite nuclear localization sequences such as the sequence KRKGDEVDGVDEVAKKKS KK (SEQ ID NO: 897) of the human poly(ADP-ribose) polymerase or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 898) of the steroid hormone receptors (human) glucocorticoid.
  • the AAV-Cas9 vector may comprise additional elements to facilitate packaging of the vector and expression of the Cas9.
  • the AAV- Cas9 vector may comprise a polyA sequence.
  • the polyA sequence may be a mini-polyA sequence.
  • the AAV-Cas9 vector may comprise a transposable element.
  • the AAV-Cas9 vector may comprise a regulator element.
  • the regulator element is an activator or a repressor.
  • the AAV-Cas9 vector may contain one or more promoters.
  • the one or more promoters drive expression of the Cas9.
  • the one or more promoters are muscle-specific promoters.
  • Exemplary muscle-specific promoters include myosin light chain-2 promoter, the a-actin promoter, the troponin 1 promoter, the Na + /Ca 2+ exchanger promoter, the dystrophin promoter, the a7 integrin promoter, the brain natriuretic peptide promoter, the aB-crystallin/small heat shock protein promoter, a- myosin heavy chain promoter, the ANF promoter, the CK8 promoter and the CK8e promoter.
  • the AAV-Cas9 vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in human cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in a bacculovirus expression system.
  • the AAV-Cas9 vector comprises a sequence of SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 2599, as shown in Table 3, or a sequence at least 90%, at least 95%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto.
  • Table 3 Exemplary AAV-Cas9 Expression Cassette (from ITR to ITR)
  • the construct comprises or consists of a promoter sequence and a sequence encoding a nuclease.
  • the construct comprises or consists of a CK8e promoter and a sequence encoding a Cas9 nuclease.
  • the construct comprises or consists of a CK8e promoter and a sequence encoding a Cas9 nuclease isolated or derived from Staphylococcus pyogenes (“SpCas9”).
  • the CK8e promoter comprises or consists of a nucleotide sequence of TGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTAGAATTAACCCAGACATGTGGCTGCCCC CCCCCCCAACACCTGCTGCCICTAAAAATAACCCTGCATGCCATGTGCCCGGCGAAGGGCCAGCTGTCCCG CCAGCTAGACTCAGCACGTAGTTTAGGAACCAGTGAGCAAGTCAGCCCGTGGGGCAGCCCATACAAGGCCATGGG GCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACGGTGCCCGGGCAACGAGCTGAAAGCTCATCTGCTCTCA GGGGCCCCTCCCTGGGGACAGCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGGCACAG GGGCTGCCCTCATTCTACCACCACCTCCACAGCACAGACAGACACTCAGGAGCCAGCCAG (SEQ ID NO: 2590).
  • SEQ ID NO: 2590 a
  • the construct comprising a promoter and a sequence encoding a nuclease further comprises at least two inverted terminal repeat (ITR) sequences.
  • the construct comprising a promoter and a sequence encoding a nuclease further comprises at least two ITR sequences isolated or derived from an AAV of serotype 2 (AAV2).
  • the construct comprising a promoter and a sequence encoding a nuclease further comprises at least two ITR sequences, wherein the first ITR sequence comprises or consists of a nucleotide sequence of
  • TGGCCACTCCCTCTCTGCGCIC GCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTG CCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT (SEQ ID NO: 2584) and the second ITR sequence comprises or consists of a nucleotide sequence of
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a sequence encoding a nuclease and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a sequence encoding a SpCas9 nuclease and a second AAV2 ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a sequence encoding a nuclease and a second ITR, further comprises a poly A sequence.
  • the polyA sequence comprises or consists of a minipolyA sequence.
  • Exemplary minipolyA sequences of the disclosure comprise or consist of a nucleotide sequence of lAGCAATAAAGGATCGTGTATTITCATTGGAAGCGTGTGITGGTTITTGGATCAGGCGCG (SEQ ID NO: 2680).
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a sequence encoding a nuclease, a minipoly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a sequence encoding a SpCas9 nuclease, a minipoly A sequence and a second AAV2
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR, and further comprises at least one nuclear localization signal.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR, and further comprises at least two nuclear localization signals.
  • Exemplary nuclear localization signals of the disclosure comprise or consist of a nucleotide sequence of CCAAAGAAGAAGCGGAAGGTC (SEQ ID NO: 2581), or a nucleotide sequence of
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a first nuclear localization signal, a sequence encoding a nuclease, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a first nuclear localization signal, a sequence encoding a nuclease, a second nuclear localization signal, a poly A sequence and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 2581, a sequence encoding a SpCas9 nuclease, a minipoly A sequence and a second AAV2 ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 2580, a minipoly A sequence and a second AAV2 ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 2581, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 2580, a minipoly A sequence and a second AAV2 ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a first nuclear localization signal, a sequence encoding a nuclease, a second nuclear localization signal, a poly A sequence and a second ITR, and further comprises a stop codon.
  • the stop codon may have a sequence of TAG (SEQ ID NO: 904), TAA (SEQ ID NO: 905), or TGA (SEQ ID NO: 906).
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, promoter, a first nuclear localization signal, a sequence encoding a nuclease, a second nuclear localization signal, a stop codon, a poly A sequence and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 2581, a sequence encoding a SpCas9 nuclease, a stop codon, a minipoly A sequence and a second AAV2 ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 2580, a stop codon, a minipoly A sequence and a second AAV2 ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 2581, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 2580, a stop codon a minipoly A sequence and a second AAV2 ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a first nuclear localization signal, a sequence encoding a nuclease, a second nuclear localization signal, a stop codon, a poly A sequence and a second ITR, and further comprises transposable element inverted repeats.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a promoter, a first nuclear localization signal, a sequence encoding a nuclease, a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, a regulatory sequence and a second transposable element inverted repeat.
  • the construct may further comprise one or more spacer sequences. Exemplary spacer sequences of the disclosure have length from 1-1500 nucleotides, inclusive of all ranges therebetween.
  • the spacer sequences may be located either 5’ to or 3’ to an ITR, a promoter, a nuclear localization sequence, a nuclease, a stop codon, a polyA sequence, a transposable element inverted repeat, and/or a regulator element.
  • the construct may have a sequence comprising or consisting of SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 2599.
  • an AAV expression cassette comprises a sequence encoding at least one gRNA, wherein the gRNA comprises a spacer region and a scaffold region.
  • the AAV expression cassette comprises, from 5’ to 3’, a U6 promoter, a sequence encoding a first sgRNA comprising a first spacer region and a first scaffold region, a HI promoter, a sequence encoding a second sgRNA comprising a second spacer region and a second scaffold region, a 7SK promoter, and a sequence encoding a third gRNA comprising a third sgRNA targeting region and third scaffold region, wherein the expression cassette is flanked by a first inverted terminal repeat (ITR) and a second ITR, wherein the first ITR has the sequence of SEQ ID NO: 2584, wherein the second ITR has the sequence of SEQ ID NO: 2583.
  • the AAV expression cassette is self-complementary.
  • the U6 promoter has the sequence of SEQ ID NO: 2589, or a sequence at least 95% identical thereto.
  • the HI promoter has the sequence of SEQ ID NO: 2586, or a sequence at least 95% identical thereto.
  • the 7SK promoter has the sequence of SEQ ID NO: 2587, or a sequence at least 95% identical thereto.
  • each of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are the same. In some embodiments, at least two of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are different. In some embodiments, each of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are different.
  • the sequences of first spacer region, the second spacer region, and the third spacer region are each independently selected from any one of SEQ ID NO: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036-1043, 1052-1063, 1494-1499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668.
  • the sequences encoding the first, the second, and the third spacer regions are the same, and the sequences are each SEQ ID NO: 2668.
  • the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are the same. In some embodiments, at least two of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are different. In some embodiments, each of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are different. In some embodiments, at least one of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region has the sequence of SEQ ID NO: 2672, or a sequence at least 95% identical thereto. [0148] In some embodiments, the expression cassette further comprises a fourth sgRNA comprising a fourth spacer region and a fourth scaffold region. In some embodiments, the expression cassette further comprises a fifth sgRNA comprising a fifth spacer region and a fifth scaffold region.
  • the expression cassette further comprises a stuffer sequence.
  • the expression cassette is less than about 2 kb, less than about 1.8 kb, less than about 1.6 kb, or less than about 1.4kb.
  • an expression cassette comprising a sequence encoding at least one gRNA may be packaged into an AAV vector.
  • the AAV vector is a wildtype AAV vector.
  • the AAV vector contains one or more mutations.
  • the AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVRh.74, AAV2i8, AAVRh.lO, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof.
  • the AAV vector may be self-complementary. In some embodiments, the AAV vector may be non-self-complementary.
  • Exemplar ⁇ ' AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising one or more sgRNA sequences.
  • the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.lO, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof.
  • the ITRs are isolated or derived from an AAV vector of a first serotype and a sequence encoding a capsid protein of the AAV-sgRNA vector is isolated or derived from an AAV vector of a second serotype, In some embodiments, the first serotype and the second serotype are the same. In some embodiments, the first serotype and the second serotype are not the same.
  • the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.lO, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.
  • the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.lO, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.
  • the first serotype is AAV2 and the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.
  • the first serotype is AAV2 and the second serotype is AAV9.
  • Exemplar ⁇ ' AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising one or more gRNA sequences.
  • the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof.
  • a first ITR is isolated or derived from an AAV vector of a first serotype
  • a second ITR is isolated or derived from an AAV vector of a second serotype
  • a sequence encoding a capsid protein of the AAV-sgRNA vector is isolated or derived from an AAV vector of a third serotype.
  • the first serotype and the second serotype are the same.
  • the first serotype and the second serotype are not the same.
  • the first serotype, the second serotype, and the third serotype are the same.
  • the first serotype, the second serotype, and the third serotype are not the same.
  • the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.
  • the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.
  • the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.
  • the first serotype is AAV2
  • the second serotype is AAV4
  • the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.
  • the first serotype is AAV2
  • the second serotype is AAV4
  • the third serotype is AAV9.
  • Exemplar ⁇ ' AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the sgRNA sequences.
  • the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.lO, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof.
  • the ITRs comprise or consist of full- length and/or wildtype sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, deletion, insertion, inversion, or transposition.
  • the ITRs comprise or consist of at least 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs.
  • the ITRs comprise or consist of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs.
  • the ITRs have a length of 110 ⁇ 10 base pairs.
  • the ITRs have a length of 120 + 10 base pairs. In some embodiments, the ITRs have a length of 130 ⁇ 10 base pairs. In some embodiments, the ITRs have a length of 140 + 10 base pairs. In some embodiments, the ITRs have a length of 150 + 10 base pairs. In some embodiments, the ITRs have a length of 115, 145, or 141 base pairs. In some embodiments, at least one of the ITRs has a sequence selected from SEQ ID NO: 2582, SEQ ID NO: 2583, SEQ ID NO: 2584, SEQ ID NO: 2585 or SEQ ID NO: 2679.
  • the AAV-sgRNA vector may comprise additional elements to facilitate packaging of the vector and expression of the sgRNA.
  • the AAV-sgRNA vector may comprise a transposable element.
  • the AAV- sgRNA vector may comprise a regulatory element.
  • the regulatory element comprises an activator or a repressor.
  • the AAV-sgRNA sequence may comprise a non-functional or “stuffer” sequence. Exemplary stuffer sequences of the disclosure may have some (a non-zero percentage of) identity or homology to a genomic sequence of a mammal (including a human).
  • exemplary stuffer sequences of the disclosure may have no identify or homology to a genomic sequence of a mammal (including a human).
  • Exemplary stuffer sequences of the disclosure may comprise or consist of naturally occurring non-coding sequences or sequences that are neither transcribed nor translated following administration of the AAV vector to a subject.
  • the AAV-sgRNA vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV-sgRNA vector may be optimized for expression in human cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in a bacculovirus expression system.
  • the AAV-sgRNA vector comprises at least one promoter. In some embodiments, the AAV-sgRNA vector comprises at least two promoters. In some embodiments, the AAV-sgRNA vector comprises at least three promoters. In some embodiments, the AAV-sgRNA vector comprises at least four promoters. In some embodiments, the AAV-sgRNA vector comprises at least five promoters. Exemplar ⁇ ' promoters include, for example, immunoglobulin light chain, immunoglobulin heavy chain,
  • T-cell receptor HLA DQ a and/or DQ b, b-interferon, interleukin-2, interleukin-2 receptor, MHC class II, MHC class II HLA-Dra, b-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, a-fetoprotein, t- globin, b-globin, c-fos, c-HA-ra.s, insulin, neural cell adhesion molecule (NCAM), ⁇ Xi- antitrypain, H2B (TH2B) histone, mouse and/or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), Duchenne muscular dystrophy, SV40, polyoma, retroviruse
  • the sequence encoding the gRNA spacer comprises a sequence selected from any one of SEQ ID Nos: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036- 1043, 1052-1063, 1494-1499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668.
  • the AAV expression cassette or vector comprises a first sequence encoding a gRNA and a second sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA and a second promoter drives expression of the second sequence encoding a gRNA.
  • the first and second promoters are the same. In some embodiments, the first and second promoters are different. In some embodiments, the first and second promoters are selected from the HI promoter, the U6 promoter, and the 7SK promoter.
  • the first sequence encoding a gRNA and the second sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA and the second sequence encoding a gRNA are not identical.
  • the AAV expression cassette or vector comprises a first sequence encoding a gRNA, a second sequence encoding a gRNA, and a third sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA, a second promoter drives expression of the second sequence encoding a gRNA, and a third promoter drives expression of a third sequence encoding a gRNA.
  • a first promoter drives expression of the first sequence encoding a gRNA
  • a second promoter drives expression of the second sequence encoding a gRNA
  • a third promoter drives expression of a third sequence encoding a gRNA.
  • at least two of the first, second, and third promoters are the same.
  • each of the first, second, and third promoters are different.
  • the first, second, and third promoters are selected from the HI promoter, the U6 promoter, and the 7SK promoter.
  • the first promoter is the U6 promoter.
  • the second promoter is the HI promoter.
  • the third promoter is the 7SK promoter.
  • the first promoter is the U6 promoter, the second promoter is the HI promoter, and the third promoter is the 7SK promoter.
  • the first sequence encoding a gRNA, the second sequence encoding a gRNA, and the third sequence encoding a gRNA are identical.
  • the first sequence encoding a gRNA, the second sequence encoding a gRNA, and the third sequence encoding a gRNA are not identical.
  • the AAV vector comprises a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, and a fourth sequence encoding a gRNA
  • a first promoter drives expression of the first sequence encoding a gRNA
  • a second promoter drives expression of the second sequence encoding a gRNA
  • a third promoter drives expression of the third sequence encoding a gRNA
  • a fourth promoter drives expression of the fourth sequence encoding a gRNA.
  • at least two of the first, second, third, and fourth promoters are the same.
  • each of the first, second, third, and fourth promoters are different. In some embodiments, each of the first, second, third and fourth promoters are selected from the HI promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, and the fourth sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, and the fourth sequence encoding a gRNA are not identical.
  • the AAV vector comprises a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, a fourth sequence encoding a gRNA, and a fifth sequence encoding a gRNA
  • a first promoter drives expression of the first sequence encoding a gRNA
  • a second promoter drives expression of the second sequence encoding a gRNA
  • a third promoter drives expression of the third sequence encoding a gRNA
  • a fourth promoter drives expression of the fourth sequence encoding a gRNA
  • a fifth promoter drives expression of the fifth sequence encoding a gRNA.
  • first, second, third, fourth, and fifth promoters are the same. In some embodiments, each of the first, second, third, fourth, and fifth promoters are different. In some embodiments, each of the first, second, third, and fourth promoters are different. In some embodiments, each of the first, second, third, fourth and fifth promoters are selected from the HI promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, the fourth sequence encoding a gRNA, and the fifth sequence encoding a gRNA are identical.
  • the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, the fourth sequence encoding a gRNA, and the fifth sequence encoding a gRNA are not identical.
  • the AAV-sgRNA vector comprises a sequence of SEQ ID NO: 2597, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.
  • SEQ ID NO: 2597 is provided in Table 4.
  • Table 4 Exemplary AAV-sgRNA vector.
  • the construct comprises or consists of a first promoter, a first sequence encoding a gRNA (spacer + scaffold), a second promoter, and a second sequence encoding a gRNA (spacer + scaffold), a third promoter, and a third sequence encoding a gRNA (spacer + scaffold).
  • Exemplary gRNA spacer sequences include SEQ ID NO: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036-1043, 1052-1063, 1494-1499, 1500- 1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668.
  • Exemplary sequences encoding gRNA scaffold sequences are SEQ ID NO: 2672 to 2678.
  • the sequence encoding the gRNA spacer is GGCTTACAGGAACICCAGGA (SEQ ID NO: 2668).
  • the sequence encoding the gRNA spacer is CACTAGAGT AAC AG T c T G Ac (SEQ ID NO: 708).
  • the sequence encoding the gRNA spacer is AICTTACAGGAACTCCAGGA (SEQ ID NO: 762). In some embodiments, the sequence encoding the gRNA spacer is CACCAGAGTAACAGTCTGAG (SEQ ID NO: 714). In some embodiments, the sequence encoding the gRNA spacer is CACCAGAGTAACAGTCTGAC (SEQ ID NO: 863).
  • the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a second promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 2668, a third promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 2668.
  • the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a second promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 708, a third promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 708.
  • the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, a second promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 714, a third promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 714.
  • the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, a second promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 863, a third promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 863.
  • the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a second promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 762, a third promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 762.
  • Exemplary promoters of the disclosure include the U6 promoter having a sequence of
  • the first, second, and third promoter are each individually selected from the U6 promoter (SEQ ID NO: 2589), the H1 promoter (SEQ ID NO: 2586), and the 7SK promoter (SEQ ID NO: 2587). In some embodiments, the first, second, and third promoter are each individually selected from the U6 promoter (SEQ ID NO: 2589), and the HI promoter (SEQ ID NO: 2586).
  • the construct comprises, from 5’ to 3’, a U6 promoter, a first sequence encoding a gRNA, a HI promoter, a second sequence encoding a gRNA, a 7SK promoter, and a third sequence encoding a gRNA.
  • the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, the HI promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 2668, the 7SK promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 2668.
  • the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, the HI promoter, a second sequence encoding a gRNA comprising SEQ ID NO: 714, the 7SK promoter, and a third sequence encoding a gRNA of SEQ ID NO: 714.
  • the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, the HI promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 708, the 7SK promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 708.
  • the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, the HI promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 762, the 7SK promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 762.
  • the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, the HI promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 863, the 7SK promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 863.
  • the construct comprising a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, and a third sequence encoding a gRNA further comprises at least two inverted terminal repeat (ITR) sequences.
  • the construct comprising a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, and a third sequence encoding a gRNA further comprises at least two ITR sequences isolated or derived from an AAV of serotype 2 (AAV2).
  • the construct comprising a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, and a third sequence encoding a gRNA further comprises at least two ITR sequences, wherein the first ITR sequence is isolated or derived from an AAV of serotype 4 (AAV4) and the second ITR sequence is isolated or derived from an AAV of serotype 2 (AAV2).
  • AAV4 AAV4
  • AAV2 AAV2
  • the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, a first sequence encoding a gRNA, and a second ITR.
  • the construct comprises or encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, a third sequence consists of, from 5’ to 3’, a first ITR, a U6 promoter, a first sequence encoding a gRNA, a HI promoter, and a second sequence encoding a gRNA, a 7SK promoter, a third sequence encoding a gRNA, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a second promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a second promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 762, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a second promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, a second promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, a second promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a HI promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 708, a 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, a Hl promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 714, a 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a HI promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a HI promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 762, a 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, and a second ITR.
  • the construct comprising, from 5’ to 3’ a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, and a second ITR, further comprises a poly A sequence.
  • the poly A sequence comprises or consists of a minipolyA sequence.
  • Exemplary minipolyA sequences of the disclosure comprise or consist of a nucleotide sequence of
  • the constmct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding sgRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, a first sequence encoding a gRNA, the HI promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a minipolyA sequence, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a second promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a minipolyA sequence, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a second promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a minipolyA sequence, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a minipolyA sequence, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA spacer of SEQ ID NO.: 714, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, a minipolyA sequence, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, a minipolyA sequence, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, the HI promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, the 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a minipolyA sequence, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, the HI promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, the 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a minipolyA sequence, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, the HI promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a minipolyA sequence, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, the HI promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, the 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, a minipolyA sequence, and a second ITR.
  • a nucleic acid comprises a sequence encoding a first guide RNA targeting a first genomic target sequence, a sequence encoding a second guide RNA targeting a second genomic target sequence, a first promoter wherein the first promoter drives expression of the sequence encoding the first guide RNA, and a second promoter wherein the first promoter drives expression of the sequence encoding the second guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a genomic locus of interest.
  • the genomic locus of interest is a splice acceptor or splice donor site.
  • a nucleic acid comprises a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a first promoter wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a second promoter wherein the first promoter drives expression of the sequence encoding the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor site.
  • the first promoter and the second promoter are identical. In some embodiments, the first promoter and the second promoter not identical. In some embodiments, the first promoter and the second promoter share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity.
  • the first genomic target sequence and the second genomic target sequence are identical. In some embodiments, the first genomic target sequence and the second genomic target sequence are not identical. In some embodiments, the first genomic target sequence and the second genomic target sequence share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, the first genomic target sequence and the second genomic target sequence are complementary.
  • the nucleic acid further comprises a sequence encoding a third DMD guide RNA targeting a third genomic target sequence, and a third promoter wherein the third promoter drives expression of the sequence encoding the third DMD guide RNA, and wherein the third genomic target sequence comprises a dystrophin splice acceptor site.
  • the first promoter, the second promoter, and the third promoter are identical. In some embodiments, at least two of the first promoter, the second promoter, and the third promoter are not identical.
  • At least two of the first promoter, the second promoter, and the third promoter share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence are identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence are not identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence are complementary.
  • the nucleic acid further comprises a sequence encoding a fourth DMD guide RNA targeting a fourth genomic target sequence, and a fourth promoter, wherein the fourth promoter drives expression of the fourth sequence encoding a DMD guide RNA, wherein the fourth genomic target sequence comprises a dystrophin splice acceptor site.
  • the first promoter, the second promoter, the third promoter, and the fourth promoter are identical. In some embodiments, at least two of the first promoter, the second promoter, the third promoter, and the fourth promoter are not identical.
  • At least two of the first promoter, the second promoter, the third promoter, and the fourth promoter share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence are identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence are not identical.
  • At least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence are complementary.
  • the nucleic acid further comprises a sequence encoding a fifth DMD guide RNA targeting a fifth genomic target sequence, and a fifth promoter, wherein the fifth promoter drives expression of the sequence encoding the fifth DMD guide RNA, wherein the fifth genomic target sequence comprises a dystrophin splice acceptor site.
  • the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter are identical. In some embodiments, at least two of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter are not identical.
  • At least two of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence are identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence are not identical.
  • At least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence are complementary.
  • the nucleic acid further comprises at least one sequence encoding an additional DMD guide RNA targeting a genomic target sequence, and at least one additional promoter, wherein the additional promoter drives expression of the additional DMD guide RNA, wherein the additional genomic target sequence comprises a dystrophin splice acceptor site.
  • the dystrophin splice acceptor site comprises the 5’ splice acceptor site of exon 51.
  • the first promoter or the second promoter comprises a constitutive promoter. In some embodiments, the first promoter or the second promoter comprises a constitutive promoter.
  • At least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a sequence encoding a constitutive promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a constitutive promoter. In some embodiments, the first promoter or the second promoter comprises an inducible promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises an inducible promoter. In some embodiments, the first promoter or the second promoter comprises a cell-type specific promoter.
  • At least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a cell-type specific promoter.
  • the cell type specific promoter comprises a muscle-specific promoter.
  • the first promoter or the second promoter comprises a U6 promoter, an HI promoter, or a 7SK promoter.
  • at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a U6 promoter, an HI promoter, or a 7SK promoter.
  • At least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a U6 promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises an HI promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a 7SK promoter.
  • the sequence encoding the first DMD guide RNA, the sequence encoding the second DMD guide RNA, and sequence encoding the third DMD guide RNA are identical, and the 5’ splice acceptor site comprises a 5’ splice acceptor site of exon 51.
  • the first promoter comprises a U6 promoter
  • the second promoter comprises a HI promoter
  • the third promoter comprises a 7SK promoter.
  • the nucleic acid comprises a DNA sequence.
  • the nucleic acid comprises an RNA sequence.
  • the nucleic acid further comprises one or more sequences encoding an inverted terminal repeat (ITR). In some embodiments, the nucleic acid further comprises a sequence encoding a 5’ inverted terminal repeat (ITR) and a sequence encoding a 3’ ITR. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno- associated vims (AAY). In some embodiments, the sequence encoding the 5 !
  • inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated vims (AAV) of serotype 2 (AAV2).
  • the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV2.
  • the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV) of serotype 4 (AAV4).
  • the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV4. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3 ! ITR comprises or consists of 145 nucleotides. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 115 nucleotides. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 141 nucleotides.
  • the nucleic acid further comprises a polyadenosine (poly A) sequence.
  • the poly A sequence is a mini poly A sequence.
  • the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises the sequence of any one of SEQ ID NOs: 60-382, 706- 708, 712-714, 718-789, 863, 947, 1036-1043, 1052-1063, 1494-1499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668.
  • the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises the sequence of SEQ ID NO: 714.
  • a vector comprising a nucleic acid comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a first promoter wherein the first promoter drives expression of the first DMD guide RNA, and a second promoter wherein the second promoter drives expression of the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor site.
  • the vector further comprises an inverted terminal repeat of a transposable element.
  • the transposable element is a transposon.
  • the transposon is a Tn7 transposon.
  • the vector is a non-viral vector.
  • the non-viral vector is a plasmid.
  • the vector is a viral vector.
  • the viral vector is an adeno-associated viral (AAV) vector.
  • the AAV vector is replication- defective or conditionally replication defective.
  • the AAV vector is a recombinant AAV vector.
  • the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11), AAVrh74, AAVrhlO or any combination thereof.
  • the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 9 (AAV9).
  • the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 2 (AAV2).
  • the AAV vector comprises a sequence isolated or derived from an AAV2 and a sequence isolated or derived from an AAV9. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV4 and a sequence isolated or derived from an AAV9. In some embodiments, the vector is optimized for expression in mammalian cells. In some embodiments, the vector is optimized for expression in human cells. [0181] Also provided is a nucleic acid comprising a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the promoter comprises a muscle-specific promoter. In some embodiments, the muscle-specific promoter comprises a CK8 promoter.
  • the muscle-specific promoter comprises a CK8e promoter.
  • the sequence encoding the Cas9 or the nuclease domain thereof is isolated or derived from a sequence encoding an S. pyogenes Cas9 or a nuclease domain thereof.
  • the sequence encoding the Cas9 or the nuclease domain thereof is isolated or derived from a sequence encoding S. aureus Cas9 or a nuclease domain thereof.
  • the sequence encoding the Cas9 or the nuclease domain thereof is codon optimized for expression in a mammal.
  • the sequence encoding the Cas9 or the nuclease domain thereof is codon optimized for expression in a human.
  • the nucleic acid comprising a promoter and a sequence encoding a Cas9 or a nuclease domain thereof further comprises a polyA sequence.
  • the polyA sequence is a mini polyA sequence.
  • the nucleic acid further comprises one or more sequences encoding an inverted terminal repeat (ITR).
  • the nucleic acid further comprises a sequence encoding a 5’ inverted terminal repeat (ITR) and a sequence encoding a 3’ ITR.
  • the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV) of serotype 2 (AAV2). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV2.
  • the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated vims (AAV) of serotype 4 (AAV4).
  • the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV4.
  • the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 145 nucleotides, 115 nucleotides, or 141 nucleotides.
  • the nucleic acid further comprises a nuclear localization signal. In some embodiments, the nucleic acid is optimized for expression in am alian cells. In some embodiments, the nucleic acid is optimized for expression in human cells.
  • a vector comprising a nucleic acid comprising a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the promoter comprises a muscle-specific promoter such as the CK8 or CK8e promoter.
  • the vector further comprises an inverted terminal repeat (ITR) of a transposable element. In some embodiments, the transposable element is a transposon.
  • the transposon is a Tn7 transposon.
  • the vector further comprises a sequence encoding a 5’ ITR of a T7 transposon and a sequence encoding a 3’ ITR of a T7 transposon.
  • the vector is a non- viral vector.
  • the non-viral vector is a plasmid.
  • the vector is a viral vector.
  • the viral vector is an adeno-associated viral (AAV) vector.
  • the AAV vector is replication-defective or conditionally replication defective.
  • the AAV vector is a recombinant AAV vector.
  • the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11), AAVrh74, AAVrhlO or any combination thereof.
  • the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 9 (AAV9).
  • the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 2 (AAV2).
  • the AAV vector comprises a sequence isolated or derived from an AAV2 and a sequence isolated or derived from an AAV9. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 4 (AAV4). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV4 and a sequence isolated or derived from an AAV9. In some embodiments, wherein the vector is optimized for expression in mammalian cells. In some embodiments, the vector is optimized for expression in human cells.
  • compositions comprising one or more vectors, expression cassettes and/or nucleic acids of the disclosure.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • compositions are prepared in a form appropriate for the intended application. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • Appropriate salts and buffers are used to render drugs, proteins or delivery vectors stable and allow for uptake by target cells.
  • Aqueous compositions of the present disclosure comprise an effective amount of the dmg, vector or proteins, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • “pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is not incompatible with the active ingredients of the present disclosure, its use in therapeutic compositions may be used. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or cells of the compositions.
  • the active compositions of the present disclosure may include classic pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any common route so long as the target tissue is available via that route, but generally including systemic administration ⁇ This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into muscle tissue. Such compositions would normally be administered as pharmaceutically acceptable compositions, as described supra.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • these preparations are sterile and fluid to the extent that easy injectability exists.
  • Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above.
  • the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions of the present disclosure are formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like). Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine) and the like.
  • inorganic acids e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like).
