EP3735462A1 - Compositions crispr/cas9 thérapeutiques et méthodes d'utilisation - Google Patents

Compositions crispr/cas9 thérapeutiques et méthodes d'utilisation

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Publication number
EP3735462A1
EP3735462A1 EP19705415.8A EP19705415A EP3735462A1 EP 3735462 A1 EP3735462 A1 EP 3735462A1 EP 19705415 A EP19705415 A EP 19705415A EP 3735462 A1 EP3735462 A1 EP 3735462A1
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European Patent Office
Prior art keywords
vector
composition
sequence encoding
sequence
promoter
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EP19705415.8A
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German (de)
English (en)
Inventor
Leonela AMOASII
Eric N. Olson
Yi-Li Min
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University of Texas System
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University of Texas System
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Publication of EP3735462A1 publication Critical patent/EP3735462A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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 using an exon-skipping and/or refraining approach.
  • MMD Muscular dystrophies
  • DMD Duchenne muscular dystrophy
  • ⁇ DMD dystrophin ⁇ DMD
  • compositions comprising a DMD guide RNA-Cas9 complex for disrupting a dystrophin splice acceptor site