  • Salts formed with the free carboxyl groups of the protein can also be derived
  • solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations may easily be administered in a variety of dosage forms such as injectable solutions, dmg release capsules and the like.
  • the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose.
  • aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration ⁇
  • sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure.
  • a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
  • the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • a first vector and a second vector are administered to a patient.
  • the first vector comprises a nucleic acid comprising a first sequence encoding a first DMD guide RNA targeting a first genomic target sequence; a sequence encoding a second DMD guide RNA targeting a second genomic target sequence; a first promoter, wherein the first promoter drives expression of the first DMD guide RNA; and a second promoter, wherein the second promoter drives expression of the second DMD guide RNA.
  • the second vector comprise a nucleic acid comprising a sequence encoding a Cas9 or a nuclease domain thereof; a muscle-specific promoter, wherein the muscle-specific promoter drives expression of the Cas9 or a nuclease domain thereof.
  • a first vector and a second vector are administered to a patient in a therapeutically effective ratio.
  • ratio may refer to a concentration ratio (i.e., a ratio of the amount of vectors in a composition), a dose ratio (/. e.
  • a ratio of the amount delivered to a patient a bioavailability ratio (i.e ., a ratio of the amount available to a therapeutic site), a copy number ratio (i.e., a ratio of the amount expressed by a target cell), an efficacy ratio (i.e., a ratio of the amount of modifications made), a DNA ratio (i.e., a ratio of the amount of DNA), or a coding sequences ratio (i.e. a ratio of the number of coding sequences, e.g., sequences encoding a gRNA or a Cas9).
  • the ratio of the first vector and the second vector is between 1 : 1 and 1:30. In other embodiments, the ratio of the first vector and the second vector is between 30:1 and 1:1. In some embodiments, the ratio of the first vector and the second vector is any one of the ratios shown in Table 5 below.
  • the ratio of the amount of the first vector and amount of the second vector is between 1:1 and 1:30. In other embodiments, the ratio of the amount of the first vector and amount of the second vector is between 30:1 and 1:1. In some embodiments, the ratio of the amount of first vector and the amount of the second vector is any one of the ratios shown in Table 5.
  • the first vector is an AAV-Cas9 vector of the disclosure and the second vector is an AAV-sgRNA vector of the disclosure.
  • the ratio of the AAV-Cas9 vector to the AAV-sgRNA vector is any one of the ratios shown in Table 5. [0198] In some embodiments, the ratio of the first vector to the second vector is greater than
  • the ratio of the first vector to the second vector may be about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 50:1, about 75:1, or about 100:1.
  • the ratio of an AAV-sgRNA vector to an AAV-Cas9 vector is greater than 10:1; for example, the ratio may be about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 50:1, about 75:1, or about 100:1.
  • between 4 x 10 12 viral genomes (vg)/kilogram (kg) and 3 x 10 13 vg/kg, inclusive of the endpoints, of the first and/or the second vector are administered to the patient. In some embodiments, between 4 x 10 12 viral genomes (vg)/kilogram (kg) and 3 x 10 13 vg/kg, inclusive of the endpoints, of the first and/or the second vector are administered to the patient.
  • At least 5 x 10 12 viral genomes (vg)/kilogram (kg), 6 x 10 12 viral genomes (vg)/kilogram (kg), 1 x 10 13 viral genomes (vg)/kilogram (kg), 2 x 10 13 viral genomes (vg)/kilogram (kg), 3 x 10 13 viral genomes (vg)/kilogram (kg), 5 x 10 13 viral genomes (vg)/kilogram (kg), 1 x 10 14 viral genomes (vg)/kilogram (kg), 2 x 10 14 viral genomes (vg)/kilogram (kg), 3 x 10 14 viral genomes (vg)/kilogram (kg), or 4 x 10 14 viral genomes (vg)/kilogram (kg) of the first and/or the second vector are administered to the patient.
  • the Cas9 or Cpfl and gRNAs described herein may be delivered to the patient using adoptive cell transfer (ACT).
  • adoptive cell transfer one or more expression constructs are provided ex vivo to cells which have originated from the patient (autologous) or from one or more individual(s) other than the patient (allogeneic). The cells are subsequently introduced or reintroduced into the patient.
  • one or more nucleic acids encoding Cas9 or Cpfl and a guide RNA that targets a dystrophin splice site are provided to a cell ex vivo before the cell is introduced or reintroduced to a patient.
  • a composition comprises (i) a first nucleic acid sequence comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a first promoter wherein the first promoter drives expression of the first DMD guide RNA, and a second promoter wherein the second promoter drives expression of the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor and (ii) a second nucleic acid sequence comprising a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the comprises a muscle-specific promoter such as the CK8 or CK8e promoter.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • a composition comprises (i) a first vector comprising a nucleic acid sequence comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a first promoter wherein the first promoter drives expression of the first DMD guide RNA, and a second promoter wherein the second promoter drives expression of the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor and (ii) a second vector comprising a nucleic acid sequence comprising a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the promoter comprises a sequence encoding a muscle- specific promoter such as the CK8 or CK8e promoter.
  • the composition comprises (i) a first vector comprising a nucleic acid sequence comprising a sequence encoding a first DMD guide RNA
  • a cell comprising one or more nucleic acids of the disclosure.
  • the cell is a human cell.
  • the cell is a muscle cell or satellite cell.
  • the cell is an induced pluripotent stem (iPS) cell.
  • a composition comprising a cell comprising one or more nucleic acids of the disclosure.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • a cell comprising a composition comprising one or more vectors of the disclosure.
  • the cell is a human cell.
  • the cell is a muscle cell or satellite cell.
  • the cell is an induced pluripotent stem (iPS) cell.
  • kits for correcting a gene defect in a cell comprising contacting an AAV vector comprising an AAV expression cassette of the disclosure with the cell.
  • the methods comprise contacting the cell with a first AAV vector comprising an AAV-sgRNA expression cassette and a second AAV vector comprising an AAV-Cas9 expression cassette of the disclosure.
  • the AAV-sgRNA expression cassette is self-complementary.
  • the AAV- Cas9 expression cassette is not self-complementary.
  • the AAV-sgRNA expression cassette is self-complementary and the AAV-Cas9 expression cassette is not selfcomplementary.
  • Also provided herein is method of treating a subject in need thereof comprising administering to the subject a first AAV vector comprising a self-complementary AAV-sgRNA expression cassette of the disclosure.
  • the subject may be a mammal, such as a human.
  • the subject suffers from a genetic disease.
  • the subject suffers from a monogenic disease.
  • the subject suffers from a polygenic disease.
  • the subject suffers from a genetic muscle disease.
  • the subject suffers from Duchenne Muscular Dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss dystrophy, myotonic dystrophy, limb-girdle muscular dystrophy (LGMD), oculopharyngeal muscular dystrophy, congential dystrophy, congenital myopathy, familial periodic paralysis.
  • DMD Duchenne Muscular Dystrophy
  • BMD Becker muscular dystrophy
  • Emery-Dreifuss dystrophy myotonic dystrophy
  • LGMD limb-girdle muscular dystrophy
  • oculopharyngeal muscular dystrophy congential dystrophy
  • congenital myopathy familial periodic paralysis
  • the subject suffers from mitochondrial oxidative phosphorylation disorder or a glycogen storage disease (e.g., von Gierke’s disease, Pompe’s disease, Forbes-Cori disease, Andersen’s disease, McArdle’s disease, Hers’ disease, Tarui’s disease, or Fanconi-Bickel syndrome).
  • a glycogen storage disease e.g., von Gierke’s disease, Pompe’s disease, Forbes-Cori disease, Andersen’s disease, McArdle’s disease, Hers’ disease, Tarui’s disease, or Fanconi-Bickel syndrome.
  • the subject suffers from Duchenne Muscular Dystrophy (DMD).
  • the method also comprises administering to the subject a second AAV vector comprising an AAV-Cas9 expression cassette.
  • the expression cassette for the Cas9 nuclease is not self-complimentary.
  • dystrophin expression is at least partially restored in skeletal muscle in the patient. In some embodiments, dystrophin expression is at least partially restored in heart muscle in the patient. In some embodiments, the dosage of the first AAV required to at least partially restore dystrophin expression is at least about 20-fold lower than the dosage that would be required to achieve the same level of dystrophin expression if the expression cassette of the first AAV was not self-complimentary.
  • a method of treating a subject in need thereof comprises administering to the patient a therapeutically effective amount of an AAV-sgRNA vector of the disclosure, or a composition comprising the same, and a AAV-Cas9 vector of the disclosure, or a composition comprising the same, wherein the subject suffers from Duchenne Muscular Dystrophy (DMD).
  • DMD Duchenne Muscular Dystrophy
  • a method for correcting a dystrophin defect comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the first DMD guide RNA, the second DMD guide RNA and the Cas9 protein or a nuclease domain thereof, wherein at least one of first DMD guide RNA or the second DMD guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one DMD guide RNA-Cas9 complex, wherein the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping of a DMD exon and/or refraining.
  • the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces a reframing of a dystrophin reading frame. In some embodiments, the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and produces an insertion which restores the dystrophin protein reading frame. In some embodiments, the insertion comprises an insertion of a single adenosine.
  • Also provided is a method for inducing selective skipping and/or reframing of a DMD exon comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the first DMD guide RNA, the second DMD guide RNA and the Cas9 protein or a nuclease domain thereof, wherein at least one of first DMD guide RNA or the second DMD guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one DMD guide RNA-Cas9 complex, wherein the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping and/or refraining of a DMD exon.
  • a method for inducing a reframing event in the dystrophin reading frame comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the first DMD guide RNA, the second DMD guide RNA and the Cas9 protein or a nuclease domain thereof, wherein at least one of first DMD guide RNA or the second DMD guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one DMD guide RNA-Cas9 complex, wherein the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping and/or reframing of a DMD exon.
  • the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping and/or reframing of exon 51 of a human DMD gene.
  • compositions of the disclosure comprising administering to the subject a therapeutically effective amount of one or more compositions of the disclosure.
  • the composition is administered locally.
  • the composition is administered directly to a muscle tissue.
  • the composition is administered by an intramuscular infusion or injection.
  • the muscle tissue comprises a tibialis anterior tissue, a quadriceps tissue, a soleus tissue, a diaphragm tissue, or a heart tissue.
  • the composition is administered by an intra-cardiac injection.
  • the composition is administered systemically.
  • the composition is administered by an intravenous infusion or injection.
  • the subject following administration of the composition, the subject exhibits normal dystrophin-positive myofibers, and mosaic dystrophin-positive myofibers containing centralized nuclei, or a combination thereof. In some embodiments, following administration of the composition, the subject exhibits an emergence or an increase in a level of abundance of normal dystrophin-positive myofibers when compared to an absence or a level of abundance of normal dystrophin-positive myofibers prior to administration of the composition.
  • the subject following administration of the composition, the subject exhibits an emergence or an increase in a level of abundance of mosaic dystrophin-positive myofibers containing centralized nuclei when compared to an absence or an level of abundance of mosaic dystrophin-positive myofibers containing centralized nuclei prior to administration of the composition.
  • the subject following administration of the composition, the subject exhibits a decreased semm CK level when compared to a serum CK level prior to administration of the composition.
  • the subject following administration of the composition, the subject exhibits improved grip strength when compared to a grip strength prior to administration of the composition.
  • the subject is a neonate, an infant, a child, a young adult, or an adult.
  • the subject has muscular dystrophy. In some embodiments, the subject is a genetic carrier for muscular dystrophy. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject appears to be asymptomatic and a genetic diagnosis reveals a mutation in one or both copies of a DMD gene that impairs function of the DMD gene product. In some embodiments, the subject presents an early sign or symptom of muscular dystrophy. In some embodiments, the early sign or symptom of muscular dystrophy comprises loss of muscle mass or proximal muscle weakness. In some embodiments, the loss of muscle mass or proximal muscle weakness occurs in one or both leg(s) and/or a pelvis, followed by one or more upper body muscle(s).
  • the early sign or symptom of muscular dystrophy further comprises pseudohypertrophy, low endurance, difficulty standing, difficulty walking, difficulty ascending a staircase or a combination thereof.
  • the subject presents a progressive sign or symptom of muscular dystrophy.
  • the progressive sign or symptom of muscular dystrophy comprises muscle tissue wasting, replacement of muscle tissue with fat, or replacement of muscle tissue with fibrotic tissue.
  • the subject presents a later sign or symptom of muscular dystrophy.
  • the later sign or symptom of muscular dystrophy comprises abnormal bone development, curvature of the spine, loss of movement, and paralysis.
  • the subject presents a neurological sign or symptom of muscular dystrophy.
  • the neurological sign or symptom of muscular dystrophy comprises intellectual impairment and paralysis.
  • administration of the composition occurs prior to the subject presenting one or more progressive, later or neurological signs or symptoms of muscular dystrophy.
  • the subject is less than 10 years old, less than 5 years old, or less than 2 years old.
  • compositions of the disclosure for treating muscular dystrophy in a subject in need thereof.
  • Tables 6-10, 13, and 15-17 provide exemplary guide RNA spacer sequences for use in connection with the compositions and methods disclosed herein.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.
  • the expression vector comprises a genetically engineered form of adenovirus.
  • retrovirus the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its midsized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
  • ITRs inverted repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
  • the El region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off.
  • the products of the late genes are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP).
  • MLP major late promoter
  • the MLP (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5 ‘-tripartite leader (TPL) sequence which makes them preferred mRNAs for translation.
  • TPL tripartite leader
  • recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two pro viral vectors, wild- type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of vims from an individual plaque and examine its genomic structure.
  • adenovirus generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins. Since the E3 region is dispensable from the adenovirus genome, the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions. In nature, adenovirus can package approximately 105% of the wild-type genome, providing capacity for about 2 extra kb of DNA.
  • the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector- borne cytotoxicity. Also, the replication deficiency of the El -deleted virus is incomplete.
  • Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.
  • the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g. , Vero cells or other monkey embryonic mesenchymal or epithelial cells.
  • the preferred helper cell line is 293.
  • the adenoviruses of the disclosure are replication defective, or at least conditionally replication defective.
  • the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present disclosure.
  • the typical vector according to the present disclosure is replication defective and will not have an adenovims El region.
  • the position of insertion of the construct within the adenovirus sequences is not critical.
  • the polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors, or in the E4 region where a helper cell line or helper virus complements the E4 defect.
  • Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo.
  • This group of viruses can be obtained in high titers, e.g. , 10 9 -10 12 plaque-forming units per ml, and they are highly infective.
  • the life cycle of adenovirus does not require integration into the host cell genome.
  • the foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus, demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
  • Adenovirus vectors have been used in eukaryotic gene expression and vaccine development. Animal studies suggested that recombinant adenovirus could be used for gene therapy. Studies in administering recombinant adenovirus to different tissues include trachea instillation, muscle injection, peripheral intravenous injections and stereotactic inoculation into the brain.
  • the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse- transcription.
  • the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
  • Two long terminal repeat (LTR) sequences are present at the 5‘ and 3‘ ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome.
  • a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed.
  • the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media.
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells. [0229] A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification could permit the specific infection of hepatocytes via sialoglycoprotein receptors.
  • a different approach to targeting of recombinant retroviruses may be used, in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor are used.
  • the antibodies are coupled via the biotin components by using streptavidin.
  • streptavidin Using antibodies against major histocompatibility complex class I and class II antigens, it has been demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro.
  • retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes.
  • Another concern with the use of defective retrovirus vectors is the potential appearance of wild- type replication-competent virus in the packaging cells. This can result from recombination events in which the intact- sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome.
  • packaging cell lines may be selected to greatly decrease the likelihood of recombination.
  • viral vectors may be employed as expression constructs in the present disclosure.
  • Vectors derived from viruses such as vaccinia virus adeno-associated virus (AAV) and herpesviruses may be employed. They offer several attractive features for various mammalian cells.
  • AAV vaccinia virus adeno-associated virus
  • herpesviruses may be employed. They offer several attractive features for various mammalian cells.
  • the AAV vector is replication-defective or conditionally replication defective.
  • the AAV vector is a recombinant AAV vector.
  • the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof.
  • a single viral vector is used to deliver a nucleic acid encoding a Cas9 or a Cpfl and at least one gRNA to a cell.
  • Cas9 or Cpfl is provided to a cell using a first viral vector and at least one gRNA is provided to the cell using a second viral vector.
  • a single viral vector is used to deliver a nucleic acid encoding Cas9 or Cpfl and at least one gRNA to a cell.
  • Cas9 or Cpfl is provided to a cell using a first viral vector and at least one gRNA is provided to the cell using a second viral vector.
  • the expression construct must be delivered into a cell.
  • the cell may be a muscle cell, a satellite cell, a mesangioblast, a bone marrow derived cell, a stromal cell or a mesenchymal stem cell.
  • the cell is a cardiac muscle cell, a skeletal muscle cell, or a smooth muscle cell.
  • the cell is a cell in the tibialis anterior, quadriceps, soleus, diaphragm or heart.
  • the cell is an induced pluripotent stem cell (iPSC) or inner cell mass cell (iCM).
  • iPSC induced pluripotent stem cell
  • iCM inner cell mass cell
  • the cell is a human iPSC or a human iCM.
  • human iPSCs or human iCMs of the disclosure may be derived from a cultured stem cell line, an adult stem cell, a placental stem cell, or from another source of adult or embryonic stem cells that does not require the destruction of a human embryo.
  • Delivery to a cell may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states.
  • One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.
  • the nucleic acid encoding the gene of interest may be positioned and expressed at different sites.
  • the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.
  • a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them.
  • Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force.
  • the microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
  • the expression construct is delivered directly to the liver, skin, and/or muscle tissue of a subject. This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i. e. , ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present disclosure.
  • the expression construct may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated are lipofectamine-DNA complexes.
  • Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful.
  • a reagent known as Lipofectamine 2000TM is widely used and commercially available.
  • the liposome may be complexed with a hemagglutinating vims (HVJ) to facilitate fusion with the cell membrane and promote cell entry of liposome- encapsulated DNA.
  • HVJ hemagglutinating vims
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1).
  • HMG-1 nuclear non-histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a particular gene into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific.
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor- specific ligand and a DNA-binding agent.
  • ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (AS OR) and transferrin.
  • AS OR asialoorosomucoid
  • transferrin A synthetic neoglycoprotein, which recognizes the same receptor as AS OR, has been used as a gene delivery vehicle and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells.
  • EGF epidermal growth factor
  • Duchenne muscular dystrophy is a recessive X-linked form of muscular dystrophy, affecting around 1 in 5000 boys, which results in muscle degeneration and premature death.
  • the disorder is caused by a mutation in the gene dystrophin (see GenBank Accession NO. NC_000023.11), located on the human X chromosome, which codes for the protein dystrophin (GenBank Accession No. AAA53189; SEQ ID NO: 5).
  • dystrophin mRNA contains 79 exons.
  • Dystrophin mRNA is known to be alternatively spliced, resulting in various isoforms.
  • Exemplary dystrophin isoforms are listed in Table 1.
  • the murine dystrophin protein has the following amino acid sequence (Uniprot Accession No. P11531, SEQ ID. NO: 869):
  • Dystrophin is an important component within muscle tissue that provides structural stability to the dystroglycan complex (DGC) of the cell membrane. While both sexes can carry the mutation, females are rarely affected with the skeletal muscle form of the disease.
  • DGC dystroglycan complex
  • compositions and methods of the disclosure there is no current cure for DMD.
  • treatment was generally aimed at controlling the onset of symptoms to maximize the quality of life.
  • therapies may be used in combination with the compositions and methods of the disclosure and may include the following:
  • Corticosteroids such as prednisolone and deflazacort increase energy and strength and defer severity of some symptoms.
  • beta-2-agonists increase muscle strength but do not modify disease progression.
  • follow-up time for most RCTs on beta2-agonists is only around 12 months and hence results cannot be extrapolated beyond that time frame.
  • Orthopedic appliances may improve mobility and the ability for self-care.
  • Form-fitting removable leg braces that hold the ankle in place during sleep can defer the onset of contractures.
  • the examples disclose packaging Cas9 nuclease in conventional single-stranded AAV (ssAAV) and CRISPR single guide RNAs in double-stranded self-complementary AAV (scAAV) to deliver this dual AAV system into a mouse model of DMD harboring an exon 44 deletion.
  • the doses of scAAV required for efficient gene editing were 20- fold lower than with ssAAV.
  • Mice receiving systemic treatment showed restoration of dystrophin expression in all skeletal muscle groups and the heart, reduced DMD pathological phenotypes, and improved muscle contractility.
  • the present application discloses a strategy for permanent and efficient correction of mutations in the endogenous DMD gene that may provide an ultimate cure for DMD.
  • application of the CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR- associated proteins) system for engineering site-specific DNA double- stranded breaks (DSB) provides simplicity and precision in mammalian genome editing.
  • the CRISPR-Cas system can be used to efficiently correct missense mutations in subjects of DMD by homology directed repair (HDR)-based germline editing or non- homologous end joining (NHEJ)-based postnatal editing.
  • HDR homology directed repair
  • NHEJ non- homologous end joining
  • CRISPR-Cas system can be deployed to correct diverse genetic mutations that cause DMD and offer the prospect of a potential gene therapy for the permanent correction of DMD.
  • Recombinant AAV is a nonenveloped virus with a single- stranded linear DNA viral genome.
  • AAV 5.5 x 10 14 to 1.8 x 10 15 vg/kg
  • systemic administration of high doses of AAV may cause acute liver toxicity.
  • the efficiency of in vivo CRISPR/Cas9- mediated genome editing may be, in some embodiments, highly dose-dependent, and elevating the dose of sgRNA AAV relative to Cas9 AAV may enhance the efficiency of genome editing.
  • elevating the dose of sgRNA AAV relative to Cas9 AAV may enhance the efficiency of genome editing.
  • the sgRNA AAV genome is preferentially depleted after systemic delivery of CRISPR-Cas9 genome editing components.
  • sgRNA AAV self-complementary AAV
  • recombinant AAV was chosen as a delivery vector for use with the compositions and methods disclosed herein.
  • the Cas9 nuclease is encoded by conventional ssAAV while sgRNAs are expressed by double-stranded scAAV.
  • scAAV- expressed sgRNAs (4 x 10 12 vg/kg) is sufficient to restore 18%, 14% and 50% of dystrophin protein in TA, triceps and diaphragm, respectively, representing a 20-fold improvement in efficiency compared with the ssAAV-packaged sgRNA vector.
  • the scAAV undergoes concatemerization more rapidly than ssAAV because scAAV-based concatemerization bypasses second-strand synthesis, which is a rate-limiting step for ssAAV.
  • scAAV monomeric viral genome degradation is significantly slower in scAAV transduced skeletal muscle compared with ssAAV. Therefore, scAAV is more stable than ssAAV during initial viral transduction, leading to higher episomal persistence in the long-term.
  • DNA DSBs in post-mitotic cells are repaired by the classical NHEJ pathway, which requires the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs).
  • DNA-PKcs is required for AAV viral genome concatemerization in AAV transduced skeletal muscle.
  • scAAV-packaged sgRNA leads to a higher incidence of DNA DSB at the target site. This may induce higher DNA-PKcs expression, which in turn facilitates AAV concatemerization and long-term gene expression.
  • higher viral genome persistence of both sgRNA vector and Cas9 vector was found in mice treated with scAAV-packaged sgRNA.
  • the scAAV-sgRNA delivery system has many appealing features, including stable persistence of AAV viral genomes, higher INDEL frequency at the targeted genomic locus, and highly efficient genome editing in vitro and in vivo at low viral dose.
  • the CRISPR/Cas9-mediated “single-cut” repair strategy has unique advantages, including predictable DNA repair outcome, minimum genomic modification at a precise location, and low frequency of off-target effects.
  • a low dose of scAAV-delivered CRISPR-Cas genome editing components is sufficient to restore dystrophin protein expression, reduce DMD pathological phenotypes, and improve muscle function in a DMD mouse model. Therefore, this robust scAAV delivery system combined with the efficient CRISPR-Cas9 genome editing technology represents a promising therapy for permanent correction of diverse genetic mutations in neuromuscular diseases.
  • upper case letters represent nucleotides that align to the exon sequence of the gene.
  • Lower case letters represent nucleotides that align to the intron sequence of the gene.
  • Table 8 sgRNA spacers targeting human Dmd Exon 51
  • Table 9 sgRNA spacers targeting sites in various human Dmd Exons
  • Table 13 Exon 43 & 45 gRNA spacer sequences
  • Table 14 Sequence of primers for cloning sgRNA spacers targeting DMD and Dmd exon 45 splicing acceptor site
  • the sgRNA targeting mouse Dmd exon 45 was first cloned into a modified TRISPR-sgRNA-CK8e-GFP plasmid, using Golden Gate Assembly (New England Biolabs).
  • the sgRNA expression cassette containing three copies of same sgRNA driven by the U6, HI, and 7SK promoter was PCR amplified and subcloned into the pSJG selfcomplementary AAV plasmid (scAAV plasmid), or into the pSSV9 single-stranded AAV plasmid (ssAAV plasmid), using In-Fusion Cloning Kit (Takara Bio).
  • a 2.3 kb staffer sequence was cloned into the ssAAV plasmid for optimal viral packaging.
  • Both the scAAV and the ssAAV genome contain the same sgRNA expression cassette, consisting of three copies of sgRNA sequence driven by three RNA polymerase III promoters.
  • the sequence of the sgRNA spacer sequence used was ggcttacagGAACTCCAGGA (SEQ ID NO: 2668).
  • Cloning primers are listed in FIG 3A. All AAV viral plasmids were column purified and digested with Sma I and Ahd I to check ITR integrity. AAVs were packaged by Boston Children’s Hospital Viral Core and serotype 9 was chosen for capsid assembly.
  • AAV titers were determined by Droplet Digital PCR (ddCPR) (Bio-Rad Laboratories) according to the manufacturer’s protocol. Primers and probes used for titration are listed in FIG. 3A. Alkaline agarose gel electrophoresis
  • AAV virus (2 x 10 11 vg) was equalized with water to 13 m ⁇ and digested with 10 m ⁇ DNase solution (lOmM Tris-HCl, pH 7.5, lOmM CaCh, lOmM MgCh, O.lmg/ml DNase) at 37°C for 1 hour, followed by chelating Mg 2+ and Ca 2+ with 5 m ⁇ 0.5M EDTA. Then the capsid was denatured by adding 2 m ⁇ 10% SDS. The reaction mixture was mixed with 6 ul 6X alkaline agarose gel loading dye (Alfa Aesar) and loaded into 1% alkaline agarose gel.
  • 6 ul 6X alkaline agarose gel loading dye Alfa Aesar
  • Denaturing gel electrophoresis was performed in a cold room at 50 V for 15 hours.
  • the gel was neutralized with neutralization buffer (0.5 M Tris-HCl pH 7.5, 1M NaCl) and stained with SYBR Gold (Thermo Fisher Scientific) for visualization.
  • Cas9-expressing C2C12 myoblasts were cultured in 96-well dishes with growth medium (DMEM with 10% FBS) until reaching 90% confluency. Then, the myoblasts were allowed to differentiate in myotube in differentiation medium (DMEM with 2% horse serum) for 5 days. Two-hours before viral transduction, myotubes were treated with Vibrio cholerae Neuraminidase (50 mu/ml) (Sigma- Aldrich), followed by washing with differentiation medium twice. Myotubes were incubated with varying doses of scAAV or ssAAV and centrifuged at 1,000 x g at 4°C for 1.5 hour. After spin transduction, the virus was aspirated and the myotubes were washed with differentiation medium three times. The myotubes were cultured in differentiation medium for an additional week prior genomic DNA isolation for INDEL analysis.
  • Genomic DNA of mouse C2C12 myotubes, skeletal muscles and heart was isolated using DirectPCR (cell) lysis reagent (Viagen Biotech) according to the manufacturer’s protocol.
  • Total RNA of skeletal muscles and heart was isolated using miRNeasy (QIAGEN) according to the manufacturer’s protocol.
  • cDNA was reverse-transcribed from total RNA using Superscript III First-Strand Synthesis SuperMix (Thermo Fisher Scientific) according to the manufacturer’s protocol. Primer sequences are listed in FIG. 3A.
  • INDEFs in genomic DNA and cDNA were analyzed using Tracking of INDEFs by Decomposition (TIDE) software package (deskgen.com). Briefly, the sgRNA sequence targeting mouse Dmd exon 45 was first uploaded to the software to define SpCas9-mediated DSB site. Then, the CRISPR/Cas9-edited sequence and non-edited control sequence were uploaded and aligned using Smith-Waterman local alignment algorithm. The percentage of INDEFs was calculated based on the relative abundance of aberrant nucleotides over the length of the whole sequence trace.
  • TIDE Tracking of INDEFs by Decomposition
  • PCR of genomic DNA was performed using primers designed against the DMD exon 45 region. A second round of PCR was performed to add Illumina flow cell binding sequence and barcodes. All primer sequences are listed in FIG. 3B. Deep sequencing analysis was performed.
  • the AAV viral genome copy number was determined by quantitative PCR using custom-designed primers (FIG. 3A).
  • the primer sets used in AAV-sgRNA and AAV-Cas9 viral genome quantification anneal to the 7SK promoter and Cas9 gene, respectively.
  • the threshold cycle value of each reaction was converted to the viral genome copy number by measuring against the copy number standard curve of the AAV plasmids used for AAV packaging in this study.
  • Mouse 18S ribosomal RNA gene was used as the reference gene to calibrate genomic DNA quantity.
  • EXAMPLE 2 sgRNA design and scAAV production
  • exon 44 of the human DMD gene generates a premature stop codon in exon 45 and represents one of most common mutations of DMD.
  • a sgRNA was designed to target the splice acceptor region of exon 45 (FIG. 1A). This sgRNA recognizes a 5’-TGG-3’ protospacer adjacent motif (PAM) in exon 45 and generates insertions and deletions (INDELs) 7 base pairs (bp) downstream of the 5 ’-AG-3’ splice acceptor site (FIG. IB).
  • PAM protospacer adjacent motif
  • NHEJ-mediated DNA repair events can restore the open reading frame (ORF) of the Dmd gene. These include exon 45 skipping, if the INDEL is large enough to delete the 5’ -AG-3’ splice acceptor sequence in exon 45, or reframing of exon 45 through INDELs that either insert one nucleotide (3n+l) or delete two nucleotides (3n-2) (FIG. 1C).
  • sgRNA expression cassette was cloned into a scAAV vector and the conventional ssAAV vector as a control (FIG. 1C). Alkaline denaturing gel electrophoresis was performed to confirm the integrity of both AAVs (FIG. 2).
  • the size of ssAAV-sgRNA (SEQ ID NO: 2598) is 3.9 kilo- nucleotides (knt) and remains unchanged after alkaline gel electrophoresis.
  • scAAV- sgRNA SEQ ID NO: 2597
  • SEQ ID NO: 2597 The size of scAAV- sgRNA (SEQ ID NO: 2597) is 1.4 kilo-base pairs and is doubled to 2.8 knts under denaturing conditions, indicative of the double- stranded viral genome. Primers and probes used in this example are shown in FIG. 3.
  • EXAMPLE 3 In vitro genome editing using ssAAV or scAAV-packaged sgRNA
  • INDELs required 5 x 10 8 vg/mL of scAAV and 1 x 10 10 vg/mL of ssAAV, representing a 20-fold increase in efficiency of scAAV.
  • INDELS an intermediate level of INDELS of -22%, 40-fold less scAAV (1.8 x 10 9 vg/mL) was required compared to ssAAV (7.8 x 10 10 vg/mL).
  • INDELs over 40%
  • ssAAV required 5 x 10 12 vg/mL, representing a 70-fold improvement in efficiency with scAAV.
  • the INDEL composition in myotubes transduced with ssAAV or scAAV was analyzed, and it was found that -50% of total INDEL events contained a +1 nt insertion, which can bring exon 45 in- frame with exon 43 (FIG. 4B).
  • scAAV- expressed sgRNA demonstrated enhanced efficiency by in vitro genome editing at Dmd exon 45 compared to the conventional ssAAV-expressed sgRNA. Moreover, the majority of the INDEL events (over 50%) contained a single nt insertion, which is able to restore the Dmd exon 45 ORF.
  • EXAMPLE 4 Systemic delivery of scAAV-packaged sgRNAs restores dystrophin expression in AEx44 mice
  • ssAAV-packaged SpCas9 and scAAV or ssAAV -packaged sgRNA was delivered systemically in DEc44 mice through intraperitoneal (IP) injection.
  • IP intraperitoneal
  • the AAV9 serotype was chosen because of its tropism to skeletal muscle and heart.
  • SpCas9 expression was driven by a muscle specific promoter containing key regulatory elements derived from creatine kinase promoter and enhancer, restricting its expression to striated muscles.
  • AAV -packaged sgRNA is the rate limiting factor for in vivo genome editing in dystrophic mouse models.
  • ssAAV-packaged SpCas9 was kept at a constant dose of 8 x 10 13 vg/kg while titrating scAAV or ssAAV-packaged sgRNA at multiple doses.
  • skeletal muscles and heart of CRISPR/Cas9-edited DEc44 mice were harvested for analysis. By immunohistochemistry, it was found that dystrophin restoration in skeletal muscles was dose-dependent (FIG. 5 and FIG. 6).
  • mice receiving lowest dose of ssAAV-packaged sgRNA (4 x 10 12 vg/kg) showed less than 5% dystrophin-positive myofibers in TA and triceps; diaphragm and heart showed 52% and 61% dystrophin-positive myocytes, respectively (FIG. 6 and FIG. 7B).
  • dose of scAAV-packaged sgRNA was increased to 1.6 x 10 13 vg/kg, virtually all myofibers and cardiomyocytes were dystrophin-positive (FIG. 5 and FIG. 7A).
  • mice receiving the highest dose of ssAAV (8 x 10 13 vg/kg) showed only 13%, 16% and 30% of normal dystrophin protein levels in TA, triceps and diaphragm, respectively (FIG. 10A-C and FIG. 11).
  • EXAMPLE 5 Systemic delivery of scAAV-packaged sgRNAs restores muscle integrity and improves muscle function in AEx44 mice
  • H&E hematoxylin and eosin staining of skeletal muscles and heart isolated from DEc44 mice was performed four weeks after CRISPR/Cas9-mediated genome editing.
  • mice receiving the same dose of ssAAV-packaged sgRNA still showed over 70% of regenerating myofibers with central nuclei, together with signs of muscle necrosis and inflammatory infiltration (FIG. 13 and FIG. 14B).
  • skeletal muscles isolated from mice receiving the highest dose of scAAV-packaged sgRNA (8 x 10 13 vg/kg) were virtually indistinguishable from those of wild- type (WT) littermates, whereas the ssAAV-treated cohort still showed 30% central nuclei in the TA and triceps (FIGS. 12-14).
  • muscle-specific force of the EDL was increased from 54% to 83% and to 82% for the middle and high doses; in contrast, for the ssAAV-treated cohort, muscle- specific force of the EDL was only increased from 54% to 62% and to 66% for the middle and high doses (FIG. 15A).
  • muscle-specific force was increased from 58% to 93% and to 96% after receiving the middle and high doses of scAAV-packaged sgRNA; in contrast, for the ssAAV-treated cohort, only high dose treatment was able to improve muscle-specific force of the soleus to 85%, while no improvement was observed with the middle dose (FIG.
  • the maximal tetanic force of the soleus was improved to over 90% of WT after receiving the middle or high doses of scAAV-packaged sgRNA; high dose of ssAAV- packaged sgRNA improved maximal tetanic force of the soleus to 85% of WT, while the middle dose did not provide any improvement (FIG. 15D).
  • serum creatine kinase (CK) levels in the DEc44 mice were reduced by 87% and 95%, respectively, compared with DEc44 mice without treatment (FIG. 16).
  • EXAMPLE 6 The scAAV system induces significant TNDELs within Dmd exon 45 and maintains higher copies of the viral genome in vivo
  • TIDE analysis was also performed on dystrophin cDNA transcripts isolated from skeletal muscles and heart.
  • Total cDNA INDEL rate and +1 nt insertion events at exon 45 followed similar ascending patterns seen in the genomic TIDE analysis while the absolute percentage increased significantly (FIG. 17B), indicating enrichment of the reframed cDNA transcript after nonsense-mediated decay of unedited transcript with a premature stop codon in exon 45.
  • DMD Duchenne muscular dystrophy
  • CRISPR-Cas9 corrects Duchenne muscular dystrophy exon 44 deletion mutations in mice and human cells. Sci Adv 5, eaav4324 (2019).

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Abstract

CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. Here, CRISPR/Cas9 compositions and methods for the treatment and/or prevention of genetic diseases are provided.

Description

DESCRIPTION
SELF-COMPLEMENTARY AAV DELIVERY SYSTEM FOR CRISPR/CAS9
PRIORITY CLAIM
[0001] This application claims benefit of priority to U.S. Provisional Application Serial No. 62/956,726, filed January 3, 2020, the entire contents of which are hereby incorporated by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant numbers HL 130253 and AR-067294 awarded by the NIH. The government has certain rights in the invention.
INCORPORATION OF THE SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 28, 2020, is named UTFDP3499WO_ST25.txt and is 1,337 kilobytes in size.
FIELD OF THE DISCLOSURE
[0004] The present disclosure relates to the fields of molecular biology, medicine and genetics. More particularly, the disclosure relates to compositions and uses thereof for genome editing to correct mutations in vivo.
BACKGROUND
[0005] With recent advancements in gene editing technologies, there is great promise for treating human genetic diseases using in vivo corrections of defective genes. For example, CRISPR/Cas systems can be used to induce double-strand DNA breaks at a specific locus in the genome, which are resolved by endogenous DNA repair mechanisms.
[0006] Safe and effective delivery of gene editing machinery into the nucleus of the desired cells is essential for therapeutic gene editing. Ideally, a delivery system should (1) target specific tissues or cells, (2) effectively enter cells, (3) avoid activation of the immune system, and (4) prevent off-target events. There remains a need in the art for safe and effective systems to deliver gene editing machinery. [0007] Provided herein are self-complementary AAV delivery vectors, and compositions comprising the same, for delivery of gene editing machinery to cells in vitro and in vivo.
SUMMARY
[0008] Despite intense research efforts, there remains no cure for many genetic diseases, including muscular dystrophy (DMD). The instant disclosure provides self-complimentary adeno-associated virus (AAV) delivery systems comprising sequences encoding a guide RNA and a CRISPR nuclease, e.g. , a Cas9. The AAV delivery systems disclosed herein may be used, for example, for disrupting a dystrophin splice acceptor site and inducing skipping and/or refraining of an exon of a DMD gene, thereby modifying a DMD gene in a cell or a subject. Compositions and methods according to the disclosure may be used to treat and/or prevent muscular dystrophy.
[0009] In some embodiments, the disclosure provides an AAV expression cassette comprising, from 5’ to 3’, a first promoter, a sequence encoding a first sgRNA comprising a first spacer region and a first scaffold region, a second promoter, a sequence encoding a second sgRNA comprising a second spacer region and a second scaffold region, a third promoter, and a sequence encoding a third gRNA comprising a third sgRNA targeting region and third scaffold region, wherein the expression cassette is flanked by a first inverted terminal repeat (ITR) and a second ITR, wherein the first ITR has the sequence of SEQ ID NO: 2584, wherein the second ITR has the sequence of SEQ ID NO: 2583, and wherein the AAV expression cassette is self-complimentary ( i.e ., a scAAV).
[0010] In some embodiments, the first promoter is a U6 promoter. The U6 promoter may have the sequence of SEQ ID NO: 2589, or a sequence at least 95% identical thereto. In some embodiments, the second promoter is the HI promoter. The HI promoter may have the sequence of SEQ ID NO: 2586, or a sequence at least 95% identical thereto. In some embodiments, the third promoter is the 7SK promoter. The 7SK promoter may have the sequence of SEQ ID NO: 2587, or a sequence at least 95% identical thereto.
[0011] In some embodiments, each of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are the same. In some embodiments, the at least two of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are different. In some embodiments, each of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are different. [0012] In some embodiments, at least one of the first spacer region, the second spacer region, and the third spacer region targets the human dystrophin gene. In some embodiments, the sequences of first spacer region, the second spacer region, and the third spacer region are each independently selected from any one of SEQ ID NO: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036-1043, 1052-1063, 1494-1499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668. In some embodiments, the sequences encoding the first, the second, and the third spacer regions are the same, and the sequences are each SEQ ID NO: 2668.
[0013] In some embodiments, the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are the same. In some embodiments, at least two of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are different. In some embodiments, each of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are different. In some embodiments, at least one of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region has the sequence of SEQ ID NO: 2672, or a sequence at least 95% identical thereto.
[0014] In some embodiments, the expression cassette further comprises a fourth sgRNA comprising a fourth spacer region and a fourth scaffold region. In some embodiments, the expression cassette further comprises a fifth sgRNA comprising a fifth spacer region and a fifth scaffold region.
[0015] In some embodiments, the expression cassette has a length less than about 2 kb, less than about 1.8 kb, less than about 1.6 kb, or less than about 1.4kb.
[0016] Also provided herein is an AAV expression cassette comprising, from 5’ to 3’, a first ITR, a promoter, a sequence encoding a Cas9 nuclease, and a second ITR. In some embodiments, the first ITR has the sequence of SEQ ID NO: 2585, wherein the second ITR has the sequence of SEQ ID NO: 2679, wherein the AAV expression cassette is not selfcomplimentary. In some embodiments, the promoter is a CK8e promoter. In some embodiments, the promoter has the sequence of SEQ ID NO: 2590, or a sequence at least 95% identical thereto. In some embodiments, the sequence encoding the Cas9 nuclease is derived from S. aureus or S. pyogenes. In some embodiments, the sequence encoding the Cas9 nuclease comprises SEQ ID NO: 2591, or a sequence at least 95% identical thereto. In some embodiments, the expression cassette further comprises a sequence encoding a PolyA tail. In some embodiments, the PolyA tail comprises a sequence of SEQ ID NO: 2593, or a sequence at least 95% identical thereto.
[0017] Also provided herein is a composition comprising an AAV expression cassette of the disclosure.
[0018] Also provided herein is a vector comprising the AAV expression cassette of the disclosure. In some embodiments, the vector is a non- viral vector, such as a plasmid. In some embodiments, the vector is a viral vector, such as an AAV vector. In some embodiments, the AAV vector is a self-complimentary AAV (scAAV). In some embodiments, the AAV vector is a recombinant AAV (rAAV). In some embodiments, the AAV vector comprises a capsid protein isolated or derived from an AAV vector of serotype 9 (AAV9). In some embodiments, the AAV vector comprises a wild type AAV9 capsid protein.
[0019] Also provided herein is a composition comprising one or more vectors of the disclosure. In some embodiments, the composition comprises a pharmaceutically acceptable carrier or excipient.
[0020] The disclosure additionally provides a recombinant AAV comprising a capsid protein, and an AAV expression cassette of the disclosure encapsidated by the capsid protein. In some embodiments, the capsid protein is isolated or derived from a wild type AAV capsid of one or more of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the capsid protein is isolated or derived from an AAV9 capsid.
[0021] Also provided herein is a method of producing an AAV vector comprising contacting a vector comprising the AAV expression cassette of the disclosure with an AAV host cell. The AAV host cell may be a mammalian cell, such as a HEK293 cell or modified HEK293 cell. In some embodiments, the AAV host cell is an insect cell, such as a Sf9 cell or modified Sf9 cell.
[0022] The disclosure additionally provides a method of correcting a gene defect in a cell, the method comprising contacting the cell with an AAV vector comprising an AAV expression cassette of the disclosure, wherein the expression cassette comprises a sequence encoding a least one sgRNA comprising a first spacer region and a first scaffold region. In some embodiments, the cell is a human cell. In some embodiments, the gene defect is a gene defect in the dystrophin gene. In some embodiments, the method also comprises contacting the cell with an AAV vector comprising an expression cassette for a Cas9 nuclease. In some embodiments, the expression cassette for the Cas9 nuclease is not self-complimentary. In some embodiments, the expression cassette for the Cas9 nuclease is an AAV expression cassette of the disclosure.
[0023] Also provided is a method of treating a subject in need thereof, the method comprising administering to the patient a therapeutically effective amount of a vector or a composition of the disclosure, wherein the subject suffers from Duchenne Muscular Dystrophy (DMD).
[0024] Also provided is a method of treating a subject in need thereof comprising administering to the subject a first AAV vector comprising an AAV expression cassette comprising a sequence encoding at least one sgRNA comprising a first spacer region and a first scaffold region. In some embodiments, the subject is a human. In some embodiments, the subject suffers from Duchenne Muscular Dystrophy (DMD). In some embodiments, the method also comprises administering to the subject a second AAV vector comprising an expression cassette for a Cas9 nuclease. In some embodiments, the expression cassette for the Cas9 nuclease is not self-complimentary. In some embodiments, dystrophin expression is at least partially restored in skeletal muscle in the patient. In some embodiments, dystrophin expression is at least partially restored in heart muscle in the patient. In some embodiments, the dosage of the first AAV required to at least partially restore dystrophin expression is at least about 20-fold lower than the dosage that would be required to achieve the same level of dystrophin expression if the expression cassette of the first AAV was not self-complimentary.
[0025] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0027] FIG. 1A-C. Strategies for CRISPR/Cas9-mediated genome editing in Dmd exon 45. (FIG. 1A) An out-of-frame deletion of Dmd exon 44 results in splicing of exon 43 to 45, generating a premature stop codon in exon 45. A CRISPR/Cas9-mediated “single-cut” strategy was designed to restore the open reading frame (ORF) of the Dmd gene. If the genomic insertions and deletions (INDEL) produced as a result of the CRISPR/Cas9 mediated cleavage and subsequent repair is one nucleotide insertion (3n+l) or two nucleotides deletion (3n-2), exon 45 will be reframed with adjacent exon 43 and 46. If the INDEL is large enough to delete the 5 ’-AG-3’ splice acceptor sequence, exon 45 will be skipped, resulting in splicing of exon 43 to 46. (FIG. IB) Illustration of sgRNA targeting Dmd exon 45. This sgRNA recognizes a 5 ’ -TGG-3 ’ PAM in exon 45 and generates a cut 7 base pairs downstream of the 5 ’ - AG-3 ’ splice acceptor site. FIG. IB provides SEQ ID NOs: 2733 and 2734. (FIG. 1C) Illustration of AAV vectors used to deliver the sgRNA expression cassette. Three copies of the same sgRNA are driven by three RNA polymerase III promoters, U6, HI, and 7SK. The top vector produces ssAAV. A 2.3kb staffer sequence was cloned into the ssAAV vector for optimal packaging. The bottom vector produces double-stranded scAAV.
[0028] FIG. 2. Alkaline denaturing gel electrophoresis confirms integrity of AAV vectors. The viral genomes of the Cas9 vector and sgRNA vectors were analyzed by gel electrophoresis under alkaline denaturing conditions. The size of ssAAV-sgRNA and ssAAV-Cas9 is 3.9 and 5.1 kilobases, respectively, and remains unchanged after alkaline gel electrophoresis. The size of scAAV-sgRNA is 1.4 kilobases and is doubled to 2.8 kilobases under denaturing conditions, indicating its double-stranded viral genome. M, marker; knt, kilo-nucleotides.
[0029] FIG. 3A-3B. Primers used throughout experiments and probes used for titration. FIG. 3A provides SEQ ID NOs: 2648 to 2671, and FIG. 3B provides SEQ ID NOs: 2681 to 2684.
[0030] FIG. 4A-B. (FIG. 4 A) Analysis of total INDEL event in 5 days differentiated myotubes transduced with scAAV or ssAAV-packaged sgRNA at multiple doses. Data are represented as mean ± SEM (n = 3). (FIG. 4B) Analysis of +1 nt insertion event in 5 days differentiated myotubes transduced with sc AAV or ssAAV-packaged sgRNA at multiple doses. Data are represented as mean ± SEM (n = 3).
[0031] FIG. 5. Systemic AAV delivery of CRISPR/Cas9 genome editing components to DEc44 mice rescues dystrophin expression. Immunohistochemistry shows restoration of dystrophin in tibialis anterior (TA), triceps, diaphragm, and heart of DEc44 mice 4 weeks after systemic delivery of ssAAV-packaged SpCas9 and scAAV-packaged sgRNA. SpCas9 vector was kept at constant dose of 8 x 1013 vg/kg. The dose of sgRNA vector was shown in the figure. Dystrophin is shown in green, n = 5 for each muscle group. Scale bar, 100 pm.
[0032] FIG. 6. Systemic delivery of CRISPR/Cas9 genome editing components by single- stranded AAV vector to DEc44 mice rescues dystrophin expression. Immunohistochemistry shows restoration of dystrophin in tibialis anterior (TA), triceps, diaphragm, and heart of DEc44 mice 4 weeks after systemic delivery of ssAAV-packaged SpCas9 and ssAAV- packaged sgRNA. SpCas9 vector was kept at constant dose of 8 x 1013 vg/kg. The dose of sgRNA vector is shown in the figure. Dystrophin is shown in green, n = 5 for each muscle group. Scale bar, 100 pm.
[0033] FIG. 7A-B. Whole muscle scanning of immunohistochemistry of TA, triceps, diaphragm, and heart from CRISPR/Cas9-corrected DEc44 mice. Both panels show whole muscle scanning of TA, triceps, diaphragm, and heart from DEc44 mice 4 weeks after systemic delivery of ssAAV-packaged SpCas9 and scAAV-packaged sgRNA (FIG. 7A) or ssAAV- packaged sgRNA (FIG. 7B). SpCas9 vector was kept at constant dose of 8 x 1013 vg/kg. The dose of sgRNA vector was shown in the figure. Dystrophin is shown in green, n = 5 for each muscle group. Scale bar in TA, triceps, diaphragm is 500 μm, in heart is 1.5mm.
[0034] FIG. 8A-D. Western blot analysis of skeletal muscles and heart from DEc44 mice receiving systemic AAV delivery of CRISPR/Cas9 genome editing components. Western blot analysis shows restoration of dystrophin expression in the TA (FIG. 8A), triceps (FIG. 8B), diaphragm (FIG. 8C), and heart (FIG. 8D) of DEc44 mice 4 weeks after systemic delivery of ssAAV-packaged SpCas9 and scAAV-packaged sgRNA. SpCas9 vector was kept at constant dose of 8 x 1013 vg/kg. The dose of sgRNA vector is shown in the figure. Vinculin was used as the loading control (n = 3).
[0035] FIG. 9A-B. Quantitative western blot analysis of skeletal muscles and heart from DEc44 mice receiving systemic AAV delivery of CRISPR/Cas9 genome editing components. (FIG. 9A) Quantification of dystrophin expression in TA, triceps, diaphragm, and heart. Relative dystrophin intensity was calibrated with vinculin internal control before normalizing to the WT control. Data are represented as mean ± SEM. One-way ANOVA was performed with post-hoc Tukey’s multiple comparisons test. **P<0.005, ***P<0.001, ****P<0.0001 (n=3). (FIG. 9B) Quantification of Cas9 expression in TA, triceps, diaphragm, and heart. Relative Cas9 intensity was calibrated with vinculin internal control before normalizing to the group treated with lowest dose of scAAV-packaged sgRNA (4 x 1012 vg/kg). Data are represented as mean ± SEM. One-way ANOVA was performed with post-hoc Tukey’s multiple comparisons test. *P<0.05, **P<0.005 (n=3).
[0036] FIG. 10A-D. Western blot analysis of skeletal muscles and heart from DEc44 mice treated with ssAAV-packaged CRISPR/Cas9 genome editing components. Western blot analysis shows restoration of dystrophin expression in the TA (FIG. 10A), triceps (FIG. 10B), diaphragm (FIG. IOC), and heart (FIG. 10D) of DEc44 mice 4 weeks after systemic delivery of ssAAV-packaged SpCas9 and ssAAV-packaged sgRNA. SpCas9 vector was kept at constant dose of 8 x 1013 vg/kg. The dose of sgRNA vector was shown in the figure. Vinculin was used as the loading control (n = 3). Red asterisks indicates non-specific band.
[0037] FIG. 11. Quantitative western blot analysis of skeletal muscles and heart from DEc44 mice treated with ssAAV-packaged CRISPR/Cas9 genome editing components. Quantification of dystrophin expression in TA, triceps, diaphragm, and heart. Relative dystrophin intensity was calibrated with vinculin internal control before normalizing to the WT control. Data are represented as mean + SEM. One-way ANOVA was performed with post-hoc Tukey’s multiple comparisons test. *P<0.05, **P<0.005, ***P<0.001, ****P<0.0001 (n=3).
[0038] FIG. 12. Muscle histology of AEx44 mice after systemic delivery of scAAV expressing CRISPR/Cas9 genome editing components. H&E staining of TA, triceps, diaphragm, and heart of DEc44 mice 4 weeks after systemic delivery of ssAAV-packaged SpCas9 and scAAV- packaged sgRNA. SpCas9 vector was kept at constant dose of 8 x 1013 vg/kg. The dose of sgRNA vector was shown in the figure, n = 5 for each muscle group. Scale bar, 100 pm.
[0039] FIG. 13. Muscle histology of AEx44 mice after systemic delivery of ssAAV expressing CRISPR/Cas9 genome editing components. As in FIG. 12, FIG. 13 shows H&E staining of TA, triceps, diaphragm, and heart of DEc44 mice 4 weeks after systemic delivery but for ssAAV-packaged SpCas9 and ssAAV-packaged sgRNA. SpCas9 vector was kept at constant dose of 8 x f013 vg/kg. The dose of sgRNA vector was shown in the figure, n = 5 for each muscle group. Scale bar, 100 pm. [0040] FIG. 14A-B. Whole muscle scanning of H&E staining of TA, triceps, diaphragm, and heart from CRISPR/Cas9-corrected DEc44 mice. Whole muscle scanning of H&E staining of TA, triceps, diaphragm, and heart from DEc44 mice 4 weeks after systemic delivery of ssAAV- packaged SpCas9 and scAAV-packaged sgRNA (FIG. 14A) or ssAAV-packaged sgRNA (FIG. 14B). SpCas9 vector was kept at constant dose of 8 x 1013 vg/kg. The dose of sgRNA vector was shown in the figure, n = 5 for each muscle group. Scale bar in TA, triceps, diaphragm is 500 μm, in heart is 1.5mm.
[0041] FIG. 15A-D. Rescue of skeletal muscle function after systemic AAV delivery of CRISPR/Cas9 genome editing components. Specific force (mN/mm2) of the extensor digitorum longus (EDL) (FIG. 15A) and soleus (FIG. 15B) muscles in WT, AFx44 mice untreated, and DEc44 mice treated with ssAAV-packaged SpCas9 and scAAV or ssAAV- packaged sgRNA. SpCas9 vector was kept at constant dose of 8 x 1013 vg/kg. The dose of sgRNA vector was shown in the figure. Data are represented as mean ± SEM. One-way ANOVA was performed with post-hoc Tukey’s multiple comparisons test. *P<0.05, **P<0.005, ***P<0.001, ****P<0.0001 (n=6). Maximal tetanic force of the EDL (FIG. 15C) and soleus (FIG. 15D) muscles in WT, DEc44 untreated mice, and DEc44 mice treated with ssAAV-packaged SpCas9 and scAAV or ssAAV-packaged sgRNA. SpCas9 vector was kept at constant dose of 8 x 1013 vg/kg. The dose of sgRNA vector was shown in the figure. (n=6).
[0042] FIG. 16. Serum creatine kinase (CK) analysis of CRISPR/Cas9-corrected DEc44 mice. Serum CK was measured in WT, DEc44 mice untreated, and DEc44 mice 4 weeks after treatment with ssAAV-packaged SpCas9 and scAAV or ssAAV-packaged sgRNA. SpCas9 vector was kept at constant dose of 8 x 1013 vg/kg. The dose of sgRNA vector was shown in the figure. Serum CK was normalized to WT mice and shown as fold expression. Data are represented as mean ± SEM. One-way ANOVA was performed with post-hoc Tukey’s multiple comparisons test. **P<0.005, ****P<0.0001 (n=5).
[0043] FIG. 17A-B. scAAV vector induces significant INDELs at genomic and cDNA level. Genomic INDEL analysis (FIG. 17A) and Dystrophin cDNA INDEL analysis (FIG. 17B) of TA, triceps, diaphragm, and heart from DEc44 mice 4 weeks after systemic delivery of ssAAV- packaged SpCas9 and scAAV or ssAAV-packaged sgRNA. SpCas9 vector was kept at constant dose of 8 x 1013 vg/kg. The dose of sgRNA vector was shown in the figure. Data are represented as mean ± SEM. Two-way ANOVA was performed with post-hoc Tukey’s multiple comparisons test. *P<0.05, **P<0.005, ****P<0.0001 for total NHEJ event (n=3). ###P<0.001, ####P<0.0001 for +1 nt insertion event (n=3). [0044] FIG. 18A-B. DEc44 mice sustain higher copies of viral genome after systemic delivery of scAAV-packaged sgRNA. sgRNA viral genome copy number (FIG. 18 A) and Cas9 viral genome copy number (FIG. 18B) quantification from skeletal muscles and heart from AFx44 mice 4 weeks after systemic delivery of ssAAV-packaged SpCas9 and scAAV or ssAAV- packaged sgRNA. SpCas9 vector was kept at constant dose of 8 x 1013 vg/kg. The dose of sgRNA vector was shown in the figure. Data are represented as mean ± SEM. One-way ANOYA was performed with post-hoc Tukey’s multiple comparisons test. *P<0.05, **P<0.005, ****P<0.0001 (n=3).
[0045] FIG. 19A-B. DEc44 mice express more sgRNA and Cas9 transcripts after systemic delivery of scAAV-packaged sgRNA. sgRNA cDNA transcripts (FIG. 19A) and Cas9 cDNA transcripts (FIG. 19B) were detected by quantitative RT-PCR from skeletal muscles and heart from DEc44 mice 4 weeks after systemic delivery of ssAAV-packaged SpCas9 and scAAV or ssAAV-packaged sgRNA. SpCas9 vector was kept at constant dose of 8 x 1013 vg/kg. The dose of sgRNA vector was shown in the figure. Both sgRNA (FIG. 19A) and Cas9 (FIG. 19B) cDNA transcripts were calibrated with 18s internal control before normalizing to the muscle group with lowest sgRNA transcripts. In both analyses, TA muscle treated with a low dose of ssAAV- packaged sgRNA (1.6 x 1013 vg/kg) had the lowest sgRNA and Cas9 cDNA transcripts compared to other muscle groups. Data are represented as mean ± SEM. One-way ANOVA was performed with post-hoc Tukey’s multiple comparisons test. **P<0.005, ***P<0.001, ****P<0.0001 (n=3).
[0046] FIGS. 20A-B. Deep sequencing analysis performed to determine INDEL frequency at the genomic and cDNA levels.
DETAILED DESCRIPTION
[0047] The present disclosure relates to a self-complementary adeno-associated virus (scAAV) system for delivery of gene editing systems (e.g., CRISPR/Cas9 systems) to a cell or tissue of interest. In some embodiments, the scAAV delivery systems may be used for treatment or prevention of a genetic disease including, for example, a genetic muscle disease. In some embodiments, the scAAV delivery systems may be used for treatment or prevention of Duchenne Muscular Dystrophy (DMD).
[0048] Duchenne muscular dystrophy (DMD) is a lethal X-linked monogenic neuromuscular disease caused by mutations in the DMD gene, which encodes dystrophin. Dystrophin, together with dystroglycans and sarcoglycans, maintains sarcolemma integrity and stability by interacting with intracellular actin and extracellular laminin.
[0049] To date, two clinical therapies are available for DMD treatment: steroid supplementation and morpholino antisense oligomer injection. Long-term corticosteroid supplement partially alleviates DMD pathological phenotypes but cannot restore dystrophin expression. Morpholino antisense oligomers allow skipping of mutant DMD exons, but less than 1% of normal levels of dystrophin protein can be restored by this treatment.
[0050] More than 4,000 independent mutations in the DMD gene have been identified in human patients. The majority of patient mutations include deletions that cluster in a hotspot, and thus a therapeutic approach for skipping and/or reframing certain exon applies to large group of patients. The rationale of the exon skipping and/or reframing approach is based on the genetic difference between DMD and Becker muscular dystrophy (BMD) patients. In DMD patients, the reading frame of dystrophin mRNA is disrupted resulting in prematurely truncated, non-functional dystrophin proteins. BMD patients have mutations in the DMD gene that maintain the reading frame allowing the production of internally deleted, but partially functional dystrophins leading to much milder disease symptoms compared to DMD patients.
[0051] This disclosure addresses unmet needs for improved compositions and methods for delivery of gene editing systems and to treat and/or prevent Duchenne Muscular Dystrophy.
[0052] As used herein in the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
[0053] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or ” As used herein “another” may mean at least a second or more.
[0054] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, for the method being employed to determine the value, or that exists among the study subjects. Such an inherent variation may be a variation of ±10% of the stated value.
[0055] Throughout this application, nucleotide sequences are listed in the 5’ to 3’ direction, and amino acid sequences are listed in the N-terminal to C-terminal direction, unless indicated otherwise.
[0056] The terms “single guide RNA,” “guide RNA ,” “sgRNA” and “gRNA” are used interchangeably herein, and refer to a short synthetic RNA composed of a “spacer” (or “targeting”) sequence and a “scaffold” sequence. In some embodiments, the gRNA may further comprise a polyA tail.
[0057] The percent identity between two nucleotide or amino acid sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps which need to be introduced for optimal alignment and the length of each gap. Various computer programs and mathematical algorithms are available in the art to determine percentage identities between nucleotide and amino acid sequences such as GCG Wisconsin package and BLAST.
CRISPR Systems
[0058] CRISPRs (clustered regularly interspaced short palindromic repeats) are DNA loci containing short repetitions of base sequences. Each repetition is followed by short segments of “spacer DNA” from previous exposures to a virus. CRISPRs are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. CRISPRs are often associated with Cas genes that code for proteins related to CRISPRs. The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. CRISPR spacers recognize and silence these exogenous genetic elements like RNAi in eukaryotic organisms.
[0059] CRISPR repeats range in size from 24 to 48 base pairs. They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic. Repeats are separated by spacers of similar length. Some CRISPR spacer sequences exactly match sequences from plasmids and phages, although some spacers match the prokaryote’s genome (self-targeting spacers). New spacers can be added rapidly in response to phage infection. Guide RNA (gRNA or sgRNA)
[0060] As an RNA guided protein, Cas9 requires a short RNA to direct the recognition of DNA targets. Though Cas9 preferentially interrogates DNA sequences containing a PAM sequence NGG it can bind without a protospacer target. However, the Cas9-gRNA complex generally requires a close match to the gRNA to create a double strand break. CRISPR sequences in bacteria are expressed in multiple RNAs and then processed to create guide strands for RNA. Because Eukaryotic systems lack some of the proteins required to process CRISPR RNAs the synthetic construct gRNA was created to combine the essential pieces of RNA for Cas9 targeting into a single RNA expressed with the RNA polymerase type III promoter U6. Synthetic gRNAs are slightly over lOObp at the minimum length and contain a portion which is targets the 20 protospacer nucleotides immediately preceding the PAM sequence NGG; gRNAs do not contain a PAM sequence.
[0061] Single guide RNAs comprise a spacer region and a scaffold region. When a sgRNA “targets” a DNA sequence, it means that the spacer region of the sgRNA binds to the DNA sequence. In some embodiments, the sgRNA targets a site within a wildtype dystrophin gene. An exemplary wildtype dystrophin sequence includes the human sequence (see GenBank Accession NO. NC_000023.11), located on the human X chromosome, which codes for the protein dystrophin (GenBank Accession No. AAA53189; SEQ ID NO: 5), the sequence of which is reproduced below:
1 MLWWEEVEDC YEREDVQKKT FTKWVNAQFS KFGKQHIENL FSDLQDGRRL LDLLEGLTGQ
61 KLPKEKGSTR VHALNKVNKA LRVLQNNNVD LVNIGSTDIV DGNHKLTLGL IWNIILHWQV
121 KNVMKNIMAG LQQTNSEKIL LSWVRQSIRN YPQVNVINFT TSWSDGLALN ALIHSHRPDL
181 FDWNSVVCQQ SATQRLEHAF NIARYQLGIE KLLDPEDVDT TYPDKKSILM YITSLFQVLP
241 QQVSIEAIQE VEMLPRPPKV TKEEHFQLHH QMHYSQQITV SLAQGYERTS SPKPRFKSYA
301 YTQAAYVETS DPTRSPFPSQ HLEAPEDKSF GSSLMESEVN LDRYQTALEE VLSWLLSAEC
361 TLQAQGEZSN DVEWKDQFH THEGYMMDLT AHQGRVGNIL QLGSKLIGTG KLSEDEETEV
421 QEQMNLLNSR WECLRVASME KQSNLHRVLM DLQNQKLKEL NDWLTKTEER TRKMEEEPLG
481 PDLEDLKRQV QQHKVLQEDL EQEQVRVNSL THMWWDES SGDHATAALE EQLKVLGDRW
541 ANICRWTEDR WVLLQDILLK WQRLTEEQCL FSAWLSEKED AVNKIHTTGF KDQNEMLSSL
601 QKLAVLKADL EKKKQSMGKL YSLKQDLLST LKNKSVTQKT EAWLDNFARC WDNLVQKLEK
661 STAQISQAVT TTQPSLTQTT VMETVTTVTT REQILVKHAQ EELPPPPPQK KRQITVDSEI
721 RKRLDVDZTE LHSWITRSEA VLQSPEFAIF RKEGNFSDLK EKVNAIEREK AEKFRKLQDA
781 SRSAQALVEQ MVNEGVNADS IKQASEQLNS RWIEFCQLLS ERLNWLEYQN NIIAFYNQLQ
841 QLEQMTTZAE NWLKIQPTTP SEPTAIKSQL KICKDEVNRL SGLQPQIERL KIQSIALKEK
901 GQGPMFLDAD FVAFTNHFKQ VFSDVQAREK ELQTIFDTLP PMRYQETMSA IRTWVQQSET
961 KLSIPQLSVT DYEIMEQRLG ELQALQSSLQ EQQSGLYYLS TTVKEMSKKA PSEISRKYQS
1021 EFEEIEGRWK KLSSQLVEHC QKLEEQMNKL RKIQNHIQTL KKWMAEVDVF LKEEWPALGD 1081 SEILKKQLKQ CRLLVSDIQT IQPSLNSVNE GGQKIKNEAE PEEASRLETE LKELNIQWDH 1141 MCQQVYARKE ALKGGLEKTV SLQKDLSEMH EWMTQAEEEY LERDFEYKTP DELQKAVEEM 1201 KRAKEEAQQK EAKVKLLTES VNSVIAQAPP VAQEALKKEL ETLTTNYQWL CTRLNGKCKT 1261 LEEVWACWHE LLSYLEKANK WLNEVEFKLK TTENIPGGAE EI3EVLDSLE NLMRHSEDNP 1321 NQIRILAQTL TDGGVMDELI NEELETFNSR WRELHEEAVR RQKLLEQSIQ SAQETEKSLH 1381 LIQESLTFID KQLAAYIADK VDAAQMPQEA QKIQSDLTSH EI3LEEMKKH NQGKEAAQRV 1441 LSQIDVAQKK LQDVSMKFRL FQKPANFELR LQESKMILDE VKMHLPALET KSVEQEVVQS 1501 QLNHCVNLYK SLSEVKSEVE MVIKTGRQIV QKKQTENPKE LDERVTALKL HYNELGAKVT 1561 ERKQQLEKCL KLSRKMRKEM NVLTEWLAAT DMELTKRSAV EGMPSNLDSE VAWGKATQKE 1621 IEKQKVHLKS ITEVGEALKT VLGKKETLVE EKLSLLNSNW IAVTSRAEEW LNLLLEYQKH 1681 METFDQNVDH ITKWIIQADT LLDESEKKKP QQKEDVLKRL KAELNDIRPK VDSTRDQAAN 1741 LMANRGDHCR KLVEPQISEL NHRFAAISHR IKIGKASIPL KELEQFNSDI QKLLEPLEAE 1801 IQQGVNLKEE DFNKDMNEDN EGTVKELLQR GDNLQQRITD ERKREEIKIK QQLLQTKHNA 1861 LKDLRSQRRK KALEISHQWY QYKRQADDLL KCLDDZEKKL ASLPEPRDER KIKEIDRELQ 1921 KKKEELNAVR RQAEGLSEDG AAMAVEPIQI QLSKRWREIE SKFAQFRRLN FAQIHTVREE 1981 TMMVMTEDMP LEISYVPSTY LTEITHVSQA LLEVEQLLNA PDLCAKDFED LFKQEESLKN 2041 IKDSLQQSSG RIDIIHSKKT AALQSATPVE RVKLQEALSQ LDFQWEKVNK MYKDRQGRFD 2101 RSVEKWRRFH YDIKIFNQWL TEAEQFLRKT QIPENWEHAK YKWYLKELQD GIGQRQTWR 2161 TLNATGEEII QQSSKTDASI LQEKLGSLNL RWQEVCKQLS DRKKRLEEQK NILSEFQRDL 2221 NEEVLWLEEA DNIASIPLEP GKEQQLKEKL EQVKLLVEEL PLRQGILKQL NETGGPVLVS 2281 APISPEEQDK LENKLKQTNL QWIKVSRALP EKQGEZEAQI KDLGQLEKKL EDLEEQLNHL 2341 LLWLSPIRNQ LEIYNQPNQE GPFDVQETEI AVQAKQPDVE EILSKGQHLY KEKPATQPVK 2401 RKLEDLSSEW KAVNRLLQEL RAKQPDLAPG LTTIGASPTQ TVTLVTQPW TKETAISKLE 2461 MPSSLMLEVP ALADFNRAWT ELTDWLSLLD QVZKSQRVMV GDLEDINEMI IKQKAIMQDL 2521 EQRRPQLEEL ITAAQKLKNK TSNQEARTII TDRIERIQNQ WDEVQEHLQN RRQQLNEMLK 2581 DSTQWLEAKE EAEQVLGQAR AKLESWKEGP YTVDAZQKKI TETKQLAKDL RQWQTNVDVA 2641 NDLALKLLRD YSADDTRKVH MITENINASW RSIHKRVSER EAALEETHRL LQQFPLDLEK 2701 FLAWLTEAET TANVLQDATR KERLLEDSKG VKELMKQWQD LQGEIEAHTD VYHNLDENSQ 2761 KZLRSLEGSD DAVLLQRRLD NMNFKWSELR KKSLNZRSHL EA3SDQWKRL HLSLQELLVW 2821 LQLKDDELSR QAPIGGDFPA VQKQNDVHRA EKRELKTKEP VIMSTLETVR IFLTEQPLEG 2881 LEKLYQEPRE LPPEERAQNV TRLLRKQAEE VNTEWEKLNL HSADWQRKID ETLERLQELQ 2941 EATDELDLKL RQAEVIKGSW QPVGDLLIDS LQDHLEKVKA LRGEIAPLKE NVSHVNDLAR 3001 QLTTLGIQLS PYNLSTLEDL NTRWKLLQVA VEDRVRQLHE AHRDFGPASQ HFLSTSVQGP 3061 WERAZSPNKV PYYINHETQT TCWDHPKMTE LYQSLADLNN VRFSAYRTAM KLRRLQKALC 3121 LDLLSLSAAC DALDQHNLKQ NDQPMDILQI INCLTZIYDR LEQEHNNLVN VPLCVDMCLN 3181 WLLNVYDEGR TGRIRVLSFK TGIISLCKAH LEDKYRYLFK QVASSTGFCD QRRLGLLLHD 3241 SZQIPRQLGE VASFGGSNIE PSVRSCFQFA NNKPEZEAAL FLDWMRLEPQ SMVWLPVLHR 3301 VAAAETAKHQ AKCNZCKECP IIGFRYRSLK HFNYDZCQSC FF3GRVAKGH KMHYPMVEYC 3361 TPTTSGEDVR DFAKVLKNKF RTKRYFAKHP RMGYLPVQTV LEGDNMETPV TLINFWPVDS 3421 APASSPQLSH DDTHSRIEHY ASRLAEMENS NGSYLNDSIS PNESIDDEHL LIQHYCQSLN 3481 QDSPLSQPRS PAQILISLES EERGELERIL ADLEEENRNL QAEYDRLKQQ HEHKGLSPLP 3541 SPPEf4MPZSP QSPRDAELIA EAKLLRQHKG RLEARMQILE DHNKQLESQL HRLRQLLEQP 3601 QAEAKVNGTT VSSPSTSLQR SDSSQPMLLR WGSQZSDSM GEEDLLSPPQ DTSTGLEEVM 3661 EQLNNSFPSS RGRNTFGKPM REDTM.
[0062] In some embodiments, the sgRNA targets a site within a mutant dystrophin gene. In some embodiments, the sgRNA targets a dystrophin intron. In some embodiments, the sgRNA targets a dystrophin exon. In some embodiments, the sgRNA targets a site in a dystrophin exon that is expressed and is present in one or more of the dystrophin isoforms shown in Table 1. In embodiments, the sgRNA targets a dystrophin splice site. In some embodiments, the sgRNA targets a splice donor site on the dystrophin gene. In embodiments, the sgRNA targets a splice acceptor site on the dystrophin gene. Table 1: Dystrophin isoforms
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
[0063] In some embodiments, sgRNAs comprise a spacer and a scaffold region. In embodiments, the spacer targets a mutant DMD exon. In some embodiments, the spacer targets any one of exons 1-79 of the DMD gene. In some embodiments, the spacer targets at least one of exon 23, 44 or 51. In some embodiments, the spacer targets at least one of exons 1, 6, 7, 8, 23, 41, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. In embodiments, the spacer targets an intron flanking ( i.e ., immediately 5’ or 3’ to) any one of exons 1-79 of the DMD gene. In some embodiments, the spacer targets an intron flanking at least one of exon 23, 44 or 51. In some embodiments, the spacer targets an intron flanking at least one of exons 1, 6, 7, 8, 23, 41, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. In some embodiments, the spacer targets a splice donor or splice acceptor site of any one of exons 1-79 of the DMD gene. In preferred embodiments, the spacer is designed to induce skipping and/or refraining of exon 51, exon 44 or exon 23. In embodiments, the spacer is targeted to a splice acceptor site of exon 51, exon 44 or exon 23.
[0064] Exemplar}' spacers for use in various compositions and methods disclosed herein are provided as SEQ ID NOs: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036-1043, 1052- 1063, 1494-1499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668, and in Tables 5-9, 12, and 14-16. In some embodiments, the spacer is selected from any one of SEQ ID No: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036-1043, 1052-1063, 14944499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668.
[0065] In some embodiments, the spacers of the disclosure comprise a sequence that is complementary to a target sequence within a coding sequence or a non-coding sequence corresponding to the DMD gene, and, therefore, hybridize to the target sequence.
[0066] The scaffold sequence is the sequence within the gRNA that is responsible for nuclease (e.g., Cas9) binding. The scaffold sequence does not include the spacer/targeting sequence.
[0067] In some embodiments, the scaffold may be about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, or about 120 to about 130 nucleotides in length. In some embodiments, the scaffold may be about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, or about 125 nucleotides in length. In some embodiments, the scaffold may be at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, or at least 125 nucleotides in length. In some embodiments, the scaffold may be 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125 nucleotides in length.
[0068] In some embodiments, the scaffold may have a sequence of any one of SEQ ID NO: 2672-2678 (shown in Table 2 below), or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.
Table 2: Exemplary Scaffold Sequences
Figure imgf000026_0001
[0069] In some embodiments, the scaffold may comprise a sequence of GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTA TCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT (SEQ ID NO: 2672), or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.
[0070] In some embodiments, a gRNA (spacer + scaffold) comprises a spacer of any one of SEQ ID NO: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036-1043, 1052-1063, 1494- 1499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668, and a scaffold of SEQ ID NO: 2672. In some embodiments, a nucleic acid may comprise one or more sequences encoding a gRNA. In some embodiments, a nucleic acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 sequences encoding a gRNA. In some embodiments, all of the sequences encode the same gRNA. In some embodiments, all of the sequences encode different gRNAs. In some embodiments, at least 2 of the sequences encode the same gRNA, for example at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the sequences encode the same gRNA. Nucleases
[0071] The CRISPR systems disclosed herein may comprise a nuclease or a nucleic acid encoding the same. In some embodiments, the nuclease is a Type II, Type V-A, Type V-B, Type V-C, Type V-U, Type VI-B nuclease. In some embodiments, the nuclease is a transcription activator-like effector nuclease (TALEN), a meganuclease, or a zinc-finger nuclease. In some embodiments, the nuclease is a Cas9, Casl2a (Cpfl), Casl2b, Casl2c, Tnp- B like, Casl3a (C2c2), Casl3b, or Casl4 nuclease. For example, in some embodiments, the nuclease is a Cas9 nuclease or a Cpfl nuclease.
[0072] In some embodiments, the nuclease is a modified form or variant of a Cas9, Casl2a (Cpfl), Casl2b, Casl2c, Tnp-B like, Casl3a (C2c2), Casl3b, or Casl4 nuclease. In some embodiments, the nuclease is a modified form or variant of a TAL nuclease, a meganuclease, or a zinc-finger nuclease, A “modified” or “variant” nuclease is one that is, for example, truncated, fused to another protein (such as another nuclease), catalytically inactivated, etc. In some embodiments, the nuclease may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a naturally occurring Cas9, Casl2a (Cpfl), Casl2b, Casl2c, Tnp-B like, Casl3a (C2c2), Casl3b, Casl4 nuclease, or a TALEN, meganuclease, or zinc-finger nuclease.
[0073] In embodiments, the nuclease is a Cas9 nuclease derived from S. pyogenes (SpCas9). An exemplary SpCas9 sequence is provided in SEQ ID NO: 2591. In some embodiments, the nuclease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2591.
[0074] In embodiments, the nuclease is a Cas9 derived from S. aureus (SaCas9). An exemplar}' SaCas9 sequence is provided in SEQ ID NO: 873. In some embodiments, the nuclease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 873.
[0075] In embodiments, the Cpfl is a Cpfl enzyme from Acidaminococcus (species BV3L6, UniProt Accession No. U2UMQ6). An exemplary Acidaminococcus Cpfl sequence is provided in SEQ ID NO: 870. In some embodiments, the nuclease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 870.
[0076] In some embodiments, the Cpfl is a Cpfl enzyme from Lachnospiraceae (species ND2006, UniProt Accession No. A0A182DWE3). An exemplary Lachnospiraceae Cpfl sequence is provided in SEQ ID NO: 871. In some embodiments, the nuclease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 871.
[0077] In some embodiments, a sequence encoding the nuclease is codon optimized for expression in mammalian cells. In some embodiments, the sequence encoding the nuclease is codon optimized for expression in human cells or mouse cells.
Cas Nucleases
[0078] CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. As of 2013, more than forty different Cas protein families had been described. Of these protein families, Casl appears to be ubiquitous among different CRISPR/Cas systems. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat- associated mysterious proteins (RAMPs). More than one CRISPR subtype may occur in a single genome. The sporadic distribution of the CRISPR/Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.
[0079] Exogenous DNA is apparently processed by proteins encoded by Cas genes into small elements (-30 base pairs in length), which are then somehow inserted into the CRISPR locus near the leader sequence. RNAs from the CRISPR loci are constitutively expressed and are processed by Cas proteins to small RNAs composed of individual, exogenously-derived sequence elements with a flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Evidence suggests functional diversity among CRISPR subtypes. The Cse (Cas subtype Ecoli ) proteins (called CasA-E in E. coli ) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer- repeat units that Cascade retains. In other prokaryotes, Cas6 processes the CRISPR transcripts. Interestingly, CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Casl and Cas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. RNA-guided CRISPR enzymes are classified as type V restriction enzymes. [0080] Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix. It has been demonstrated that one or both sites can be disabled while preserving Cas9’s ability to locate its target DNA. Moreover, tracrRNA and spacer RNA can be combined into a "single-guide RNA" molecule that, mixed with Cas9, can find and cut the correct DNA targets and such synthetic guide RNAs are used for gene editing.
[0081] Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR/Cas- mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during bacterial interaction with eukaryotic hosts. For example, Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence. Delivery of Cas9 DNA sequences also is contemplated.
[0082] In some embodiments, the compositions of the disclosure include a small version of a Cas9 from the bacterium Staphylococcus aureus (UniProt Accession No. J7RUA5). The small version of the Cas9 provides advantages over wildtype or full length Cas9. In some embodiments the Cas9 is a SpCas9.
Cpfl Nucleases
[0083] Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 or CRISPR/Cpfl is a DNA-editing technology which shares some similarities with the CRISPR/Cas9 system. Cpfl is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. It prevents genetic damage from viruses. Cpfl genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpfl is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations.
[0084] Cpfl appears in many bacterial species. The ultimate Cpfl endonuclease that was developed into a tool for genome editing was taken from one of the first 16 species known to harbor it.
[0085] In embodiments, the Cpfl is a Cpfl enzyme from Acidaminococcus (species BV3L6, UniProt Accession No. U2UMQ6; SEQ ID NO: 870), having the sequence set forth below: 1 MIQFEGFZNL YQVSKTLRFE LIPQGKTLKH IQEQGFIEED KARKDHYKEL KPIIDRIYKT
61 YAEQCLQLVQ LDWENLSAAI DSYRKEKTEE TRNALIEEQA TYRNAIHDYF ZGRTDNLTDA
121 INKRHAEZYK GLFKAELFNG KVLKQLGTVT TTEHENALLR SFDKFTTYFS GFYENRKNVF
181 SAEDISTAIP HRIVQDNFPK FKENCHIFTR LITAVPSLRE HFENVKKAIG IFVSTSIEEV
241 FSFPFYNQLL TQTQIDLYNQ LLGGISREAG TEKIKGLNEV LNLAIQKNDE TAHIIASLPH
301 RFIPLFKQIL SDRNTLSFIL EEFKSDEEVI QSFCKYKTLL RNENVLETAE ALFNELNSIC
361 LTHIFISHKK LETISSALCD HWDTLRNALY ERRISELTGK ITKSAKEKVQ RSLKHEDINL
421 QEIISAAGKE LSEAFKQKTS EILSHAHAAL DQPLPFTLKK QEEKEILKSQ LDSLLGLYHL
481 LDWFAVDESN EVDPEFSARL TGIKLEMEPS LSFYNKARNY ATKKPYSVEK FKLNFQMPTL
541 ASGWDVNKEK NNGAILFVKN GLYYLGIMPK QKGRYKALSF EPTEKTSEGF DKMYYDYFPC
601 AAKMIPKCST QLKAVTAHFQ THTTPILLSN NFIEPLEITK ElYDLNNPEK EPKKFQTAYA
661 KKTGDQKGYR EALCKWIDFT RDFLSKYTKT TSIDLSSLRP SSQYKDLGEY YAELNPLLYH
721 ISFQRIAEKE IMDAVETGKL YLFQIYNKDF AKGHHGKPNL HTLYWTGLFS PENLAKTSIK
781 LNGQAELFYR PKSRMKRMAH RLGEKMLNKK LKDQKEPIPD TLYQELYDYV NHRLSHDLSD
841 EARALLPNVI TKEVSHEIIK DRRFTSDKFF FHVPIELNYQ AANSPSKFNQ RVNAYLKEHP
901 ETPIIGIDRG ERNLIYITVI DSTGKILEQR SLNTIQQFDY QKKLDNREKE RVAARQAWSV
961 VGTIKDLKQG YLSQVIHEIV DLMIHYQAW VLENLNFGFK SKRTGIAEKA VYQQFEKMLI
1021 DKLNCLVLKD YPAEKVGGVL NPYQLTDQFT SFAKMGTQSG FLFYVPAPYT SKIDPLTGFV
1081 DPFVWKTZKN HESRKHFLEG FDFLHYDVKT GDFZLHFKMN RNLSFQRGLP GFMPAWDZVF
1141 EKNETQFDAK GTPFZAGKRZ VPVZENHRFT GRYRDLYPAN ELZALLEEKG ZVFRDGSNZL
1201 PKLLENDDSH AZDTMVALZR SVLQMRNSNA ATGEDYZNSP VRDLNGVCFD SRFQNPEWPM
1261 DADANGAYHZ ALKGQLLLNH LKESKDLKLQ NGZSNQDWLA YZQELRN.
[0086] In some embodiments, the Cpfl is a Cpfl enzyme from Lachnospiraceae (species ND2006, UniProt Accession No. A0A182DWE3; SEQ ID NO: 871), having the sequence set forth below:
1 AASKLEKFTN CYSLSKTLRF KAZPVGKTQE NZDNKRLLVE DEKRAEDYKG VKKLLDRYYL
61 SFZNDVLHSZ KLKNLNNYZS LFRKKTRTEK ENKELENLEZ NLRKEZAKAF KGAAGYKSLF
121 KKDZZETZLP EAADDKDEZA LVNSFNGFIT AETGFFDNRE NMF3EEAKSI SZAFRCZNEN
181 LTRYZSNMDZ FEKVDAZFDK HEVQEZKEKZ LNSDYDVEDF FEGEFFNFVL TQEGZDVYNA
241 ZZGGFVTESG EKZKGLNEYZ NLYNAKTKQA LPKFKPLYKQ VLSDRESLSF YGEGYTSDEE
301 VLEVFRNTLN KNSEZFSSZK KLEKLFKNFD EYSSAGZFVK NGPAZSTZSK DZFGEWNLZR
361 DKWNAEYDDZ HLKKKAWTE KYEDDRRKSF KKZGSFSLEQ LQEYADADLS WEKLKEZZZ
421 QKVCEZYKVY GSSEKLFDAD FVLEKSLKKN DAWAZMKDL LDSVKSFENY ZKAFFGEGKE
481 TNRDESFYGD FVLAYDZLLK VDHZYDAZRN YVTQKPYSKD KFKLYFQNPQ FMGGWDKDKE
541 TDYRATZLRY GSKYYLAZMD KKYAKCLQKZ DKDDVNGNYE KZNYKLLPGP NKMLPKVFFS
601 KKWMAYYNPS EDZQKZYKNG TFKKGDMFNL NDCHKLZDFF KDSZSRYPKW SNAYDFNFSE
661 TEKYKDZAGF YREVEEQGYK VSFESASKKE VCKLVEEGKL YMFQZYNKDF SDKSHGTPNL
721 HTMYFKLLFD ENNHGQZRLS GGAELFMRRA SLKKEELVVH PAN3PZANKN PDNPKKTTTL
781 SYDVYKDKRF SEDQYELHZP ZAZNKCPKNZ FKZNTEVRVL LKHDDNPYVZ GZDRGERNLL
841 YZVWDGKGN ZVEQYSLNEZ ZNNFNGZRZK TDYHSLLDKK EKERFEARQN WTSZENZKEL
901 KAGYZSQWH KZCELVEKYD AVZALEDLNS GFKNSRVKVE KQVYQKFEKM LZDKLNYMVD
961 KKSNPCATGG ALKGYQZTNK FESFKSMSTQ NGFZFYZPAW LTSKZDPSTG FVNLLKTKYT
1021 SZADSKKFZS SFDRZMYVPE EDLFEFALDY KNFSRTDADY ZKKWKLYSYG NRZRZFAAAK
1081 KNNVFAWEEV CLTSAYKELF NKYGZNYQQG DZRALLCEQS DKAFYSSFMA LMSLMLQMRN
1141 SZTGRTDVDF LZSPVKNSDG ZFYDSRNYEA QENAZLPKNA DANGAYNZAR KVLWAZGQFK
1201 KAECEKLDKV KZAZSNKEWL EYAQTSVK.
[0087] In some embodiments, the Cpfl is codon optimized for expression in mammalian cells. In some embodiments, the Cpfl is codon optimized for expression in human cells or mouse cells. [0088] The Cpfl locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain. The Cpfl protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cpfl does not have a HNH endonuclease domain, and the N-terminal of Cpfl does not have the alpha-helical recognition lobe of Cas9.
[0089] Cpfl CRISPR-Cas domain architecture shows that Cpfl is functionally unique, being classified as Class 2, type V CRISPR system. The Cpfl loci encode Casl, Cas2 and Cas4 proteins more similar to types I and III than from type II systems. Database searches suggest the abundance of Cpfl -family proteins in many bacterial species.
[0090] Functional Cpfl does not require a tracrRNA, therefore, only crRNA is required. This benefits genome editing because Cpfl is not only smaller than Cas9, but also it has a smaller sgRNA molecule (approximately half as many nucleotides as Cas9).
[0091] The Cpfl-crRNA complex cleaves target DNA or RNA by identification of a protospacer adjacent motif 5'-YTN-3' (where "Y" is a pyrimidine and "N" is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cpfl introduces a sticky-end-like DNA double- stranded break of 4 or 5 nucleotides overhang.
[0092] The CRISPR/Cpfl system consists of a Cpfl enzyme and a guide RNA that finds and positions the complex at the correct spot on the double helix to cleave target DNA. CRISPR/Cpfl systems activity has three stages: 1) Adaptation, during which Casl and Cas2 proteins facilitate the adaptation of small fragments of DNA into the CRISPR array; 2) Formation of crRNAs: processing of pre-cr-RNAs producing of mature crRNAs to guide the Cas protein; and 3) Interference, in which the Cpfl is bound to a crRNA to form a binary complex to identify and cleave a target DNA sequence.
CRISPR -mediated gene editing
[0093] The first step in editing the DMD gene using CRISPR/Cpfl or CRISPR/Cas9 is to identify the genomic target sequence. The genomic target for the gRNAs of the disclosure can be any ~24 nucleotide DNA sequence, provided that the sequence is unique compared to the rest of the genome. In some embodiments, the genomic target sequence corresponds to a sequence within exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene. In some embodiments, the genomic target sequence is a 5’ or 3’ splice site of exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene. In some embodiments, the genomic target sequence corresponds to a sequence within an intron immediately upstream or downstream of exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene.
[0094] The next step in editing the DMD gene is to identify all Protospacer Adjacent Motif (PAM) sequences within the genetic region to be targeted. The target sequence must be immediately upstream of a PAM. Once all possible PAM sequences and putative target sites have been identified, the next step is to choose which site is likely to result in the most efficient on-target cleavage. The gRNA targeting sequence needs to match the target sequence, and the gRNA targeting sequence must not match additional sites within the genome. In preferred embodiments, the gRNA targeting sequence has perfect homology to the target with no homology elsewhere in the genome. In some embodiments, a given gRNA targeting sequence will have additional sites throughout the genome where partial homology exists. These sites are called “off-targets” and should be considered when designing a gRNA. In general, off- target sites are not cleaved as efficiently when mismatches occur near the PAM sequence, so gRNAs with no homology or those with mismatches close to the PAM sequence will have the highest specificity. In addition to “off-target activity”, factors that maximize cleavage of the desired target sequence (“on-target activity”) must be considered. It is known to those of skill in the art that two gRNA targeting sequences, each having 100% homology to the target DNA may not result in equivalent cleavage efficiency. In fact, cleavage efficiency may increase or decrease depending upon the specific nucleotides within the selected target sequence. Close examination of predicted on-target and off-target activity of each potential gRNA targeting sequence is necessary to design the best gRNA. Several gRNA design programs have been developed that are capable of locating potential PAM and target sequences and ranking the associated gRNAs based on their predicted on-target and off-target activity (e.g. CRISPRdirect, available at www.crispr.dbcls.jp).
[0095] The next step is to synthesize and clone desired gRNAs. Targeting oligos can be synthesized, annealed, and inserted into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, the exact cloning strategy will depend on the gRNA vector that is chosen. The gRNAs for Cpfl are notably simpler than the gRNAs for Cas9, and only consist of a single crRNA containing direct repeat scaffold sequence followed by -24 nucleotides of guide sequence. [0096] Each gRNA should then be validated in one or more target cell lines. For example, after the Cas9 or Cpfl and the gRNA are delivered to the cell, the genomic target region may be amplified using PCR and sequenced according to methods known to those of skill in the art.
[0097] In some embodiments, gene editing may be performed in vitro or ex vivo. In some embodiments, cells are contacted in vitro or ex vivo with a Cas9 or a Cpfl and a gRNA that targets a dystrophin splice site. In some embodiments, the cells are contacted with one or more nucleic acids encoding the Cas9 or Cpfl and the guide RNA. In some embodiments, the one or more nucleic acids are introduced into the cells using, for example, lipofection or electroporation. Gene editing may also be performed in zygotes. In embodiments, zygotes may be injected with one or more nucleic acids encoding Cas9 or Cpfl and a gRNA that targets a dystrophin splice site. The zygotes may subsequently be injected into a host.
[0098] In some embodiments, the Cas9 or Cpfl is provided on a vector. In embodiments, the vector contains a Cas9 derived from S. pyogenes (SpCas9, SEQ ID NO: 872). In embodiments, the vector contains a Cas9 derived from S. aureus (SaCas9, SEQ ID NO: 873). In embodiments, the vector contains a Cpfl sequence derived from a Lachnospiraceae bacterium. See, for example, Uniprot Accession No. A0A182DWE3; SEQ ID NO: 871. In embodiments, the vector contains a Cpfl sequence derived from an Acidaminococcus bacterium. See, for example, Uniprot Accession No. U2UMQ6; SEQ ID NO: 870. In some embodiments, the Cas9 or Cpfl sequence is codon optimized for expression in human cells or mouse cells. In some embodiments, the vector further contains a sequence encoding a fluorescent protein, such as GFP, which allows Cas9 or Cpfl -expressing cells to be sorted using fluorescence activated cell sorting (FACS). In some embodiments, the vector is a viral vector such as an adeno-associated viral vector.
[0099] In some embodiments, the gRNA is provided on a vector. In some embodiments, the vector is a viral vector such as an adeno-associated viral vector. In embodiments, the Cas9 or Cpfl and the guide RNA are provided on the same vector. In embodiments, the Cas9 or Cpfl and the guide RNA are provided on different vectors.
[0100] In some embodiments, the cells are additionally contacted with a single-stranded DMD oligonucleotide to effect homology directed repair. In some embodiments, small INDELs restore the protein reading frame of dystrophin (“reframing” strategy). When the reframing strategy is used, the cells may be contacted with a single gRNA. In embodiments, a splice donor or splice acceptor site is disrupted, which results in exon skipping and restoration of the protein reading frame (“exon skipping” strategy). When the exon skipping strategy is used, the cells may be contacted with two or more gRNAs.
[0101] Efficiency of in vitro or ex vivo Cas9 or Cpfl -mediated DNA cleavage may be assessed using techniques known to those of skill in the art, such as the T7 El assay. Restoration of DMD expression may be confirmed using techniques known to those of skill in the art, such as RT-PCR, western blotting, and immunocytochemistry.
[0102] In some embodiments, in vitro or ex vivo gene editing is performed in a muscle or satellite cell. In some embodiments, gene editing is performed in iPSC or iCM cells. In embodiments, the iPSC cells are differentiated after gene editing. For example, the iPSC cells may be differentiated into a muscle cell or a satellite cell after editing. In embodiments, the iPSC cells are differentiated into cardiac muscle cells, skeletal muscle cells, or smooth muscle cells. In embodiments, the iPSC cells are differentiated into cardiomyocytes. iPSC cells may be induced to differentiate according to methods known to those of skill in the art.
[0103] In some embodiments, contacting the cell with the Cas9 or the Cpfl and the gRNA restores dystrophin expression. In embodiments, cells which have been edited in vitro or ex vivo , or cells derived therefrom, show levels of dystrophin protein that are comparable to wildtype cells. In embodiments, the edited cells, or cells derived therefrom, express dystrophin at a level that is 50%, 60%, 70%, 80%, 90%, 95% or any percentage in between of wildtype dystrophin expression levels. In embodiments, the cells which have been edited in vitro or ex vivo, or cells derived therefrom, have a mitochondrial number that is comparable to that of wildtype cells. In embodiments the edited cells, or cells derived therefrom, have 50%, 60%, 70%, 80%, 90%, 95% or any percentage in between as many mitochondria as wildtype cells. In embodiments, the edited cells, or cells derived therefrom, show an increase in oxygen consumption rate (OCR) compared to non-edited cells at baseline.
Nucleic Acid Expression Vectors
[0104] As discussed above, in certain embodiments, expression cassettes are employed to express a transcription factor product, either for subsequent purification and delivery to a cell/subject, or for use directly in a genetic -based delivery approach. Provided herein are expression vectors which contain one or more nucleic acids encoding Cas9 or Cpfl and at least one DMD guide RNA that targets a dystrophin splice site. In some embodiments, a nucleic acid encoding Cas9 or Cpf 1 and a nucleic acid encoding at least one guide RNA are provided on the same vector. In further embodiments, a nucleic acid encoding Cas9 or Cpfl and a nucleic acid encoding least one guide RNA are provided on separate vectors.
[0105] Expression requires that appropriate signals be provided in the vectors, and include various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
[0106] Throughout this application, the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i.e., is under the control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. An “expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.
Regulatory Elements
[0107] The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
[0108] At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
RNA Polymerase and Pol III Promoters
[0109] In eukaryotes, RNA polymerase III (also called Pol III) transcribes DNA to synthesize ribosomal 5S rRNA, tRNA and other small RNAs. The genes transcribed by RNA Pol III fall in the category of “housekeeping” genes whose expression is required in all cell types and most environmental conditions. Therefore, the regulation of Pol III transcription is primarily tied to the regulation of cell growth and the cell cycle, thus requiring fewer regulatory proteins than RNA polymerase II. Under stress conditions however, the protein Mail represses Pol III activity.
[0110] In the process of transcription (by any polymerase) there are three main stages: (i) initiation, requiring construction of the RNA polymerase complex on the gene's promoter; (ii) elongation, the synthesis of the RNA transcript; and (iii) termination, the finishing of RNA transcription and disassembly of the RNA polymerase complex.
[0111] Promoters under the control of RNA Pol III include those for ribosomal 5S rRNA, tRNA and few other small RNAs such as U6 spliceosomal RNA, RNase P and RNase MRP RNA, 7SL RNA (the RNA component of the signal recognition particles), Vault RNAs, Y RNA, SINEs (short interspersed repetitive elements), 7SK RNA, two microRNAs, several small nucleolar RNAs and several few regulatory antisense RNAs.
Additional Promoters and Elements
[0112] In some embodiments, the Cas9 or Cpfl constructs of the disclosure are expressed by a cell type-specific promoter. In some embodiments, the Cas9 or Cpfl constructs of the disclosure are expressed by a muscle cell-specific promoter. This cell type-specific promoter (e.g., the muscle-specific promoter) may be constitutively active or may be an inducible promoter.
[0113] Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
[0114] In certain embodiments, viral promoters such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma vims long terminal repeat, rat insulin promoter and glyceraldehyde- 3 -phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.
[0115] Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
[0116] Below is a list of promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct. [0117] The promoter and/or enhancer may be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and/or DQ b, b-interferon, interleukin-2, interleukin-2 receptor, MHC class II, MHC class II HLA-Dra, b-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, oc-fetoprotein, t-globin, b-globin, c-fos, c-HA-mv, insulin, neural cell adhesion molecule (NCAM), ai-antitrypain, H2B (TH2B) histone, mouse and/or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), Duchenne muscular dystrophy, SV40, polyoma, retroviruses, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon ape leukemia vims.
[0118] In some embodiments, inducible elements may be used. In some embodiments, the inducible element is, for example, MTII, MMTV (mouse mammary tumor vims), b-interferon, adenovirus 5 E2, collagenase, stromelysin, SV40, murine MX gene, GRP78 gene, oc-2- macroglobulin, vimentin, MHC class I gene H-2Kb, HSP70, proliferin, tumor necrosis factor, and/or thyroid stimulating hormone a gene. In some embodiments, the inducer is phorbol ester (TFA), heavy metals, glucocorticoids, poly(rI)x, poly(rc), E1A, phorbol ester (TPA), interferon, Newcastle Disease Virus, A23187, IL-6, semm, interferon, SV40 large T antigen, PMA, and/or thyroid hormone. Any of the inducible elements described herein may be used with any of the inducers described herein.
[0119] Of particular interest are muscle specific promoters. These include the myosin light chain-2 promoter, the a-actin promoter, the troponin 1 promoter; the Na+/Ca2+ exchanger promoter, the dystrophin promoter, the a7 integrin promoter, the brain natriuretic peptide promoter and the aB-crystallin/small heat shock protein promoter, a-myosin heavy chain promoter and the ANF promoter.
[0120] In some embodiments, the muscle specific promoter is the CK8 promoter. The CK8 promoter has the following sequence (SEQ ID NO: 874):
1 CTAGACTAGC AGGCTGCCCA TGTAAGGAGG CAAGGCCTGG GGACACCCGA
GATGCCTGGT
61 TATAAGTAAC CCAGACATGT GGCTGCCCCC CCCCCCCCAA CACCTGCTGC
CTCTAAAAAT
121 AACCCGGCAT GCCATGTTCC CGGCGAAGGG CCAGCTGTCC CCCGCCAGCT
AGACTCAGCA
181 CTTAGGTTAG GAACCAGTGA GCAAGTCAGC CCTTGGGGCA GCCCATACAA
GGCCATGGGG
241 CTGGGCAAGC TGCACGCCTG GGTCCGGGGT GGGCACGGTG CCCGGGCAAC
GAGCTGAAAG 301 CTCATCTGCT CGCAGGGGCC CCTCCCTGGG GACAGCCCCT CCTGGCTAGT
CACACCCTGT
361 AGGCTCCTCT AGATAACCCA GGGGCACAGG GGCTGCCCTC ATTCTACCAC
CACCTCCACA
421 GCACAGACAG ACACTCAGGA GCCAGCCAGC.
[0121] In some embodiments, the muscle-cell cell specific promoter is a variant of the CK8 promoter, called CK8e. The CK8e promoter has the following sequence (SEQ ID NO. 2590):
1 TGCCCATGTA AGGAGGCAAG GCCTGGGGAC ACCCGAGATG CCTGGTTATA AGTAACCCAG
61 ACATGTGGCT GCCCCCCCCC CCCCAACACC TGCTGCCTCT AAAAATAACC CTGCATGCCA
121 TGTTCCCGGC GAAGGGCCAG CTGTCCCCCG CCAGCTAGAC TCAGCACTTA GTTTAGGAAC
181 CAGTGAGCAA GTCAGCCCTT GGGGCAGCCC ATACAAGGCC ATGGGGCTGG GCAAGCTGCA
241 CGCCTGGGGC CGGGGTGGGC ACGGTGCCCG GGCAACGAGC TGAAAGCTCA TCTGCTCTCA
301 GGGGCCCCGC CCTGGGGACA GCCCCTCCTG GCTAGTCACA CCCIGTAGGC TCCTCTATAT
361 AACCCAGGGG CAGAGGGGCT GCCCTCATTC TACCACCACC TCCACAGCAC AGACAGACAC
421 TCAGGAGCCA GCGAGC.
[0122] Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. Any polyadenylation sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
Self-cleaving peptides
[0123] In some embodiments of self-cleaving peptides of the disclosure, the self-cleaving peptide is a 2A peptide. In some embodiments, a 2A-like self-cleaving domain from the insect virus Thosea asigna (TaV 2A peptide) (SEQ ID NO: 876, EGRGSLLTCGDVEENPGP) is used. These 2A-like domains have been shown to function across eukaryotes and cause cleavage of amino acids to occur co-translationally within the 2A-like peptide domain. Therefore, inclusion of T aV 2A peptide allows the expression of multiple proteins from a single mRNA transcript. Importantly, the domain of TaV when tested in eukaryotic systems has shown greater than 99% cleavage activity. Other acceptable 2A-like peptides include, but are not limited to, equine rhinitis A virus (ERAV) 2A peptide (SEQ ID NO: 877; QCTN Y ALLKL AGD VESNPGP) , porcine teschovirus-1 (PTV1) 2A peptide (SEQ ID NO: 878; ATNFSLLKQAGDVEENPGP) and foot and mouth disease vims (FMDV) 2 A peptide (SEQ ID NO: 879; PVKQLLNFDLLKLAGDVESNPGP) or modified versions thereof.
[0124] In some embodiments, the 2A peptide is used to express a reporter and a Cas9 or a Cpfl simultaneously. The reporter may be, for example, GFP. [0125] Other self-cleaving peptides that may be used include, but are not limited to nuclear inclusion protein a (Nia) protease, a PI protease, a 3C protease, an L protease, a 3C-like protease, or modified versions thereof.
Therapeutic Compositions
AAV-Cas9 expression cassettes and vectors
[0126] In some embodiments, an AAV expression cassette comprises a sequence encoding a Cas9 nuclease. In some embodiments, the AAV expression cassette comprises, from 5’ to 3’, a promoter and a sequence encoding a Cas9 nuclease, wherein the expression cassette is flanked by a first inverted terminal repeat (ITR) and a second ITR, wherein the first ITR has the sequence of SEQ ID NO: 2585, wherein the second ITR has the sequence of SEQ ID NO: 2679. In some embodiments, the AAV expression cassette is self-complementary. In some embodiments, the AAV expression cassette is not self-complimentary.
[0127] In some embodiments, the promoter is a CK8e promoter. In some embodiments, the promoter has the sequence of SEQ ID NO: 2590, or a sequence at least 95% identical thereto.
[0128] In some embodiments, the sequence encoding the Cas9 nuclease is derived from S. aureus or S. pyogenes. In some embodiments, the sequence encoding the Cas9 nuclease comprises SEQ ID NO: 2591, or a sequence at least 95% identical thereto.
[0129] In some embodiments, the expression cassette further comprises a sequence encoding a PolyA tail. In some embodiments, the PolyA tail comprises a sequence of SEQ ID NO: 2593, or a sequence at least 95% identical thereto.
[0130] In some embodiments, an expression cassette encoding a Cas9 may be packaged into an AAV vector. In some embodiments, the AAV vector is a wildtype AAV vector. In some embodiments, the AAV vector contains one or more mutations. In some embodiments, the AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, A AVI 2, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof.
[0131] Exemplar}' AAV-Cas9 vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the Cas9 sequence. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, A AVI 2, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof. In some embodiments, the ITRs comprise or consist of full-length and/or wildtype sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, deletion, insertion, inversion, or transposition. In some embodiments, the ITRs comprise or consist of at least 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs comprise or consist of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs have a length of 110 ± 10 base pairs. In some embodiments, the ITRs have a length of 120 ± 10 base pairs. In some embodiments, the ITRs have a length of 130 ± 10 base pairs. In some embodiments, the ITRs have a length of 140 ± 10 base pairs. In some embodiments, the ITRs have a length of 150 ± 10 base pairs. In some embodiments, the ITRs have a length of 115, 145, or 141 base pairs. In some embodiments, the ITRs have a sequence selected from SEQ ID NO: 2584, SEQ ID NO: 2583, SEQ ID NO: 2585, SEQ ID NO: 2679 and SEQ ID. NO: 2582.
[0132] In some embodiments, the AAV-Cas9 vector may contain one or more nuclear localization signals (NLS). In some embodiments, the AAV-Cas9 vector contains 1, 2, 3, 4, or 5 nuclear localization signals. Exemplary NLS include the c-myc NLS (SEQ ID NO: 884), the SV40 NLS (SEQ ID NO: 885), the hnRNPAI M9 NLS (SEQ ID NO: 886), the nucleoplasmin NLS (SEQ ID NO: 887), the sequence
RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 888) of the IBB domain from importin-alpha, the sequences VSRKRPRP (SEQ ID NO: 889) and PPKKARED (SEQ ID NO: 890) of the myoma T protein, the sequence PQPKKKPL (SEQ ID NO: 891) of human p53, the sequence SALIKKKKKMAP (SEQ ID NO: 892) of mouse c-abl IV, the sequences DRLRR (SEQ ID NO: 893) and KQKKRK (SEQ ID NO: 894) of the influenza virus NS1, the sequence RKLKKKIKKL (SEQ ID NO: 895) of the Hepatitis virus delta antigen and the sequence REKKKFLKRR (SEQ ID NO: 896) of the mouse Mxl protein. Further acceptable nuclear localization signals include bipartite nuclear localization sequences such as the sequence KRKGDEVDGVDEVAKKKS KK (SEQ ID NO: 897) of the human poly(ADP-ribose) polymerase or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 898) of the steroid hormone receptors (human) glucocorticoid.
[0133] In some embodiments, the AAV-Cas9 vector may comprise additional elements to facilitate packaging of the vector and expression of the Cas9. In some embodiments, the AAV- Cas9 vector may comprise a polyA sequence. In some embodiments, the polyA sequence may be a mini-polyA sequence. In some embodiments, the AAV-Cas9 vector may comprise a transposable element. In some embodiments, the AAV-Cas9 vector may comprise a regulator element. In some embodiments, the regulator element is an activator or a repressor.
[0134] In some embodiments, the AAV-Cas9 vector may contain one or more promoters. In some embodiments, the one or more promoters drive expression of the Cas9. In some embodiments, the one or more promoters are muscle-specific promoters. Exemplary muscle- specific promoters include myosin light chain-2 promoter, the a-actin promoter, the troponin 1 promoter, the Na+/Ca2+ exchanger promoter, the dystrophin promoter, the a7 integrin promoter, the brain natriuretic peptide promoter, the aB-crystallin/small heat shock protein promoter, a- myosin heavy chain promoter, the ANF promoter, the CK8 promoter and the CK8e promoter.
[0135] In some embodiments, the AAV-Cas9 vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in human cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in a bacculovirus expression system.
[0136] In some embodiments, the AAV-Cas9 vector comprises a sequence of SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 2599, as shown in Table 3, or a sequence at least 90%, at least 95%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto. Table 3: Exemplary AAV-Cas9 Expression Cassette (from ITR to ITR)
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
[0137] In some embodiments of the AAV-Cas9 cassettes of the disclosure, including those embodiments encompassing SEQ ID NOs: 2599, the construct comprises or consists of a promoter sequence and a sequence encoding a nuclease. In some embodiments, the construct comprises or consists of a CK8e promoter and a sequence encoding a Cas9 nuclease. In some embodiments, the construct comprises or consists of a CK8e promoter and a sequence encoding a Cas9 nuclease isolated or derived from Staphylococcus pyogenes (“SpCas9”). In some embodiments, the CK8e promoter comprises or consists of a nucleotide sequence of TGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTAGAATTAACCCAGACATGTGGCTGCCCC CCCCCCCCCAACACCTGCTGCCICTAAAAATAACCCTGCATGCCATGTGCCCGGCGAAGGGCCAGCTGTCCCCCG CCAGCTAGACTCAGCACGTAGTTTAGGAACCAGTGAGCAAGTCAGCCCGTGGGGCAGCCCATACAAGGCCATGGG GCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACGGTGCCCGGGCAACGAGCTGAAAGCTCATCTGCTCTCA GGGGCCCCTCCCTGGGGACAGCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGGCACAG GGGCTGCCCTCATTCTACCACCACCTCCACAGCACAGACAGACACTCAGGAGCCAGCCAG (SEQ ID NO: 2590). In some embodiments, the SpCas9 nuclease comprises or consists of a nucleotide sequence of
GACAAGAAGTACAGCATCGGCCIGGACATCGGCACCAACTCTGTGGGCGGGGCCGTGATCACCGACGAGTACAAG GTGCCCAGCAAGAAATTCAAGGIGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTG CTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAG AACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTG GAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTGGGCAACATCGTGGACGAGGTG
GCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTG CGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACGTCCTGATCGAGGGCGACCTGAACCCC GACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATC AACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATC GCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCGCAAC TTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGAC AACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTG CTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGAC GAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTC TTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATC AAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAAGAGAGAGGACCTGCTGCGGAAG CAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAG GAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTAC GTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGG AACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAAC CTGCCCAACGAGAAGGTGCTGCGCAAGCACAGCCTGCTGTACGAGTACGTCACGGTGTATAACGAGCTGAGCAAA GTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTG CTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACGACTTCAAGAAAATCGAGTGCTTCGAC TCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATC AAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTT GAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGGTCGACGACAAAGTGATGAAGCAGCTG AAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGGATCCGGGACAAGCAGTGCGGC AAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACGTCATGCAGCTGATCCACGACGACAGC CTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAAT CTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATG GGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAAC AGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTG GAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAG GAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCC ATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTG AAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACC AAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAG ATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGGACGACGAGAATGACAAGCTGATCCGG GAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGC GAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTAC CCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAG CAGGAAATCGGCAAGGCGACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACC CTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACGGGGGAGATCGTGTGGGATAAG GGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAG
ACAGGCGGCTTCAGCAAAGAGTGTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGG GACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAG
GGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAG AATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTAC TCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTG GCCCTGCCCTCCAAATAGGTGAACTICCIGTACCTGGCCAGCCACIATGAGAAGCTGAAGGGCTCCCCCGAGGAT AATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTC TCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCC ATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTAC TTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGC ATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGAC (SEQ ID NO:2591). In SOOie embodiments, the construct comprising a promoter and a sequence encoding a nuclease further comprises at least two inverted terminal repeat (ITR) sequences. In some embodiments, the construct comprising a promoter and a sequence encoding a nuclease further comprises at least two ITR sequences isolated or derived from an AAV of serotype 2 (AAV2). In some embodiments, the construct comprising a promoter and a sequence encoding a nuclease further comprises at least two ITR sequences, wherein the first ITR sequence comprises or consists of a nucleotide sequence of
TGGCCACTCCCTCTCTGCGCGCICGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTG CCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT (SEQ ID NO: 2584) and the second ITR sequence comprises or consists of a nucleotide sequence of
AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGG ICGCCCGACGCCCGGGCGTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCA (SEQ
ID NO: 2679). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a sequence encoding a nuclease and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a sequence encoding a SpCas9 nuclease and a second AAV2 ITR.
[0138] In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a sequence encoding a nuclease and a second ITR, further comprises a poly A sequence. In some embodiments, the polyA sequence comprises or consists of a minipolyA sequence. Exemplary minipolyA sequences of the disclosure comprise or consist of a nucleotide sequence of lAGCAATAAAGGATCGTGTATTITCATTGGAAGCGTGTGITGGTTITTGGATCAGGCGCG (SEQ ID NO: 2680). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a sequence encoding a nuclease, a minipoly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a sequence encoding a SpCas9 nuclease, a minipoly A sequence and a second AAV2
ITR.
[0139] In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR, and further comprises at least one nuclear localization signal.
[0140] In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR, and further comprises at least two nuclear localization signals. Exemplary nuclear localization signals of the disclosure comprise or consist of a nucleotide sequence of CCAAAGAAGAAGCGGAAGGTC (SEQ ID NO: 2581), or a nucleotide sequence of
AAGCGTCCIGCIGCIACGAAGAAAGCTGGTCAAGCTAAGAAAAAGAAA (SEQ ID NO: 885). In Some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a first nuclear localization signal, a sequence encoding a nuclease, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a first nuclear localization signal, a sequence encoding a nuclease, a second nuclear localization signal, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 2581, a sequence encoding a SpCas9 nuclease, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 2580, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 2581, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 2580, a minipoly A sequence and a second AAV2 ITR.
[0141] In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a first nuclear localization signal, a sequence encoding a nuclease, a second nuclear localization signal, a poly A sequence and a second ITR, and further comprises a stop codon. The stop codon may have a sequence of TAG (SEQ ID NO: 904), TAA (SEQ ID NO: 905), or TGA (SEQ ID NO: 906). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, promoter, a first nuclear localization signal, a sequence encoding a nuclease, a second nuclear localization signal, a stop codon, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 2581, a sequence encoding a SpCas9 nuclease, a stop codon, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 2580, a stop codon, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 2581, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 2580, a stop codon a minipoly A sequence and a second AAV2 ITR.
[0142] In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a promoter, a first nuclear localization signal, a sequence encoding a nuclease, a second nuclear localization signal, a stop codon, a poly A sequence and a second ITR, and further comprises transposable element inverted repeats. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a promoter, a first nuclear localization signal, a sequence encoding a nuclease, a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, a regulatory sequence and a second transposable element inverted repeat. In some embodiments, the construct may further comprise one or more spacer sequences. Exemplary spacer sequences of the disclosure have length from 1-1500 nucleotides, inclusive of all ranges therebetween. In some embodiments, the spacer sequences may be located either 5’ to or 3’ to an ITR, a promoter, a nuclear localization sequence, a nuclease, a stop codon, a polyA sequence, a transposable element inverted repeat, and/or a regulator element.
[0143] In some embodiments, the construct may have a sequence comprising or consisting of SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 2599.
AAV-sgRNA expression cassettes and vectors
[0144] In some embodiments, an AAV expression cassette comprises a sequence encoding at least one gRNA, wherein the gRNA comprises a spacer region and a scaffold region. In some embodiments, the AAV expression cassette comprises, from 5’ to 3’, a U6 promoter, a sequence encoding a first sgRNA comprising a first spacer region and a first scaffold region, a HI promoter, a sequence encoding a second sgRNA comprising a second spacer region and a second scaffold region, a 7SK promoter, and a sequence encoding a third gRNA comprising a third sgRNA targeting region and third scaffold region, wherein the expression cassette is flanked by a first inverted terminal repeat (ITR) and a second ITR, wherein the first ITR has the sequence of SEQ ID NO: 2584, wherein the second ITR has the sequence of SEQ ID NO: 2583. In some embodiments, the AAV expression cassette is self-complementary. In some embodiments, the AAV expression cassette is not self-complementary.
[0145] In some embodiments, the U6 promoter has the sequence of SEQ ID NO: 2589, or a sequence at least 95% identical thereto. In some embodiments, the HI promoter has the sequence of SEQ ID NO: 2586, or a sequence at least 95% identical thereto. In some embodiments, the 7SK promoter has the sequence of SEQ ID NO: 2587, or a sequence at least 95% identical thereto.
[0146] In some embodiments, each of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are the same. In some embodiments, at least two of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are different. In some embodiments, each of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are different.
[0147] In some embodiments, at least one of the first spacer region, the second spacer region, and the third spacer region targets the human dystrophin gene. In some embodiments, the sequences of first spacer region, the second spacer region, and the third spacer region are each independently selected from any one of SEQ ID NO: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036-1043, 1052-1063, 1494-1499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668. In some embodiments, the sequences encoding the first, the second, and the third spacer regions are the same, and the sequences are each SEQ ID NO: 2668. In some embodiments, the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are the same. In some embodiments, at least two of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are different. In some embodiments, each of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are different. In some embodiments, at least one of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region has the sequence of SEQ ID NO: 2672, or a sequence at least 95% identical thereto. [0148] In some embodiments, the expression cassette further comprises a fourth sgRNA comprising a fourth spacer region and a fourth scaffold region. In some embodiments, the expression cassette further comprises a fifth sgRNA comprising a fifth spacer region and a fifth scaffold region.
[0149] In some embodiments, the expression cassette further comprises a stuffer sequence.
[0150] In some embodiments, wherein the expression cassette is less than about 2 kb, less than about 1.8 kb, less than about 1.6 kb, or less than about 1.4kb.
[0151] In some embodiments, an expression cassette comprising a sequence encoding at least one gRNA may be packaged into an AAV vector. In some embodiments, the AAV vector is a wildtype AAV vector. In some embodiments, the AAV vector contains one or more mutations. In some embodiments, the AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVRh.74, AAV2i8, AAVRh.lO, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof. In some embodiments, the AAV vector may be self-complementary. In some embodiments, the AAV vector may be non-self-complementary.
[0152] Exemplar}' AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising one or more sgRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.lO, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof. In some embodiments, the ITRs are isolated or derived from an AAV vector of a first serotype and a sequence encoding a capsid protein of the AAV-sgRNA vector is isolated or derived from an AAV vector of a second serotype, In some embodiments, the first serotype and the second serotype are the same. In some embodiments, the first serotype and the second serotype are not the same. In some embodiments, the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.lO, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.lO, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the first serotype is AAV2 and the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the first serotype is AAV2 and the second serotype is AAV9.
[0153] Exemplar}' AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising one or more gRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof. In some embodiments, a first ITR is isolated or derived from an AAV vector of a first serotype, a second ITR is isolated or derived from an AAV vector of a second serotype and a sequence encoding a capsid protein of the AAV-sgRNA vector is isolated or derived from an AAV vector of a third serotype. In some embodiments, the first serotype and the second serotype are the same. In some embodiments, the first serotype and the second serotype are not the same. In some embodiments, the first serotype, the second serotype, and the third serotype are the same. In some embodiments, the first serotype, the second serotype, and the third serotype are not the same. In some embodiments, the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV9. Exemplar}' AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the sgRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.lO, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof. In some embodiments, the ITRs comprise or consist of full- length and/or wildtype sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, deletion, insertion, inversion, or transposition. In some embodiments, the ITRs comprise or consist of at least 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs comprise or consist of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs have a length of 110 ± 10 base pairs. In some embodiments, the ITRs have a length of 120 + 10 base pairs. In some embodiments, the ITRs have a length of 130 ± 10 base pairs. In some embodiments, the ITRs have a length of 140 + 10 base pairs. In some embodiments, the ITRs have a length of 150 + 10 base pairs. In some embodiments, the ITRs have a length of 115, 145, or 141 base pairs. In some embodiments, at least one of the ITRs has a sequence selected from SEQ ID NO: 2582, SEQ ID NO: 2583, SEQ ID NO: 2584, SEQ ID NO: 2585 or SEQ ID NO: 2679.
[0154] In some embodiments, the AAV-sgRNA vector may comprise additional elements to facilitate packaging of the vector and expression of the sgRNA. In some embodiments, the AAV-sgRNA vector may comprise a transposable element. In some embodiments, the AAV- sgRNA vector may comprise a regulatory element. In some embodiments, the regulatory element comprises an activator or a repressor. In some embodiments, the AAV-sgRNA sequence may comprise a non-functional or “stuffer” sequence. Exemplary stuffer sequences of the disclosure may have some (a non-zero percentage of) identity or homology to a genomic sequence of a mammal (including a human). Alternatively, exemplary stuffer sequences of the disclosure may have no identify or homology to a genomic sequence of a mammal (including a human). Exemplary stuffer sequences of the disclosure may comprise or consist of naturally occurring non-coding sequences or sequences that are neither transcribed nor translated following administration of the AAV vector to a subject.
[0155] In some embodiments, the AAV-sgRNA vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV-sgRNA vector may be optimized for expression in human cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in a bacculovirus expression system.
[0156] In some embodiments, the AAV-sgRNA vector comprises at least one promoter. In some embodiments, the AAV-sgRNA vector comprises at least two promoters. In some embodiments, the AAV-sgRNA vector comprises at least three promoters. In some embodiments, the AAV-sgRNA vector comprises at least four promoters. In some embodiments, the AAV-sgRNA vector comprises at least five promoters. Exemplar}' promoters include, for example, immunoglobulin light chain, immunoglobulin heavy chain,
T-cell receptor, HLA DQ a and/or DQ b, b-interferon, interleukin-2, interleukin-2 receptor, MHC class II, MHC class II HLA-Dra, b-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, a-fetoprotein, t- globin, b-globin, c-fos, c-HA-ra.s, insulin, neural cell adhesion molecule (NCAM), <Xi- antitrypain, H2B (TH2B) histone, mouse and/or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), Duchenne muscular dystrophy, SV40, polyoma, retroviruses, papilloma virus, hepatitis B vims, human immunodeficiency vims, cytomegalovirus (CMV), and gibbon ape leukemia virus. Further exemplary promoters include the U6 promoter, the HI promoter, and/or the 7SK promoter.
[0157] In some embodiments, the sequence encoding the gRNA spacer comprises a sequence selected from any one of SEQ ID Nos: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036- 1043, 1052-1063, 1494-1499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668.
[0158] In some embodiments, the AAV expression cassette or vector comprises a first sequence encoding a gRNA and a second sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA and a second promoter drives expression of the second sequence encoding a gRNA. In some embodiments, the first and second promoters are the same. In some embodiments, the first and second promoters are different. In some embodiments, the first and second promoters are selected from the HI promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first sequence encoding a gRNA and the second sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA and the second sequence encoding a gRNA are not identical.
[0159] In some embodiments, the AAV expression cassette or vector comprises a first sequence encoding a gRNA, a second sequence encoding a gRNA, and a third sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA, a second promoter drives expression of the second sequence encoding a gRNA, and a third promoter drives expression of a third sequence encoding a gRNA. In some embodiments, at least two of the first, second, and third promoters are the same. In some embodiments, each of the first, second, and third promoters are different. In some embodiments, the first, second, and third promoters are selected from the HI promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first promoter is the U6 promoter. In some embodiments, the second promoter is the HI promoter. In some embodiments, the third promoter is the 7SK promoter. In some embodiments, the first promoter is the U6 promoter, the second promoter is the HI promoter, and the third promoter is the 7SK promoter. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, and the third sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, and the third sequence encoding a gRNA are not identical.
[0160] In some embodiments, the AAV vector comprises a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, and a fourth sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA, a second promoter drives expression of the second sequence encoding a gRNA, a third promoter drives expression of the third sequence encoding a gRNA, and a fourth promoter drives expression of the fourth sequence encoding a gRNA. In some embodiments, at least two of the first, second, third, and fourth promoters are the same. In some embodiments, each of the first, second, third, and fourth promoters are different. In some embodiments, each of the first, second, third and fourth promoters are selected from the HI promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, and the fourth sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, and the fourth sequence encoding a gRNA are not identical.
[0161] In some embodiments, the AAV vector comprises a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, a fourth sequence encoding a gRNA, and a fifth sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA, a second promoter drives expression of the second sequence encoding a gRNA, a third promoter drives expression of the third sequence encoding a gRNA, a fourth promoter drives expression of the fourth sequence encoding a gRNA, and a fifth promoter drives expression of the fifth sequence encoding a gRNA. In some embodiments, at least two of the first, second, third, fourth, and fifth promoters are the same. In some embodiments, each of the first, second, third, fourth, and fifth promoters are different. In some embodiments, each of the first, second, third, and fourth promoters are different. In some embodiments, each of the first, second, third, fourth and fifth promoters are selected from the HI promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, the fourth sequence encoding a gRNA, and the fifth sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, the fourth sequence encoding a gRNA, and the fifth sequence encoding a gRNA are not identical.
[0162] In some embodiments, the AAV-sgRNA vector comprises a sequence of SEQ ID NO: 2597, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. SEQ ID NO: 2597 is provided in Table 4.
Table 4. Exemplary AAV-sgRNA vector.
Figure imgf000062_0001
[0163] In some embodiments of the AAV-sgRNA cassettes of the disclosure, including those embodiments encompassing SEQ ID NO: 2597, the construct comprises or consists of a first promoter, a first sequence encoding a gRNA (spacer + scaffold), a second promoter, and a second sequence encoding a gRNA (spacer + scaffold), a third promoter, and a third sequence encoding a gRNA (spacer + scaffold). Exemplary gRNA spacer sequences include SEQ ID NO: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036-1043, 1052-1063, 1494-1499, 1500- 1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668. Exemplary sequences encoding gRNA scaffold sequences are SEQ ID NO: 2672 to 2678. In some embodiments, the sequence encoding the gRNA spacer is GGCTTACAGGAACICCAGGA (SEQ ID NO: 2668). In some embodiments, the sequence encoding the gRNA spacer is CACTAGAGT AAC AG T c T G Ac (SEQ ID NO: 708). In some embodiments, the sequence encoding the gRNA spacer is AICTTACAGGAACTCCAGGA (SEQ ID NO: 762). In some embodiments, the sequence encoding the gRNA spacer is CACCAGAGTAACAGTCTGAG (SEQ ID NO: 714). In some embodiments, the sequence encoding the gRNA spacer is CACCAGAGTAACAGTCTGAC (SEQ ID NO: 863). [0164] In some embodiments, the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a second promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 2668, a third promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 2668. In some embodiments, the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a second promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 708, a third promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 708. In some embodiments, the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, a second promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 714, a third promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 714. In some embodiments, the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, a second promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 863, a third promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 863. In some embodiments, the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a second promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 762, a third promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 762.
[0165] Exemplary promoters of the disclosure include the U6 promoter having a sequence of
GAGGGCCTATTTCCCATGATTCGTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAAT
ITGACTGTAAACACAAAGATATIAGIACAAAATACGTGACGTAGAAAGGAATAATTICTIGGGTAGTTTGCAGIT
TTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATAT
ATCTTGTGGAAAGGAC (SEQ ID NO: 2589), the HI promoter having a sequence of
ATATTTGCATGICGCTAGGTGTICTGGGAAAICACCATAAACGTGAAAGGTCTITGGATITGGGAATCTTATAAC- TTCTGTATGAGACCAC (SEQ ID. NO: 2586), and the 7SK promoter having a sequence of
ATTTGCTATGCTGGTTAAAT TAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGTAACTTGACCTA AGTGTAAAGTTGAGATTGCCTTCAGGTTiATATAGCTTGiGCGCCGCCGG (SEQ ID NO: 2587). In some embodiments, the first, second, and third promoter are each individually selected from the U6 promoter (SEQ ID NO: 2589), the H1 promoter (SEQ ID NO: 2586), and the 7SK promoter (SEQ ID NO: 2587). In some embodiments, the first, second, and third promoter are each individually selected from the U6 promoter (SEQ ID NO: 2589), and the HI promoter (SEQ ID NO: 2586). In some embodiments, the construct comprises, from 5’ to 3’, a U6 promoter, a first sequence encoding a gRNA, a HI promoter, a second sequence encoding a gRNA, a 7SK promoter, and a third sequence encoding a gRNA. In some embodiments, the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, the HI promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 2668, the 7SK promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 2668. In some embodiments, the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, the HI promoter, a second sequence encoding a gRNA comprising SEQ ID NO: 714, the 7SK promoter, and a third sequence encoding a gRNA of SEQ ID NO: 714. In some embodiments, the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, the HI promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 708, the 7SK promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 708. In some embodiments, the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, the HI promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 762, the 7SK promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 762. In some embodiments, the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, the HI promoter, a second sequence encoding a gRNA spacer of SEQ ID NO: 863, the 7SK promoter, and a third sequence encoding a gRNA spacer of SEQ ID NO: 863.
[0166] In some embodiments, the construct comprising a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, and a third sequence encoding a gRNA further comprises at least two inverted terminal repeat (ITR) sequences. In some embodiments, the construct comprising a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, and a third sequence encoding a gRNA further comprises at least two ITR sequences isolated or derived from an AAV of serotype 2 (AAV2). In some embodiments, the construct comprising a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, and a third sequence encoding a gRNA further comprises at least two ITR sequences, wherein the first ITR sequence is isolated or derived from an AAV of serotype 4 (AAV4) and the second ITR sequence is isolated or derived from an AAV of serotype 2 (AAV2). Exemplary ITR sequences are
CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGGCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGG (SEQ ID NO: 2584) and
AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGG TCGCCCGACGCCCGGGCGTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGGCGTAATAGCGAAGAGG CCCGCACCGATCGCCCTGC (SEQ ID NO: 2583). In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, a first sequence encoding a gRNA, and a second ITR. In some embodiments, the construct comprises or encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, a third sequence consists of, from 5’ to 3’, a first ITR, a U6 promoter, a first sequence encoding a gRNA, a HI promoter, and a second sequence encoding a gRNA, a 7SK promoter, a third sequence encoding a gRNA, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a second promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a second promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 762, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a second promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, a second promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, a second promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, and a second ITR.
[0167] In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a HI promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 708, a 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, a Hl promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 714, a 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a HI promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a HI promoter, and the sequence encoding a gRNA spacer of SEQ ID NO: 762, a 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, and a second ITR.
[0168] In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, and a second ITR, further comprises a poly A sequence. In some embodiments, the poly A sequence comprises or consists of a minipolyA sequence. Exemplary minipolyA sequences of the disclosure comprise or consist of a nucleotide sequence of
TAGCAATAAAGGATCGTGTATTTTCATTGGAAGCGTGTGITGGTTTTTGGATCAGGCGCG (SEQ ID NO: 903). In some embodiments, the constmct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding sgRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, a first sequence encoding a gRNA, the HI promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a second promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a second promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA spacer of SEQ ID NO.: 714, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 863, a minipolyA sequence, and a second ITR.
[0169] In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, the HI promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, the 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 2668, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, the HI promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, the 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 762, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, the HI promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 708, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, the HI promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, the 7SK promoter, the sequence encoding a gRNA spacer of SEQ ID NO: 714, a minipolyA sequence, and a second ITR.
Exemplary Therapeutic Nucleic Acids and Vectors
[0170] In some embodiments, a nucleic acid comprises a sequence encoding a first guide RNA targeting a first genomic target sequence, a sequence encoding a second guide RNA targeting a second genomic target sequence, a first promoter wherein the first promoter drives expression of the sequence encoding the first guide RNA, and a second promoter wherein the first promoter drives expression of the sequence encoding the second guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a genomic locus of interest. In some embodiments, the genomic locus of interest is a splice acceptor or splice donor site.
[0171] In some embodiments, a nucleic acid comprises a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a first promoter wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a second promoter wherein the first promoter drives expression of the sequence encoding the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor site.
[0172] In some embodiments, the first promoter and the second promoter are identical. In some embodiments, the first promoter and the second promoter not identical. In some embodiments, the first promoter and the second promoter share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity.
[0173] In some embodiments, the first genomic target sequence and the second genomic target sequence are identical. In some embodiments, the first genomic target sequence and the second genomic target sequence are not identical. In some embodiments, the first genomic target sequence and the second genomic target sequence share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, the first genomic target sequence and the second genomic target sequence are complementary.
[0174] In some embodiments, the nucleic acid further comprises a sequence encoding a third DMD guide RNA targeting a third genomic target sequence, and a third promoter wherein the third promoter drives expression of the sequence encoding the third DMD guide RNA, and wherein the third genomic target sequence comprises a dystrophin splice acceptor site. In some embodiments, at least two of the first promoter, the second promoter, and the third promoter are identical. In some embodiments, at least two of the first promoter, the second promoter, and the third promoter are not identical. In some embodiments, at least two of the first promoter, the second promoter, and the third promoter share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence are identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence are not identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence are complementary.
[0175] In some embodiments, the nucleic acid further comprises a sequence encoding a fourth DMD guide RNA targeting a fourth genomic target sequence, and a fourth promoter, wherein the fourth promoter drives expression of the fourth sequence encoding a DMD guide RNA, wherein the fourth genomic target sequence comprises a dystrophin splice acceptor site. In some embodiments, at least two of the first promoter, the second promoter, the third promoter, and the fourth promoter are identical. In some embodiments, at least two of the first promoter, the second promoter, the third promoter, and the fourth promoter are not identical. In some embodiments, at least two of the first promoter, the second promoter, the third promoter, and the fourth promoter share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence are identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence are not identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence are complementary.
[0176] In some embodiments, the nucleic acid further comprises a sequence encoding a fifth DMD guide RNA targeting a fifth genomic target sequence, and a fifth promoter, wherein the fifth promoter drives expression of the sequence encoding the fifth DMD guide RNA, wherein the fifth genomic target sequence comprises a dystrophin splice acceptor site. In some embodiments, at least two of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter are identical. In some embodiments, at least two of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter are not identical. In some embodiments, at least two of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence are identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence are not identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence are complementary.
[0177] In some embodiments, the nucleic acid further comprises at least one sequence encoding an additional DMD guide RNA targeting a genomic target sequence, and at least one additional promoter, wherein the additional promoter drives expression of the additional DMD guide RNA, wherein the additional genomic target sequence comprises a dystrophin splice acceptor site. In some embodiments, the dystrophin splice acceptor site comprises the 5’ splice acceptor site of exon 51. In some embodiments, the first promoter or the second promoter comprises a constitutive promoter. In some embodiments, the first promoter or the second promoter comprises a constitutive promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a sequence encoding a constitutive promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a constitutive promoter. In some embodiments, the first promoter or the second promoter comprises an inducible promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises an inducible promoter. In some embodiments, the first promoter or the second promoter comprises a cell-type specific promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a cell-type specific promoter. In some embodiments, the cell type specific promoter comprises a muscle-specific promoter. In some embodiments, the first promoter or the second promoter comprises a U6 promoter, an HI promoter, or a 7SK promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a U6 promoter, an HI promoter, or a 7SK promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a U6 promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises an HI promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a 7SK promoter.
[0178] In some embodiments, the sequence encoding the first DMD guide RNA, the sequence encoding the second DMD guide RNA, and sequence encoding the third DMD guide RNA are identical, and the 5’ splice acceptor site comprises a 5’ splice acceptor site of exon 51. In some embodiments, the first promoter comprises a U6 promoter, the second promoter comprises a HI promoter, and the third promoter comprises a 7SK promoter. In some embodiments, the nucleic acid comprises a DNA sequence. In some embodiments, the nucleic acid comprises an RNA sequence.
[0179] In some embodiments, the nucleic acid further comprises one or more sequences encoding an inverted terminal repeat (ITR). In some embodiments, the nucleic acid further comprises a sequence encoding a 5’ inverted terminal repeat (ITR) and a sequence encoding a 3’ ITR. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno- associated vims (AAY). In some embodiments, the sequence encoding the 5! inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated vims (AAV) of serotype 2 (AAV2). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV2. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV) of serotype 4 (AAV4). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV4. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3! ITR comprises or consists of 145 nucleotides. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 115 nucleotides. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 141 nucleotides. In some embodiments, the nucleic acid further comprises a polyadenosine (poly A) sequence. In some embodiments, the poly A sequence is a mini poly A sequence. In some embodiments, the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises the sequence of any one of SEQ ID NOs: 60-382, 706- 708, 712-714, 718-789, 863, 947, 1036-1043, 1052-1063, 1494-1499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, and 2668. In some embodiments, the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises the sequence of SEQ ID NO: 714.
[0180] Also provided is a vector comprising a nucleic acid comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a first promoter wherein the first promoter drives expression of the first DMD guide RNA, and a second promoter wherein the second promoter drives expression of the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor site. In some embodiments, the vector further comprises an inverted terminal repeat of a transposable element. In some embodiments, the transposable element is a transposon. In some embodiments, the transposon is a Tn7 transposon. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a plasmid. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector is replication- defective or conditionally replication defective. In some embodiments, the AAV vector is a recombinant AAV vector. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11), AAVrh74, AAVrhlO or any combination thereof. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 9 (AAV9). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 2 (AAV2). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV2 and a sequence isolated or derived from an AAV9. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV4 and a sequence isolated or derived from an AAV9. In some embodiments, the vector is optimized for expression in mammalian cells. In some embodiments, the vector is optimized for expression in human cells. [0181] Also provided is a nucleic acid comprising a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the promoter comprises a muscle-specific promoter. In some embodiments, the muscle-specific promoter comprises a CK8 promoter. In some embodiments, the muscle-specific promoter comprises a CK8e promoter. In some embodiments, the sequence encoding the Cas9 or the nuclease domain thereof is isolated or derived from a sequence encoding an S. pyogenes Cas9 or a nuclease domain thereof. In some embodiments, the sequence encoding the Cas9 or the nuclease domain thereof is isolated or derived from a sequence encoding S. aureus Cas9 or a nuclease domain thereof. In some embodiments, the sequence encoding the Cas9 or the nuclease domain thereof is codon optimized for expression in a mammal. In some embodiments, the sequence encoding the Cas9 or the nuclease domain thereof is codon optimized for expression in a human.
[0182] In some embodiments, the nucleic acid comprising a promoter and a sequence encoding a Cas9 or a nuclease domain thereof further comprises a polyA sequence. In some embodiments, the polyA sequence is a mini polyA sequence. In some embodiments, the nucleic acid further comprises one or more sequences encoding an inverted terminal repeat (ITR). In some embodiments, the nucleic acid further comprises a sequence encoding a 5’ inverted terminal repeat (ITR) and a sequence encoding a 3’ ITR. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV) of serotype 2 (AAV2). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV2. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated vims (AAV) of serotype 4 (AAV4). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV4. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 145 nucleotides, 115 nucleotides, or 141 nucleotides. In some embodiments, the nucleic acid further comprises a nuclear localization signal. In some embodiments, the nucleic acid is optimized for expression in am alian cells. In some embodiments, the nucleic acid is optimized for expression in human cells. [0183] Also provided is a vector comprising a nucleic acid comprising a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the promoter comprises a muscle-specific promoter such as the CK8 or CK8e promoter. In some embodiments, the vector further comprises an inverted terminal repeat (ITR) of a transposable element. In some embodiments, the transposable element is a transposon. In some embodiments, the transposon is a Tn7 transposon. In some embodiments, the vector further comprises a sequence encoding a 5’ ITR of a T7 transposon and a sequence encoding a 3’ ITR of a T7 transposon. In some embodiments, the vector is a non- viral vector. In some embodiments, the non-viral vector is a plasmid. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector is replication-defective or conditionally replication defective. In some embodiments, the AAV vector is a recombinant AAV vector. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11), AAVrh74, AAVrhlO or any combination thereof. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 9 (AAV9). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 2 (AAV2). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV2 and a sequence isolated or derived from an AAV9. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 4 (AAV4). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV4 and a sequence isolated or derived from an AAV9. In some embodiments, wherein the vector is optimized for expression in mammalian cells. In some embodiments, the vector is optimized for expression in human cells.
Pharmaceutical Compositions and Delivery Methods
[0184] Also provided herein are compositions comprising one or more vectors, expression cassettes and/or nucleic acids of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.
[0185] For clinical applications, pharmaceutical compositions are prepared in a form appropriate for the intended application. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. [0186] Appropriate salts and buffers are used to render drugs, proteins or delivery vectors stable and allow for uptake by target cells. Aqueous compositions of the present disclosure comprise an effective amount of the dmg, vector or proteins, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is not incompatible with the active ingredients of the present disclosure, its use in therapeutic compositions may be used. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or cells of the compositions.
[0187] In some embodiments, the active compositions of the present disclosure may include classic pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any common route so long as the target tissue is available via that route, but generally including systemic administration· This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into muscle tissue. Such compositions would normally be administered as pharmaceutically acceptable compositions, as described supra.
[0188] The active compounds may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.
[0189] The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
[0190] Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0191] In some embodiments, the compositions of the present disclosure are formulated in a neutral or salt form. Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like). Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine) and the like.
[0192] Upon formulation, solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, dmg release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration· Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
[0193] In some embodiments, a first vector and a second vector are administered to a patient. In some embodiments, the first vector comprises a nucleic acid comprising a first sequence encoding a first DMD guide RNA targeting a first genomic target sequence; a sequence encoding a second DMD guide RNA targeting a second genomic target sequence; a first promoter, wherein the first promoter drives expression of the first DMD guide RNA; and a second promoter, wherein the second promoter drives expression of the second DMD guide RNA. In some embodiments, the second vector comprise a nucleic acid comprising a sequence encoding a Cas9 or a nuclease domain thereof; a muscle-specific promoter, wherein the muscle-specific promoter drives expression of the Cas9 or a nuclease domain thereof.
Therapeutically-Effecitve Ratios
[0194] In some embodiments, a first vector and a second vector are administered to a patient in a therapeutically effective ratio. As used herein, the term “ratio” may refer to a concentration ratio (i.e., a ratio of the amount of vectors in a composition), a dose ratio (/. e. , a ratio of the amount delivered to a patient), a bioavailability ratio ( i.e ., a ratio of the amount available to a therapeutic site), a copy number ratio (i.e., a ratio of the amount expressed by a target cell), an efficacy ratio (i.e., a ratio of the amount of modifications made), a DNA ratio (i.e., a ratio of the amount of DNA), or a coding sequences ratio (i.e. a ratio of the number of coding sequences, e.g., sequences encoding a gRNA or a Cas9).
[0195] In some embodiments, the ratio of the first vector and the second vector is between 1 : 1 and 1:30. In other embodiments, the ratio of the first vector and the second vector is between 30:1 and 1:1. In some embodiments, the ratio of the first vector and the second vector is any
Figure imgf000078_0001
one of the ratios shown in Table 5 below.
Figure imgf000079_0001
[0196] In some embodiments, the ratio of the amount of the first vector and amount of the second vector is between 1:1 and 1:30. In other embodiments, the ratio of the amount of the first vector and amount of the second vector is between 30:1 and 1:1. In some embodiments, the ratio of the amount of first vector and the amount of the second vector is any one of the ratios shown in Table 5.
[0197] In some embodiments, the first vector is an AAV-Cas9 vector of the disclosure and the second vector is an AAV-sgRNA vector of the disclosure. In some embodiments, the ratio of the AAV-Cas9 vector to the AAV-sgRNA vector is any one of the ratios shown in Table 5. [0198] In some embodiments, the ratio of the first vector to the second vector is greater than
10:1. For example, the ratio of the first vector to the second vector may be about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 50:1, about 75:1, or about 100:1. In some embodiments, the ratio of an AAV-sgRNA vector to an AAV-Cas9 vector is greater than 10:1; for example, the ratio may be about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 50:1, about 75:1, or about 100:1.
[0199] In some embodiments, between 4 x 1012 viral genomes (vg)/kilogram (kg) and 3 x 1013 vg/kg, inclusive of the endpoints, of the first and/or the second vector are administered to the patient. In some embodiments, between 4 x 1012 viral genomes (vg)/kilogram (kg) and 3 x 1013 vg/kg, inclusive of the endpoints, of the first and/or the second vector are administered to the patient. In some embodiments, at least 5 x 1012 viral genomes (vg)/kilogram (kg), 6 x 1012 viral genomes (vg)/kilogram (kg), 1 x 1013 viral genomes (vg)/kilogram (kg), 2 x 1013 viral genomes (vg)/kilogram (kg), 3 x 1013 viral genomes (vg)/kilogram (kg), 5 x 1013 viral genomes (vg)/kilogram (kg), 1 x 1014 viral genomes (vg)/kilogram (kg), 2 x 1014 viral genomes (vg)/kilogram (kg), 3 x 1014 viral genomes (vg)/kilogram (kg), or 4 x 1014 viral genomes (vg)/kilogram (kg) of the first and/or the second vector are administered to the patient.
[0200] In some embodiments, the Cas9 or Cpfl and gRNAs described herein may be delivered to the patient using adoptive cell transfer (ACT). In adoptive cell transfer, one or more expression constructs are provided ex vivo to cells which have originated from the patient (autologous) or from one or more individual(s) other than the patient (allogeneic). The cells are subsequently introduced or reintroduced into the patient. Thus, in some embodiments, one or more nucleic acids encoding Cas9 or Cpfl and a guide RNA that targets a dystrophin splice site are provided to a cell ex vivo before the cell is introduced or reintroduced to a patient.
[0201] In some embodiments, a composition comprises (i) a first nucleic acid sequence comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a first promoter wherein the first promoter drives expression of the first DMD guide RNA, and a second promoter wherein the second promoter drives expression of the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor and (ii) a second nucleic acid sequence comprising a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the comprises a muscle-specific promoter such as the CK8 or CK8e promoter. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.
[0202] In some embodiments, a composition comprises (i) a first vector comprising a nucleic acid sequence comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a first promoter wherein the first promoter drives expression of the first DMD guide RNA, and a second promoter wherein the second promoter drives expression of the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor and (ii) a second vector comprising a nucleic acid sequence comprising a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the promoter comprises a sequence encoding a muscle- specific promoter such as the CK8 or CK8e promoter. In some embodiments, at least one of the first vector and the second vectors are AAVs. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.
Cells and Cell Compositions
[0203] Also provided is a cell comprising one or more nucleic acids of the disclosure. In some embodiments, the cell is a human cell. In some embodiments, the cell is a muscle cell or satellite cell. In some embodiments, the cell is an induced pluripotent stem (iPS) cell. Also provided is a composition comprising a cell comprising one or more nucleic acids of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.
[0204] Also provided is a cell comprising a composition comprising one or more vectors of the disclosure. In some embodiments, the cell is a human cell. In some embodiments, the cell is a muscle cell or satellite cell. In some embodiments, the cell is an induced pluripotent stem (iPS) cell.
Therapeutic Methods and Uses
[0205] Provided herein are methods of correcting a gene defect in a cell, the methods comprising contacting an AAV vector comprising an AAV expression cassette of the disclosure with the cell. In some embodiments, the methods comprise contacting the cell with a first AAV vector comprising an AAV-sgRNA expression cassette and a second AAV vector comprising an AAV-Cas9 expression cassette of the disclosure. In some embodiments, the AAV-sgRNA expression cassette is self-complementary. In some embodiments, the AAV- Cas9 expression cassette is not self-complementary. In some embodiments, the AAV-sgRNA expression cassette is self-complementary and the AAV-Cas9 expression cassette is not selfcomplementary. [0206] Also provided herein is method of treating a subject in need thereof comprising administering to the subject a first AAV vector comprising a self-complementary AAV-sgRNA expression cassette of the disclosure. The subject may be a mammal, such as a human. In some embodiments, the subject suffers from a genetic disease. In some embodiments, the subject suffers from a monogenic disease. In some embodiments, the subject suffers from a polygenic disease. In some embodiments, the subject suffers from a genetic muscle disease. In some embodiments, the subject suffers from Duchenne Muscular Dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss dystrophy, myotonic dystrophy, limb-girdle muscular dystrophy (LGMD), oculopharyngeal muscular dystrophy, congential dystrophy, congenital myopathy, familial periodic paralysis. In some embodiments, the subject suffers from DMD. In some embodiments, the subject suffers from mitochondrial oxidative phosphorylation disorder or a glycogen storage disease (e.g., von Gierke’s disease, Pompe’s disease, Forbes-Cori disease, Andersen’s disease, McArdle’s disease, Hers’ disease, Tarui’s disease, or Fanconi-Bickel syndrome).
[0207] In some embodiments, the subject suffers from Duchenne Muscular Dystrophy (DMD). In some embodiments, the method also comprises administering to the subject a second AAV vector comprising an AAV-Cas9 expression cassette. In some embodiments, the expression cassette for the Cas9 nuclease is not self-complimentary.
[0208] In some embodiments, dystrophin expression is at least partially restored in skeletal muscle in the patient. In some embodiments, dystrophin expression is at least partially restored in heart muscle in the patient. In some embodiments, the dosage of the first AAV required to at least partially restore dystrophin expression is at least about 20-fold lower than the dosage that would be required to achieve the same level of dystrophin expression if the expression cassette of the first AAV was not self-complimentary.
[0209] In some embodiments, a method of treating a subject in need thereof comprises administering to the patient a therapeutically effective amount of an AAV-sgRNA vector of the disclosure, or a composition comprising the same, and a AAV-Cas9 vector of the disclosure, or a composition comprising the same, wherein the subject suffers from Duchenne Muscular Dystrophy (DMD).
[0210] Also provided is a method for correcting a dystrophin defect, the method comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the first DMD guide RNA, the second DMD guide RNA and the Cas9 protein or a nuclease domain thereof, wherein at least one of first DMD guide RNA or the second DMD guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one DMD guide RNA-Cas9 complex, wherein the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping of a DMD exon and/or refraining. In some embodiments, the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces a reframing of a dystrophin reading frame. In some embodiments, the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and produces an insertion which restores the dystrophin protein reading frame. In some embodiments, the insertion comprises an insertion of a single adenosine.
[0211] Also provided is a method for inducing selective skipping and/or reframing of a DMD exon, the method comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the first DMD guide RNA, the second DMD guide RNA and the Cas9 protein or a nuclease domain thereof, wherein at least one of first DMD guide RNA or the second DMD guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one DMD guide RNA-Cas9 complex, wherein the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping and/or refraining of a DMD exon.
[0212] Also provided is a method for inducing a reframing event in the dystrophin reading frame, the method comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the first DMD guide RNA, the second DMD guide RNA and the Cas9 protein or a nuclease domain thereof, wherein at least one of first DMD guide RNA or the second DMD guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one DMD guide RNA-Cas9 complex, wherein the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping and/or reframing of a DMD exon. In some embodiments, the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping and/or reframing of exon 51 of a human DMD gene.
[0213] Also provided is a method of treating muscular dystrophy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of one or more compositions of the disclosure. In some embodiments, the composition is administered locally. In some embodiments, the composition is administered directly to a muscle tissue. In some embodiments, the composition is administered by an intramuscular infusion or injection. In some embodiments, the muscle tissue comprises a tibialis anterior tissue, a quadriceps tissue, a soleus tissue, a diaphragm tissue, or a heart tissue. In some embodiments, the composition is administered by an intra-cardiac injection. In some embodiments, the composition is administered systemically. In some embodiments, the composition is administered by an intravenous infusion or injection. In some embodiments, following administration of the composition, the subject exhibits normal dystrophin-positive myofibers, and mosaic dystrophin-positive myofibers containing centralized nuclei, or a combination thereof. In some embodiments, following administration of the composition, the subject exhibits an emergence or an increase in a level of abundance of normal dystrophin-positive myofibers when compared to an absence or a level of abundance of normal dystrophin-positive myofibers prior to administration of the composition. In some embodiments, following administration of the composition, the subject exhibits an emergence or an increase in a level of abundance of mosaic dystrophin-positive myofibers containing centralized nuclei when compared to an absence or an level of abundance of mosaic dystrophin-positive myofibers containing centralized nuclei prior to administration of the composition. In some embodiments, following administration of the composition, the subject exhibits a decreased semm CK level when compared to a serum CK level prior to administration of the composition. In some embodiment, following administration of the composition, the subject exhibits improved grip strength when compared to a grip strength prior to administration of the composition. In some embodiments, the subject is a neonate, an infant, a child, a young adult, or an adult. In some embodiments, the subject has muscular dystrophy. In some embodiments, the subject is a genetic carrier for muscular dystrophy. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject appears to be asymptomatic and a genetic diagnosis reveals a mutation in one or both copies of a DMD gene that impairs function of the DMD gene product. In some embodiments, the subject presents an early sign or symptom of muscular dystrophy. In some embodiments, the early sign or symptom of muscular dystrophy comprises loss of muscle mass or proximal muscle weakness. In some embodiments, the loss of muscle mass or proximal muscle weakness occurs in one or both leg(s) and/or a pelvis, followed by one or more upper body muscle(s). In some embodiments, the early sign or symptom of muscular dystrophy further comprises pseudohypertrophy, low endurance, difficulty standing, difficulty walking, difficulty ascending a staircase or a combination thereof. In some embodiments, the subject presents a progressive sign or symptom of muscular dystrophy. In some embodiments, the progressive sign or symptom of muscular dystrophy comprises muscle tissue wasting, replacement of muscle tissue with fat, or replacement of muscle tissue with fibrotic tissue. In some embodiments, the subject presents a later sign or symptom of muscular dystrophy. In some embodiments, the later sign or symptom of muscular dystrophy comprises abnormal bone development, curvature of the spine, loss of movement, and paralysis. In some embodiments, the subject presents a neurological sign or symptom of muscular dystrophy. In some embodiments, the neurological sign or symptom of muscular dystrophy comprises intellectual impairment and paralysis. In some embodiments, administration of the composition occurs prior to the subject presenting one or more progressive, later or neurological signs or symptoms of muscular dystrophy. In some embodiments, the subject is less than 10 years old, less than 5 years old, or less than 2 years old.
[0214] Also provided is the use of a therapeutically-effective amount of one or more compositions of the disclosure for treating muscular dystrophy in a subject in need thereof.
[0215] Tables 6-10, 13, and 15-17 provide exemplary guide RNA spacer sequences for use in connection with the compositions and methods disclosed herein.
Delivery Vectors
[0216] There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals.
[0217] One of the preferred methods for in vivo delivery involves the use of an adenovirus expression vector. “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.
[0218] The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA vims, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB. In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
[0219] Adenovirus is particularly suitable for use as a gene transfer vector because of its midsized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5 ‘-tripartite leader (TPL) sequence which makes them preferred mRNAs for translation. In one system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two pro viral vectors, wild- type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of vims from an individual plaque and examine its genomic structure.
[0220] Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins. Since the E3 region is dispensable from the adenovirus genome, the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions. In nature, adenovirus can package approximately 105% of the wild-type genome, providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the E1 and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector- borne cytotoxicity. Also, the replication deficiency of the El -deleted virus is incomplete.
[0221] Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g. , Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.
[0222] Improved methods for culturing 293 cells and propagating adenovirus are known in the art. In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.
[0223] The adenoviruses of the disclosure are replication defective, or at least conditionally replication defective. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present disclosure.
[0224] As stated above, the typical vector according to the present disclosure is replication defective and will not have an adenovims El region. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the El - coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors, or in the E4 region where a helper cell line or helper virus complements the E4 defect. [0225] Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g. , 109-1012 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus, demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
[0226] Adenovirus vectors have been used in eukaryotic gene expression and vaccine development. Animal studies suggested that recombinant adenovirus could be used for gene therapy. Studies in administering recombinant adenovirus to different tissues include trachea instillation, muscle injection, peripheral intravenous injections and stereotactic inoculation into the brain.
[0227] The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse- transcription. The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5‘ and 3‘ ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome.
[0228] In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells. [0229] A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification could permit the specific infection of hepatocytes via sialoglycoprotein receptors.
[0230] A different approach to targeting of recombinant retroviruses may be used, in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor are used. The antibodies are coupled via the biotin components by using streptavidin. Using antibodies against major histocompatibility complex class I and class II antigens, it has been demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro.
[0231] There are certain limitations to the use of retrovirus vectors in all aspects of the present disclosure. For example, retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes. Another concern with the use of defective retrovirus vectors is the potential appearance of wild- type replication-competent virus in the packaging cells. This can result from recombination events in which the intact- sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, packaging cell lines may be selected to greatly decrease the likelihood of recombination.
[0232] Other viral vectors may be employed as expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus adeno-associated virus (AAV) and herpesviruses may be employed. They offer several attractive features for various mammalian cells.
[0233] In embodiments, the AAV vector is replication-defective or conditionally replication defective. In embodiments, the AAV vector is a recombinant AAV vector. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof.
[0234] In some embodiments, a single viral vector is used to deliver a nucleic acid encoding a Cas9 or a Cpfl and at least one gRNA to a cell. In some embodiments, Cas9 or Cpfl is provided to a cell using a first viral vector and at least one gRNA is provided to the cell using a second viral vector.
[0235] In some embodiments, a single viral vector is used to deliver a nucleic acid encoding Cas9 or Cpfl and at least one gRNA to a cell. In some embodiments, Cas9 or Cpfl is provided to a cell using a first viral vector and at least one gRNA is provided to the cell using a second viral vector. In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell. The cell may be a muscle cell, a satellite cell, a mesangioblast, a bone marrow derived cell, a stromal cell or a mesenchymal stem cell. In embodiments, the cell is a cardiac muscle cell, a skeletal muscle cell, or a smooth muscle cell. In embodiments, the cell is a cell in the tibialis anterior, quadriceps, soleus, diaphragm or heart. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or inner cell mass cell (iCM). In further embodiments, the cell is a human iPSC or a human iCM. In some embodiments, human iPSCs or human iCMs of the disclosure may be derived from a cultured stem cell line, an adult stem cell, a placental stem cell, or from another source of adult or embryonic stem cells that does not require the destruction of a human embryo. Delivery to a cell may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.
[0236] Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present disclosure. These include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use.
[0237] Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
[0238] In yet another embodiment, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.
[0239] In still another embodiment for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force. The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
[0240] In some embodiments, the expression construct is delivered directly to the liver, skin, and/or muscle tissue of a subject. This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i. e. , ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present disclosure.
[0241] In a further embodiment, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated are lipofectamine-DNA complexes.
[0242] Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. A reagent known as Lipofectamine 2000™ is widely used and commercially available.
[0243] In certain embodiments, the liposome may be complexed with a hemagglutinating vims (HVJ) to facilitate fusion with the cell membrane and promote cell entry of liposome- encapsulated DNA. In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present disclosure. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
[0244] Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific.
[0245] Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor- specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (AS OR) and transferrin. A synthetic neoglycoprotein, which recognizes the same receptor as AS OR, has been used as a gene delivery vehicle and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells.
Duchenne Muscular Dystrophy
[0246] Duchenne muscular dystrophy (DMD) is a recessive X-linked form of muscular dystrophy, affecting around 1 in 5000 boys, which results in muscle degeneration and premature death. The disorder is caused by a mutation in the gene dystrophin (see GenBank Accession NO. NC_000023.11), located on the human X chromosome, which codes for the protein dystrophin (GenBank Accession No. AAA53189; SEQ ID NO: 5).
[0247] In humans, dystrophin mRNA contains 79 exons. Dystrophin mRNA is known to be alternatively spliced, resulting in various isoforms. Exemplary dystrophin isoforms are listed in Table 1.
[0248] The murine dystrophin protein has the following amino acid sequence (Uniprot Accession No. P11531, SEQ ID. NO: 869):
1 MWWVDCYRDV KKITKWNASK GKHDNSDDGK RCGTGKKKGS IRVHANNVNK ARVKNNVDVN
61 GSTDVDGNHK TGWNHWVKNV MKTMAGTNSK SKVRSTRNYV NVNTSSWSDG ANAHSHRDDW
121 NSVVSHSAGR HANAKCGKDD VATTYDKKSM YTSVVSAVMR ISSKVTRHHH MHYSTVSAGY
181 TSSSKRKSYA TAAYVATSDS TSYSHARDKS DSSMTVNDSY IAV3WSADTR AGSNDWKHA 241 HGMMD1SHGV GNVGSVGKGK SDAVMNNSRW CRVASMKSKH KVMDNKKDDW TKTRTKKMGD
301 DKCVHKVDVR VNSTHMWW DSSGDHATAA KVGDRWANCR WTDRWVDKWH TCSTWSKDAM
361 KNTSGKDNMM SSHKSTKDKK KTMKSSNDSA KNKSVTKMWM NARWDNTKKS SASAVTTTST
421 TTVMTVTMVT TRMVKHAKKR TVDSRKRDVD THSWTRSAVS SAVYRKGNSD KVNAARKAKR
481 KDASRSAAVM ANGVNASRAS NSRWTCSRVN WYTNTYNMTT TANKTSTTST AKSKCKDVNR
541 SAKSKKGGMD ADVATNHNHD GVRAKKTDIM RYTMSSRTWS SKSVYSVTYM RGKASSKNGN
601 YSDTVKMAKK ASCKYSGHWK KSSVSCKHMN KRKNHKGKWM AVDVKWAGDA KKKCRVGDTS
661 NSVNGGKKSA ASRTRNTWDH CRVYTRKAKA GDKTVSKDSM HWMTAYRDYK TDTAVMKRAK
721 AKTKVKTTVN SVAHASAAKK TTTNYWCTRN GKCKTVWACW HSYKANKWNV KKTMNVAGTV
781 SNMHHSNNRA TTDGGVMDNT NSRWRHAVRK KSSAKSHSDK AAYTDKVDAA MAKSDTSHSM
841 KKHNGKDANR VSDVAKKDVS MKRKANRSKM DVKMHARKSV VSSHCVNYKS SVKSVMVKTG
901 RVKKTNKDRV TAKHYNGAKV TRKKCKSRKM RKMNVTWAAT DTTKRSAVGM SNDSVAWGKA
961 TKKKAHKSVT GSKMVGKKTV DKSNSNWAVT SRVWNYKHMT DNTKWHADDS KKKKDKRKAM
1021 NDMRKVDSGR DAAKMAKRGD HCRKVVSNRR AASHRKRGKA SKN3DKAGVN KDNKDMSDNG
1081 TVNRGDNRGD RKRKKTKHNA KDRSRRKKAS HWYYKRADDK GDKKASRDRK KDRKKKNAVR
1141 RAGSNGAAMA VTSKRWRSNA RRNAHTHTMV VTTDMDVSYV STYTSHASVD HNTCAKDDKS
1201 KNKDNSGRDH KKKTAASATS MKVKVAVAMD GKHRMYKRGR DRSVKWRHHY DMKVNWNVKK
1261 TNNWHAKYKW YKDGGRAWR TNATGSSKTD VNKGSSRWHD CKARRKRKNV SRDNVWADNA
1321 TGDKVKARGK NTGGAVVSAR DKKKKTNWKV SRAKGVHKDR DHW3RNYNSA GDKVTVHGKA
1381 DVRSKGHYKK STVKRKDRSW AVNHRRTKDR AGSTTGASAS TVTVTSWTK TVSKMSSVAA
1441 DNRAWTTDWS DRVKSRVMVG DDNMKKATDR RTAANKNKTS NARITDRRWD VNRRNMKDST
1501 WAKAVGVRGK DSWKGHTVDA KKTTKAKDRR SVDVANDAKR DYSADDTRKV HMTNNTSWGN
1561 HKRVSAATHR DKSWTATTAN VDASRKKDSR GVRMKWDGTH TDYHKDNGKR SGSDARRDNM
1621 NKWSKKSNRS HASSDWKRHS VWKDDSRAGG DAVKNDHRAK RKTKVMSTTV RTGKYRRANV
1681 TRRKAVNAWD KNRSADWRKD ARAADDKRAV KGSWVGDDSD HKVKARGAKN VNRVNDAHTT
1741 GSYNSTDNGR WRVAVDRVRH AHRDGASHST SVGWRASNKV YYNHTTTCWD HKMTYSADNN
1801 VRSAYRTAMK RRKACDSSAA CDADHNKNDM DNCTTYDRHN NVNVCVDMCN WNVYDTGRTG
1861 RRVSKTGSCK AHDKYRYKVA SSTGCDRRGH DSRGVASGGS NSVRSCANNK AADWMRSMVW
1921 VHRVAAATAK HAKCNCKCGR YRSKHNYDCS CSGRVAKGHK MHYMVYCTTT SGDVRDAKVK
1981 NKRTKRYAKH RMGYVTVGDN MTVTNWVDSA ASSSHDDTHS RHYASRAMNS NGSYNDSSNS
2041 DDHHYCSNDS SRSASSRGRA DNRNAYDRKH HKGSSMMTSS RDAAAKRHKG RARMDHNKSH
2101 RRAAKVNGGT VSSSTSRSDS SMRWGSTSS MGDSDTSTGV MNNSSSRGRN AGKMRDTM.
[0249] Dystrophin is an important component within muscle tissue that provides structural stability to the dystroglycan complex (DGC) of the cell membrane. While both sexes can carry the mutation, females are rarely affected with the skeletal muscle form of the disease.
[0250] Mutations vary in nature and frequency. Large genetic deletions are found in about 60- 70% of cases, large duplications are found in about 10% of cases, and point mutants or other small changes account for about 15-30% of cases. Bladen et al. (2015), who examined some 7000 mutations, catalogued a total of 5,682 large mutations (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions, while 199 (14%) affected the splice sites. Point mutations totaled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations).
Combination Therapies
[0251] Beyond the methods and compositions disclosed herein, there is no current cure for DMD. Prior to the developments disclosed herein, treatment was generally aimed at controlling the onset of symptoms to maximize the quality of life. These therapies may be used in combination with the compositions and methods of the disclosure and may include the following:
1. Corticosteroids such as prednisolone and deflazacort increase energy and strength and defer severity of some symptoms.
2. Randomized control trials have shown that beta-2-agonists increase muscle strength but do not modify disease progression. Follow-up time for most RCTs on beta2-agonists is only around 12 months and hence results cannot be extrapolated beyond that time frame.
3. Mild, non-jarring physical activity such as swimming is encouraged. Inactivity (such as bed rest) can worsen the muscle disease.
4. Physical therapy is helpful to maintain muscle strength, flexibility, and function.
5. Orthopedic appliances (such as braces and wheelchairs) may improve mobility and the ability for self-care. Form-fitting removable leg braces that hold the ankle in place during sleep can defer the onset of contractures.
6. Appropriate respiratory support as the disease progresses is important.
[0252] Comprehensive multi-disciplinary care standards/guidelines for DMD have been developed by the Centers for Disease Control and Prevention (CDC), and are available at treat- nmd.eu/dmd/care/diagnosis-management-DMD.
Exemplary Results of Novel scAAV Delivery System for Treatment of DMD
[0253] Using the methods and compositions described herein, the examples disclose packaging Cas9 nuclease in conventional single-stranded AAV (ssAAV) and CRISPR single guide RNAs in double-stranded self-complementary AAV (scAAV) to deliver this dual AAV system into a mouse model of DMD harboring an exon 44 deletion. The doses of scAAV required for efficient gene editing were 20- fold lower than with ssAAV. Mice receiving systemic treatment showed restoration of dystrophin expression in all skeletal muscle groups and the heart, reduced DMD pathological phenotypes, and improved muscle contractility. These findings are the first to show that the efficiency of CRISPR/Cas9-mediated genome editing can be significantly improved by using the scAAV system and represent an important advancement toward therapeutic translation of genome editing for treating neuromuscular diseases.
[0254] The present application discloses a strategy for permanent and efficient correction of mutations in the endogenous DMD gene that may provide an ultimate cure for DMD. As disclosed herein, application of the CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR- associated proteins) system for engineering site-specific DNA double- stranded breaks (DSB) provides simplicity and precision in mammalian genome editing. The CRISPR-Cas system can be used to efficiently correct missense mutations in subjects of DMD by homology directed repair (HDR)-based germline editing or non- homologous end joining (NHEJ)-based postnatal editing. While missense and nonsense substitutions only account for -20% of DMD mutations, single- or multi-exon deletions are more prevalent (-68%) in DMD populations. Injecting recombinant AAV9-packaged Cas9 nuclease and single guide RNAs (sgRNAs) can successfully rescue DMD phenotypes in subjects harboring exon 44 or 50 deletions. Accordingly, the CRISPR-Cas system can be deployed to correct diverse genetic mutations that cause DMD and offer the prospect of a potential gene therapy for the permanent correction of DMD.
[0255] Recombinant AAV is a nonenveloped virus with a single- stranded linear DNA viral genome. As the largest tissue in the human body, skeletal muscle accounts for -40% of body weight. Therefore, a high dose of AAV (5.5 x 1014 to 1.8 x 1015 vg/kg) is typically required to achieve long-term, efficient genome editing in animal models of DMD. However, several studies in large animals reported that systemic administration of high doses of AAV (> 1.5 x 1014 vg/kg) may cause acute liver toxicity. In addition, the efficiency of in vivo CRISPR/Cas9- mediated genome editing may be, in some embodiments, highly dose-dependent, and elevating the dose of sgRNA AAV relative to Cas9 AAV may enhance the efficiency of genome editing. Moreover, it has been suggested that the sgRNA AAV genome is preferentially depleted after systemic delivery of CRISPR-Cas9 genome editing components.
[0256] In order to reduce the viral dose used for gene therapy without compromising genome editing efficiency and to prevent preferential depletion of the sgRNA AAV genome, disclosed in the examples herein is the packaging of a CRISPR sgRNA expression cassette into a double- stranded AAV vector. A double- stranded AAV genome was generated by mutating the terminal resolution site sequence on one side of the inverted terminal repeats (ITR), leading to production of self-complementary AAV (scAAV). Unlike conventional ssAAV, scAAV can bypass the second-strand synthesis, which is a rate- limiting step for gene expression. Moreover, double-stranded scAAV is less prone to DNA degradation after viral transduction, thereby increasing the number of copies of stable episomes.
[0257] In the Examples herein, using embodiments of the disclosed methods and compositions, in vivo genome editing in mice with a deletion of DMD exon 44 (AEx44) was performed by coupling ssAAV-packaged SpCas9 nuclease with scAAV-expressed sgRNAs. This dual AAV delivery system provided surprisingly significant improvements in viral transduction efficiency, genome editing, and functional recovery in skeletal muscles and heart. Of note, at least 20-fold less scAAV was required to achieve these improvements compared to the ssAAV treated cohort. Thus, the scAAV system represents a promising strategy for delivering CRISPR/Cas9 genome editing components and represents an important advancement toward therapeutic translation.
[0258] Owing to non-pathogenic and low-immunogenic characteristics, recombinant AAV was chosen as a delivery vector for use with the compositions and methods disclosed herein. In the genome editing strategy disclosed herein, the Cas9 nuclease is encoded by conventional ssAAV while sgRNAs are expressed by double-stranded scAAV. After a single high dose systemic injection of this dual AAV system into DEc44 mice (8 x 1013 vg/kg of Cas9 vector, 8 x 1013 vg/kg of sgRNA vector), dystrophin protein expression in multiple muscle groups was restored by -80% and skeletal muscle function was improved by -82% in fast-twitch EDL muscle and by -96% in slow-twitch soleus muscle. Importantly, a low dose of scAAV- expressed sgRNAs (4 x 1012 vg/kg) is sufficient to restore 18%, 14% and 50% of dystrophin protein in TA, triceps and diaphragm, respectively, representing a 20-fold improvement in efficiency compared with the ssAAV-packaged sgRNA vector. Without being bound to any particular mechanism, several potential explanations may account for these observations. First, it has been widely accepted that most recombinant AAV genomes persist as double-stranded episomes in vivo, either in the form of circular or linear concatemers. During the concatemerization process, the double- stranded DNA intermediate is an indispensable prerequisite. Thus, the scAAV undergoes concatemerization more rapidly than ssAAV because scAAV-based concatemerization bypasses second-strand synthesis, which is a rate-limiting step for ssAAV. Second, it has been reported that monomeric viral genome degradation is significantly slower in scAAV transduced skeletal muscle compared with ssAAV. Therefore, scAAV is more stable than ssAAV during initial viral transduction, leading to higher episomal persistence in the long-term. Third, DNA DSBs in post-mitotic cells are repaired by the classical NHEJ pathway, which requires the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). It is known that DNA-PKcs is required for AAV viral genome concatemerization in AAV transduced skeletal muscle. In this study, it was found that scAAV-packaged sgRNA leads to a higher incidence of DNA DSB at the target site. This may induce higher DNA-PKcs expression, which in turn facilitates AAV concatemerization and long-term gene expression. Indeed, higher viral genome persistence of both sgRNA vector and Cas9 vector was found in mice treated with scAAV-packaged sgRNA. In summary, the scAAV-sgRNA delivery system has many appealing features, including stable persistence of AAV viral genomes, higher INDEL frequency at the targeted genomic locus, and highly efficient genome editing in vitro and in vivo at low viral dose.
[0259] Initial studies of Cas9-induced DNA DSBs suggested that the breakage point was blunt- ended. However, molecular dynamics simulations of the SpCas9-sgRNA-dsDNA system suggest that SpCas9-induced cleavage generates a staggered cut, producing a single nucleotide 5’ overhang at the breakage point, which is prone to be filled with one additional nucleotide by the DNA polymerase, leading to a high frequency of +1 nt insertion after NHEJ-mediated repair. Based on this mechanism, a CRISPR/Cas9-mediated “single-cut” technology was developed, and successfully restored the ORF of exon 51 in mice and dogs with exon 50 deletion, and exon 45 ORF in mice lacking exon 44. Several studies have shown that it is possible to restore the Dmd ORF by removing one or multiple exons by using two sgRNAs. However, this “double-cut” strategy is only effective when two cooperative DNA DSBs occur simultaneously. If the first DNA DSB is rapidly rejoined by NHEJ-mediated repair, the second DSB alone is not sufficient to excise the entire exon. Moreover, a high frequency of AAV ITR integration events is observed at the Cas9 target site when two sgRNAs are used to excise large genomic intervening regions. Therefore, the CRISPR/Cas9-mediated “single-cut” repair strategy has unique advantages, including predictable DNA repair outcome, minimum genomic modification at a precise location, and low frequency of off-target effects.
[0260] In summary, employing exemplary methods and compositions disclosed herein, a low dose of scAAV-delivered CRISPR-Cas genome editing components is sufficient to restore dystrophin protein expression, reduce DMD pathological phenotypes, and improve muscle function in a DMD mouse model. Therefore, this robust scAAV delivery system combined with the efficient CRISPR-Cas9 genome editing technology represents a promising therapy for permanent correction of diverse genetic mutations in neuromuscular diseases. SEQUENCES
[0261] In any of the following tables, the “T” of a disclosed DNA sequence may be changed to “U” to produce an RNA sequence. Table 6: Exemplary gRNA spacer sequences
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
* In this table, upper case letters represent nucleotides that align to the exon sequence of the gene. Lower case letters represent nucleotides that align to the intron sequence of the gene.
Table 7: sgRNA spacers for mouse Dmd Exon 51
Figure imgf000109_0001
Table 8: sgRNA spacers targeting human Dmd Exon 51
Figure imgf000109_0002
Table 9: sgRNA spacers targeting sites in various human Dmd Exons
Figure imgf000109_0003
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Table 10: sgRNA spacers targeting dog Dmd Exon 51
Figure imgf000113_0002
Table 11: Sequence of primers for in vitro transcription of sgRNA
Figure imgf000114_0001
Table 12: Sequence of primers for genotyping
Figure imgf000114_0002
Table 13: Exon 43 & 45 gRNA spacer sequences
Figure imgf000114_0003
Table 14: Sequence of primers for cloning sgRNA spacers targeting DMD and Dmd exon 45 splicing acceptor site
Figure imgf000115_0001
Table 15: Additional gRNA spacer sequences
Figure imgf000115_0002
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Table 16: Additional exemplary guide RNA spacer sequences
Figure imgf000132_0001
Figure imgf000133_0001
Table 17: Primers and Probes
Figure imgf000134_0001
EXAMPLES
EXAMPLE 1: Strategies for CRISPR/Cas9-mediated genome editing of Dmd exon 45 and vector design
Materials and Methods
[0262] The following materials and methods were employed throughout the Examples disclosed herein.
Study design
[0263] The experiments disclosed in these Examples exemplify the use of a scAAV system to deliver the CRISPR/Cas9 genome editing components for the correction of DMD mutations. These experiments also compare the efficiency of conventional ssAAV and scAAV in delivering CRISPR sgRNA for in vivo therapeutic genome editing. Exclusion, randomization, or blind approaches were not used to assign the animals for the experiments. For each experiment, sample size reflects the number of independent biological replicates and is provided in the figure legends.
AAV vector cloning and viral production
[0264] The sgRNA targeting mouse Dmd exon 45, listed in Figure 3, was first cloned into a modified TRISPR-sgRNA-CK8e-GFP plasmid, using Golden Gate Assembly (New England Biolabs). The sgRNA expression cassette containing three copies of same sgRNA driven by the U6, HI, and 7SK promoter was PCR amplified and subcloned into the pSJG selfcomplementary AAV plasmid (scAAV plasmid), or into the pSSV9 single-stranded AAV plasmid (ssAAV plasmid), using In-Fusion Cloning Kit (Takara Bio). A 2.3 kb staffer sequence was cloned into the ssAAV plasmid for optimal viral packaging. Both the scAAV and the ssAAV genome contain the same sgRNA expression cassette, consisting of three copies of sgRNA sequence driven by three RNA polymerase III promoters. The sequence of the sgRNA spacer sequence used was ggcttacagGAACTCCAGGA (SEQ ID NO: 2668). Cloning primers are listed in FIG 3A. All AAV viral plasmids were column purified and digested with Sma I and Ahd I to check ITR integrity. AAVs were packaged by Boston Children’s Hospital Viral Core and serotype 9 was chosen for capsid assembly. AAV titers were determined by Droplet Digital PCR (ddCPR) (Bio-Rad Laboratories) according to the manufacturer’s protocol. Primers and probes used for titration are listed in FIG. 3A. Alkaline agarose gel electrophoresis
[0265] AAV virus (2 x 1011 vg) was equalized with water to 13 mΐ and digested with 10 mΐ DNase solution (lOmM Tris-HCl, pH 7.5, lOmM CaCh, lOmM MgCh, O.lmg/ml DNase) at 37°C for 1 hour, followed by chelating Mg2+ and Ca2+ with 5 mΐ 0.5M EDTA. Then the capsid was denatured by adding 2 mΐ 10% SDS. The reaction mixture was mixed with 6 ul 6X alkaline agarose gel loading dye (Alfa Aesar) and loaded into 1% alkaline agarose gel. Denaturing gel electrophoresis was performed in a cold room at 50 V for 15 hours. The gel was neutralized with neutralization buffer (0.5 M Tris-HCl pH 7.5, 1M NaCl) and stained with SYBR Gold (Thermo Fisher Scientific) for visualization.
In vitro AAV viral transduction in C2C12 myotubes
[0266] Cas9-expressing C2C12 myoblasts were cultured in 96-well dishes with growth medium (DMEM with 10% FBS) until reaching 90% confluency. Then, the myoblasts were allowed to differentiate in myotube in differentiation medium (DMEM with 2% horse serum) for 5 days. Two-hours before viral transduction, myotubes were treated with Vibrio cholerae Neuraminidase (50 mu/ml) (Sigma- Aldrich), followed by washing with differentiation medium twice. Myotubes were incubated with varying doses of scAAV or ssAAV and centrifuged at 1,000 x g at 4°C for 1.5 hour. After spin transduction, the virus was aspirated and the myotubes were washed with differentiation medium three times. The myotubes were cultured in differentiation medium for an additional week prior genomic DNA isolation for INDEL analysis.
In vivo AAV delivery into AEx44 mice
[0267] Postnatal day 4 (P4) DEc44 mice were injected intraperitoneally with 80 mΐ of AAV9 viral mixture containing 8 x 1013 vg/kg AAV9-SpCas9 and varying doses of scAAV or ssAAV- packaged sgRNA using an ultrafine BD insulin syringe (Becton Dickinson). The doses of scAAV or ssAAV-packaged sgRNA are indicated in the figure legends. Four weeks after systemic delivery, DEc44 mice and WT littermates were dissected for physiological, biochemical and histological analysis. Animal work described herein has been approved and conducted under the oversight of the UT Southwestern Institutional Animal Care and Use Committee.
Genomic DNA and RNA isolation, cDNA synthesis, and PCR amplification
[0268] Genomic DNA of mouse C2C12 myotubes, skeletal muscles and heart was isolated using DirectPCR (cell) lysis reagent (Viagen Biotech) according to the manufacturer’s protocol. Total RNA of skeletal muscles and heart was isolated using miRNeasy (QIAGEN) according to the manufacturer’s protocol. cDNA was reverse-transcribed from total RNA using Superscript III First-Strand Synthesis SuperMix (Thermo Fisher Scientific) according to the manufacturer’s protocol. Primer sequences are listed in FIG. 3A.
INDELs analysis of genomic DNA and cDNA
[0269] INDEFs in genomic DNA and cDNA were analyzed using Tracking of INDEFs by Decomposition (TIDE) software package (deskgen.com). Briefly, the sgRNA sequence targeting mouse Dmd exon 45 was first uploaded to the software to define SpCas9-mediated DSB site. Then, the CRISPR/Cas9-edited sequence and non-edited control sequence were uploaded and aligned using Smith-Waterman local alignment algorithm. The percentage of INDEFs was calculated based on the relative abundance of aberrant nucleotides over the length of the whole sequence trace.
Amplicon deep sequencing analysis
[0270] PCR of genomic DNA was performed using primers designed against the DMD exon 45 region. A second round of PCR was performed to add Illumina flow cell binding sequence and barcodes. All primer sequences are listed in FIG. 3B. Deep sequencing analysis was performed.
AAV viral genome copy number quantification
[0271] The AAV viral genome copy number was determined by quantitative PCR using custom-designed primers (FIG. 3A). The primer sets used in AAV-sgRNA and AAV-Cas9 viral genome quantification anneal to the 7SK promoter and Cas9 gene, respectively. The threshold cycle value of each reaction was converted to the viral genome copy number by measuring against the copy number standard curve of the AAV plasmids used for AAV packaging in this study. Mouse 18S ribosomal RNA gene was used as the reference gene to calibrate genomic DNA quantity.
Dystrophin and SpCas9 Western blot analysis
[0272] Heart and skeletal muscles were crushed and lysed with lysis buffer [10% SDS, 62.5 mM tris (pH 6.8), 1 mM EDTA, and protease inhibitor], A total 50 pg of protein was loaded onto 4-20% Criterion™ TGX™ Precast Midi Protein Gel (Bio-Rad Faboratories). Primary antibodies used in Western blot were mouse anti-dystrophin antibody (MANDYS8, Sigma- Aldrich, D8168), mouse anti-Cas9 antibody (Clone 7A9, Millipore, MAC133), mouse anti- vinculin antibody (Sigma-Aldrich, V9131). Secondary antibodies used in Western blot were goat anti-mouse horseradish peroxidase (HRP) antibody or goat anti-rabbit HRP antibody (Bio- Rad Laboratories).
Histological analysis of skeletal muscle and heart
[0273] Skeletal muscles and heart were cryosectioned into eight-micron transverse sections, and immunohistochemistry was performed. Antibodies used in immunohistochemistry were mouse anti-dystrophin antibody (MANDYS8, Sigma-Aldrich, D8168) and M.O.M. biotinylated anti-mouse IgG (BMK-2202, Vector Laboratories).
Electrophysiological analysis of isolated EDL and soleus muscles
[0274] Four weeks after systemic AAV-CRISPR/Cas9 genome editing, EDL and soleus muscles from DEc44 mice and WT littermates were isolated for electrophysiological analysis. Specific force (mN/mm2) was calculated by normalizing contraction force to muscle cross- sectional area.
Statistics
[0275] All data are shown as means ± SEM. One-way ANOVA or two-way ANOVA was performed with post-hoc Tukey’s multiple comparisons test. A P < 0.05 value was considered statistically significant.
EXAMPLE 2: sgRNA design and scAAV production
[0276] Deletion of exon 44 of the human DMD gene generates a premature stop codon in exon 45 and represents one of most common mutations of DMD. As a strategy to correct exon 44 out-of- frame deletion mutations, a sgRNA was designed to target the splice acceptor region of exon 45 (FIG. 1A). This sgRNA recognizes a 5’-TGG-3’ protospacer adjacent motif (PAM) in exon 45 and generates insertions and deletions (INDELs) 7 base pairs (bp) downstream of the 5 ’-AG-3’ splice acceptor site (FIG. IB). Depending on the size of INDELs, two types of NHEJ- mediated DNA repair events can restore the open reading frame (ORF) of the Dmd gene. These include exon 45 skipping, if the INDEL is large enough to delete the 5’ -AG-3’ splice acceptor sequence in exon 45, or reframing of exon 45 through INDELs that either insert one nucleotide (3n+l) or delete two nucleotides (3n-2) (FIG. 1C).
[0277] To test whether double- stranded scAAV is capable of packaging sgRNAs, the sgRNA expression cassette was cloned into a scAAV vector and the conventional ssAAV vector as a control (FIG. 1C). Alkaline denaturing gel electrophoresis was performed to confirm the integrity of both AAVs (FIG. 2). The size of ssAAV-sgRNA (SEQ ID NO: 2598) is 3.9 kilo- nucleotides (knt) and remains unchanged after alkaline gel electrophoresis. The size of scAAV- sgRNA (SEQ ID NO: 2597) is 1.4 kilo-base pairs and is doubled to 2.8 knts under denaturing conditions, indicative of the double- stranded viral genome. Primers and probes used in this example are shown in FIG. 3.
EXAMPLE 3: In vitro genome editing using ssAAV or scAAV-packaged sgRNA
[0278] To compare the efficiency of ssAAV and scAAV-packaged sgRNAs in vitro, 5/>Cas9- expressing C2C12 mouse myoblasts were differentiated for 5 days to myotubes and transduced the myotubes with each of the AAVs. One week after viral transduction, Tracking of INDELs by Decomposition (TIDE) analysis was performed to detect INDELs within the Dmd exon 45 region. It was found that the total INDELs exhibited a dose-dependent curve for both ssAAV and scAAV-expressed sgRNA after one week post viral transduction (FIG. 4A). Specifically, to reach a level of 10% INDELs required 5 x 108 vg/mL of scAAV and 1 x 1010 vg/mL of ssAAV, representing a 20-fold increase in efficiency of scAAV. To reach an intermediate level of INDELS of -22%, 40-fold less scAAV (1.8 x 109 vg/mL) was required compared to ssAAV (7.8 x 1010 vg/mL). Furthermore, a high level of INDELs (over 40%) was achieved by 7.2 x 109 vg/mL of scAAV, whereas ssAAV required 5 x 1012 vg/mL, representing a 70-fold improvement in efficiency with scAAV. The INDEL composition in myotubes transduced with ssAAV or scAAV was analyzed, and it was found that -50% of total INDEL events contained a +1 nt insertion, which can bring exon 45 in- frame with exon 43 (FIG. 4B). Therefore, scAAV- expressed sgRNA demonstrated enhanced efficiency by in vitro genome editing at Dmd exon 45 compared to the conventional ssAAV-expressed sgRNA. Moreover, the majority of the INDEL events (over 50%) contained a single nt insertion, which is able to restore the Dmd exon 45 ORF.
EXAMPLE 4: Systemic delivery of scAAV-packaged sgRNAs restores dystrophin expression in AEx44 mice
[0279] To further evaluate the efficacy of the scAAV system by in vivo genome editing, ssAAV-packaged SpCas9 and scAAV or ssAAV -packaged sgRNA was delivered systemically in DEc44 mice through intraperitoneal (IP) injection. The AAV9 serotype was chosen because of its tropism to skeletal muscle and heart. Moreover, SpCas9 expression was driven by a muscle specific promoter containing key regulatory elements derived from creatine kinase promoter and enhancer, restricting its expression to striated muscles. AAV -packaged sgRNA is the rate limiting factor for in vivo genome editing in dystrophic mouse models. Therefore, ssAAV-packaged SpCas9 was kept at a constant dose of 8 x 1013 vg/kg while titrating scAAV or ssAAV-packaged sgRNA at multiple doses. Four weeks after systemic AAV delivery, skeletal muscles and heart of CRISPR/Cas9-edited DEc44 mice were harvested for analysis. By immunohistochemistry, it was found that dystrophin restoration in skeletal muscles was dose-dependent (FIG. 5 and FIG. 6). Mice receiving the lowest dose of scAAV-packaged sgRNA (4 x 1012 vg/kg) showed 40% and 32% dystrophin-positive myofibers in tibialis anterior (TA) and triceps, respectively; diaphragm and heart showed higher percentages of dystrophin-positive myocytes, reaching 95% (FIG. 5 and FIG. 7A). In contrast to the scAAV- treated cohort, DEc44 mice receiving lowest dose of ssAAV-packaged sgRNA (4 x 1012 vg/kg) showed less than 5% dystrophin-positive myofibers in TA and triceps; diaphragm and heart showed 52% and 61% dystrophin-positive myocytes, respectively (FIG. 6 and FIG. 7B). When the dose of scAAV-packaged sgRNA was increased to 1.6 x 1013 vg/kg, virtually all myofibers and cardiomyocytes were dystrophin-positive (FIG. 5 and FIG. 7A). For the ssAAV-treated cohort (1.6 x 1013 vg/kg), diaphragm and heart showed over 75% of dystrophin-positive myocytes; however, dystrophin-positive myofibers in TA and triceps were still below 18% (FIG. 6 and FIG. 7B).
[0280] Next, Western blot analysis was performed to quantitatively detect dystrophin restoration in skeletal muscles and heart after systematic delivery of scAAV or ssAAV- packaged sgRNA. The lowest dose of scAAV-packaged sgRNA (4 x 1012 vg/kg) restored 18%, 14% and 50% of dystrophin protein in TA, triceps and diaphragm, respectively (FIG. 8A-C and FIG. 9A). When the dose of scAAV-packaged sgRNA was increased to 1.6 x 1013 vg/kg, dystrophin protein restoration in each skeletal muscle group was greater than 50% (FIG. 8A-C and FIG. 9A). Of note, saturation was observed in heart because at every dose of scAAV tested, dystrophin protein restoration exceeded 70% (FIG. 8D and FIG. 9A). Interestingly, although ssAAV-packaged SpCas9 was injected at a constant dose (8 x 1013 vg/kg), DEc44 mice receiving a higher dose of scAAV-packaged sgRNA showed elevated Cas9 protein expression in skeletal muscles and heart (FIG. 8A-D and FIG. 9B). In contrast to the scAAV-treated cohort, DEc44 mice receiving ssAAV-packaged sgRNA showed significant lower efficiency in dystrophin restoration by Western blot quantification (FIG. 10 and FIG. 11). Specifically, mice receiving the highest dose of ssAAV (8 x 1013 vg/kg) showed only 13%, 16% and 30% of normal dystrophin protein levels in TA, triceps and diaphragm, respectively (FIG. 10A-C and FIG. 11). This was incomparable to the mice treated with scAAV because more than 80% of dystrophin protein was restored after receiving the same dose of scAAV-packaged sgRNA (FIG. 8A-C and FIG. 9A). Therefore, scAAV-expressed sgRNA demonstrated greater efficiency in in vivo genome editing compared to the conventional ssAAV-expressed sgRNA.
EXAMPLE 5: Systemic delivery of scAAV-packaged sgRNAs restores muscle integrity and improves muscle function in AEx44 mice
[0281] To evaluate whether systemic delivery of scAAV-packaged sgRNAs was able to rescue pathological hallmarks seen in dystrophic mice, hematoxylin and eosin (H&E) staining of skeletal muscles and heart isolated from DEc44 mice was performed four weeks after CRISPR/Cas9-mediated genome editing. The percentage of regenerating myofibers with central nuclei declined as the dose of scAAV-packaged sgRNA increased (FIGS. 12-14). Less than 5% of myofibers showed central nuclei in TA and triceps in mice receiving 1.6 x 1013 vg/kg of scAAV-packaged sgRNA (FIG. 12 and FIG. 14A). In contrast, mice receiving the same dose of ssAAV-packaged sgRNA still showed over 70% of regenerating myofibers with central nuclei, together with signs of muscle necrosis and inflammatory infiltration (FIG. 13 and FIG. 14B). In addition, skeletal muscles isolated from mice receiving the highest dose of scAAV-packaged sgRNA (8 x 1013 vg/kg) were virtually indistinguishable from those of wild- type (WT) littermates, whereas the ssAAV-treated cohort still showed 30% central nuclei in the TA and triceps (FIGS. 12-14).
[0282] To examine the effect of dystrophin restoration on muscle function after systemic delivery of scAAV or ssAAV-packaged sgRNA, electrophysiological analyses was performed on extensor digitorum longus (EDL) and soleus muscles isolated from DEc44 mice four weeks after receiving the middle dose of AAV-sgRNA (1.6 x 1013 vg/kg) or the high dose of AAV- sgRNA (8 x 1013 vg/kg). Without CRISPR/Cas9 genome editing, muscle-specific force, which was calibrated by the muscle cross-sectional area, was reduced by 46% in fast-twitch EDL muscle and by 42% in slow-twitch soleus muscle (FIG. 15A-B). After systemic delivery of scAAV-packaged sgRNA, muscle-specific force of the EDL was increased from 54% to 83% and to 82% for the middle and high doses; in contrast, for the ssAAV-treated cohort, muscle- specific force of the EDL was only increased from 54% to 62% and to 66% for the middle and high doses (FIG. 15A). For the slow-twitch soleus muscle, muscle-specific force was increased from 58% to 93% and to 96% after receiving the middle and high doses of scAAV-packaged sgRNA; in contrast, for the ssAAV-treated cohort, only high dose treatment was able to improve muscle-specific force of the soleus to 85%, while no improvement was observed with the middle dose (FIG. 15B). The maximal tetanic force of the EDL and soleus followed a similar pattern to the muscle-specific force. Specifically, DEc44 mice receiving the middle or high doses of scAAV-packaged sgRNA showed improved maximal tetanic force of the EDL muscle to over 80% of WT, whereas the ssAAV-treated cohort was only able to improve to 60% of WT (FIG. 15C). The maximal tetanic force of the soleus was improved to over 90% of WT after receiving the middle or high doses of scAAV-packaged sgRNA; high dose of ssAAV- packaged sgRNA improved maximal tetanic force of the soleus to 85% of WT, while the middle dose did not provide any improvement (FIG. 15D). After receiving the middle and high doses of scAAV-packaged sgRNA, serum creatine kinase (CK) levels in the DEc44 mice were reduced by 87% and 95%, respectively, compared with DEc44 mice without treatment (FIG. 16). In contrast, serum CK levels in the DEc44 mice receiving the same doses of ssAAV- packaged sgRNA were still 18.6- and 8.5-fold higher, respectively, than the WT littermates (FIG. 16). These findings indicate that the double- stranded scAAV vector is highly efficient in in vivo gene therapy and can significantly improve muscle integrity and function.
EXAMPLE 6: The scAAV system induces significant TNDELs within Dmd exon 45 and maintains higher copies of the viral genome in vivo
[0283] To determine the mechanism whereby the scAAV system significantly improves in vivo genome editing in DEc44 mice, deep sequencing analysis was performed to determine the INDEL frequency at the genomic and cDNA levels (FIGS. 20A-20B). The percentage of total genomic INDELs and +1 nt insertions at exon 45 correlated with ascending doses of AAV- sgRNA. DEc44 mice receiving the high dose (8 x 1013 vg/kg) of scAAV-packaged sgRNA showed more than 28% and 30% of total NHEJ events in TA, and triceps, respectively (FIG. 17A). Of note, over 60% of total NHEJ events were +1 nt insertions, which restores the Dmd exon 45 ORF. In contrast, TA and triceps from DEc44 mice receiving the same dose of ssAAV- packaged sgRNA had only 10% and 11% of total NHEJ events (FIG. 17A). No significant difference was observed between scAAV and ssAAV in inducing total NHEJ and +1 nt insertion events at the sgRNA targeting site in ssAAV- and scAAV-treated mice (FIG. 17A, FIGS. 20A-20B).
[0284] TIDE analysis was also performed on dystrophin cDNA transcripts isolated from skeletal muscles and heart. Total cDNA INDEL rate and +1 nt insertion events at exon 45 followed similar ascending patterns seen in the genomic TIDE analysis while the absolute percentage increased significantly (FIG. 17B), indicating enrichment of the reframed cDNA transcript after nonsense-mediated decay of unedited transcript with a premature stop codon in exon 45. These findings indicate that the scAAV system is highly efficient in inducing INDELs at the targeted genomic locus, and the majority of the INDEL events contain +1 nt insertions, which is able to repair the out-of-frame mutation in Dmd exon 45.
[0285] Next, quantitative PCR analysis was performed to detect viral genome copies in skeletal muscles and heart of DEc44 mice four weeks after systemic delivery of AAV-CRISPR/Cas9 genome editing components. Mice receiving scAAV treatment showed significantly higher copy numbers of sgRNA viral genomes than those receiving the same dose of ssAAV-packaged sgRNA (FIG. 18 A). Moreover, the sgRNA transcripts transcribed from the scAAV vector were also significantly higher than those transcribed from the ssAAV vector (FIG. 19A). These findings indicate that there is a significant depletion of ssAAV-packaged sgRNA vector in skeletal muscles in vivo. Interestingly, although the dose of Cas9 vector was kept constant at 8 x 1013 vg/kg during initial systemic injection, the viral genomes of Cas9 vector in TA and triceps persisted with higher copies from mice receiving scAAV-packaged sgRNA vector than those receiving same dose of ssAAV-packaged sgRNA vector (FIG. 18B). However, AAV- Cas9 viral genomes showed relatively high copies in diaphragm and heart independent of the identity of AAV-sgRNA vector (FIG. 18B). These findings are consistent with Cas9 cDNA transcript analysis (FIG. 19B). Together, these data suggest that the high efficiency of scAAV- mediated in vivo genome editing is attributed to higher viral genome persistence of the sgRNA vector and Cas9 vector.
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Claims

1. An AAV expression cassette comprising, from 5’ to 3’ : a first inverted terminal repeat (ITR); a first promoter; a sequence encoding a first sgRNA comprising a first spacer region and a first scaffold region; a second promoter; a sequence encoding a second sgRNA comprising a second spacer region and a second scaffold region; a third promoter; a sequence encoding a third gRNA comprising a third sgRNA targeting region and third scaffold region; and a second a second ITR, wherein the first ITR has the sequence of SEQ ID NO: 2584; wherein the second ITR has the sequence of SEQ ID NO: 2583; wherein the AAV expression cassette is self-complementary.
2. The AAV expression cassette of claim 1, wherein the first promoter is a U6 promoter.
3. The AAV expression cassette of claim 2, wherein the U6 promoter has the sequence of SEQ ID NO: 2589, or a sequence at least 95% identical thereto.
4. The AAV expression cassette of claim 1, wherein the second promoter is a HI promoter.
5. The AAV expression cassette of claim 4, wherein the HI promoter has the sequence of SEQ ID NO: 2586, or a sequence at least 95% identical thereto.
6. The AAV expression cassette of claim 1, wherein the third promoter is the 7SK promoter.
7. The AAV expression cassette of claim 6, wherein the 7SK promoter has the sequence of SEQ ID NO: 2587, or a sequence at least 95% identical thereto.
8. The AAV expression cassette of any one of claims 1-7, wherein the first promoter is the U6 promoter, the second promoter is the HI promoter, and the third promoter is the 7SK promoter.
9. The AAV expression cassette of any one of claims 1-8, wherein each of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are the same.
10. The AAV expression cassette of any one of claims 1-8, wherein the at least two of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are different.
11. The AAV expression cassette of any one of claims 1-8, wherein each of the sequences encoding the first sgRNA, the second sgRNA, and the third sgRNA are different.
12. The AAV expression cassette of any one of claims 1-11, wherein at least one of the first spacer region, the second spacer region, and the third spacer region targets the human dystrophin gene.
13. The AAV expression cassette of any one of claims 1-12, wherein the sequences of first spacer region, the second spacer region, and the third spacer region are each independently selected from any one of SEQ ID NO: 60-382, 706-708, 712-714, 718-789, 863, 947, 1036-1043, 1052-1063, 1494-1499, 1500-1506, 1555-1708, 1751-1994, 2045-2128, 2260-2304, 2600-2647, or 2668.
14. The AAV expression cassette of any one of claims 1-12, wherein the sequences encoding the first, the second, and the third spacer regions are the same, and wherein the sequences are each SEQ ID NO: 2668.
15. The AAV expression cassette of any one of claims 1-14, wherein the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are the same.
16. The AAV expression cassette of any one of claims 1-14, wherein at least two of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are different.
17. The AAV expression cassette of any one of claims 1-14, wherein each of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region are different.
18. The AAV expression cassette of any one of claims 1-14, wherein at least one of the sequences encoding the first scaffold region, the second scaffold region, and the third scaffold region has the sequence of SEQ ID NO: 2672, or a sequence at least 95% identical thereto.
19. The AAV expression cassette of any one of claims 1-18, wherein the expression cassette further comprises a sequence encoding a fourth sgRNA comprising a fourth spacer region and a fourth scaffold region.
20. The AAV expression cassette of claim 19, wherein the expression cassette further comprises a sequence encoding a fifth sgRNA comprising a fifth spacer region and a fifth scaffold region.
21. The AAV expression cassette of any one of claims 1-20, wherein the expression cassette has a length of less than about 2 kb, less than about 1.8 kb, less than about 1.6 kb, or less than about 1.4kb.
22. A composition comprising the AAV expression cassette of any one of claims 1-21.
23. A vector comprising the AAV expression cassette of any one of claims 1-21.
24. The vector of claim 23, wherein the vector is a non- viral vector.
25. The vector of claim 24, wherein the vector is a plasmid.
26. The vector of claim 23, wherein the vector is an AAV vector.
27. The vector of claim 23, wherein the AAV vector is a self-complimentary AAV
(scAAV).
28. The vector of claim 26 or 27, wherein the AAV vector is a recombinant AAV
(rAAV).
29. The vector of any one of claims 26-28, wherein the AAV vector comprises a capsid protein isolated or derived from an AAV vector of serotype 9 (AAV9).
30. The vector of claim 29, wherein the AAV vector comprises a wild type AAV9 capsid protein.
31. A composition comprising the vector of any one of claims 23-30.
32. The composition of claim 31, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.
33. An AAV expression cassette comprising, from 5’ to 3’ : a first inverted terminal repeat (ITR); a promoter; a sequence encoding a CRISPR nuclease, e.g., Cas9 nuclease; and a second ITR; wherein the AAV expression cassette is not self-complimentary.
34. The AAV expression cassette of claim 33, wherein the first ITR has the sequence of SEQ ID NO: 2585 and the second ITR has the sequence of SEQ ID NO: 2679.
35. The AAV expression cassette of claim 33 or 34, wherein the promoter is a CK8e promoter.
36. The AAV expression cassette of any one of claims 33-35, wherein the promoter has the sequence of SEQ ID NO: 2590, or a sequence at least 95% identical thereto.
37. The AAV expression cassette of any one of claims 33-36, wherein the sequence encoding the Cas9 nuclease is derived from S. aureus or S. pyogenes.
38. The AAV expression cassette of any one of claims 33-37, wherein the sequence encoding the Cas9 nuclease comprises SEQ ID NO: 2591, or a sequence at least 95% identical thereto.
39. The AAV expression cassette of any one of claims 33-38, wherein the expression cassette further comprises a sequence encoding a PolyA tail.
40. The AAV expression cassette of claim 39, wherein the PolyA tail comprises a sequence of SEQ ID NO: 2680, or a sequence at least 95% identical thereto.
41. A composition comprising the AAV expression cassette of any one of claims 33-40.
42. The composition of claim 41, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.
43. A vector comprising the AAV expression cassette of any one of claims 33-40.
44. The vector of claim 43, wherein the vector is a non- viral vector.
45. The vector of claim 44, wherein the vector is a plasmid.
46. The vector of claim 43, wherein the vector is an AAV vector.
47. The vector of claim 46, wherein the AAV vector is not a self-complimentary AAV
(scAAV).
48. The vector of claim 46 or 47, wherein the AAV vector is a recombinant AAV (rAAV).
49. The vector of any one of claims 46-48, wherein the AAV vector comprises a capsid protein isolated or derived from an AAV vector of serotype 9 (AAV9).
50. The vector of any one of claims 46-48, wherein the AAV vector comprises a wild type AAV9 capsid protein.
51. A composition comprising the vector of any one of claims 43-50.
52. The composition of claim 51, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.
53. A composition comprising the vector of any one of claims 23-30 and the vector of any one of claims 43-50.
54. The composition of claim 53, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.
55. A recombinant AAV comprising: a capsid protein, and the AAV expression cassette of any one of claims 1-21 or 33-40 encapsidated by the capsid protein.
56. The recombinant AAV of claim 55, wherein the capsid protein is isolated or derived from a wild type AAV capsid of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh.74, AAV2i8, AAVRh.10, AAV39, AAV43, AAVRh.8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.
57. The recombinant AAV of claim 56, wherein the capsid protein is isolated or derived from an AAV9 capsid.
58. A method of producing an AAV vector comprising contacting a vector comprising the AAV expression cassette of any one of claims 1-21 and/or 33-40 with an AAV host cell.
59. The method of claim 58, wherein the AAV host cell is a mammalian cell.
60. The method of claim 59, wherein the host cell is a HEK293 cell.
61. The method of claim 58, wherein the AAV host cell is an insect cell.
62. The method of claim 61, wherein the insect cell is a Sf9 cell.
63. A method of correcting a gene defect in a cell, the method comprising contacting an AAV vector comprising the AAV expression cassette of any one of claims 1-21 with the cell.
64. The method of claim 63, wherein the cell is a human cell.
65. The method of claim 63 or 64, wherein the gene defect is a gene defect in the dystrophin gene.
66. The method of any one of claims 63-65, wherein the method also comprises contacting the cell with an AAV vector comprising an expression cassette for a Cas9 nuclease.
67. The method of claim 66, wherein the expression cassette for the Cas9 nuclease is not self-complimentary.
68. The method of claim 66 or 67, wherein the expression cassette for the Cas9 nuclease is the AAV expression cassette of any one of claims 33-40.
69. A method of correcting a gene defect in a cell, the method comprising contacting with the cell: an AAV vector comprising the AAV expression cassette of any one of claims 1-21; and an expression cassette for a Cas9 nuclease.
70. The method of claim 69, wherein the cell is a human cell.
71. The method of claim 69 or 70, wherein the gene defect is a gene defect in the dystrophin gene.
72. The method of any one of claims 69-71, wherein the expression cassette for the Cas9 nuclease is not self-complimentary.
73. The method of any one of claims 69-72, wherein the expression cassette for the Cas9 nuclease is the AAV expression cassette of any one of claims 33-40.
74. A method of treating a subject in need thereof comprising administering to the subject a first AAV vector comprising the AAV expression cassette of any one of claims 1-21.
75. The method of claim 74, wherein the subject is a human.
76. The method of claim 74, wherein the subject suffers from a monogenic disease.
77. The method of any one of claims 74-76, wherein the subject suffers from a muscle disease.
78. The method of claim 77, wherein the muscle disease is selected from the group consisting of Duchenne Muscular Dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss dystrophy, myotonic dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular dystrophy, congential dystrophy, congenital myopathy, familial periodic paralysis.
79. The method of claim 78, wherein the muscle disease is DMD.
80. The method of claim 77, wherein the subject suffers from mitochondrial oxidative phosphorylation disorder, or glycogen storage disease.
81. The method of any one of claims 74-80, wherein the method also comprises administering to the subject a second AAV vector comprising an expression cassette for a Cas9 nuclease.
82. The method of claim 81, wherein the expression cassette for the Cas9 nuclease is not self-complimentary.
83. The method of claim 81 or 82, wherein the expression cassette for the Cas9 nuclease is the expression cassette of any one of claims 33-40.
84. The method of any one of claims 74-83, wherein dystrophin expression is at least partially restored in skeletal muscle in the patient.
85. The method of any one of claims 74-84, wherein dystrophin expression is at least partially restored in heart muscle in the patient.
86. The method of claim 84 or 85, wherein the dosage of the first AAY required to at least partially restore dystrophin expression is at least about 20-fold lower than the dosage that would be required to achieve the same level of dystrophin expression if the expression cassette of the first AAY was not self-complimentary.
87. A method of treating a subject in need thereof, the method comprising administering to the patient a therapeutically effective amount of: the vector of any one of claims 23-30 or the composition of any one of claims 31-32; and the vector of any one of claims 43-50 or the composition of any one of claims 51-52; wherein the subject suffers from Duchenne Muscular Dystrophy (DMD).
88. A nucleic acid comprising the sequence of SEQ ID NO: 2668.
89. The nucleic acid of claim 88, wherein the nucleic acid further comprises the sequence of any one of SEQ ID NO: 2672-2678.
90. The nucleic acid of claim 89, wherein the nucleic acid further comprises the sequence of SEQ ID NO: 2672.
91. A method of treating a subject in need thereof, the method comprising administering to the patient a therapeutically effective amount of the nucleic acid of any one of claims 88 to 90, or an AAV vector comprising the same.
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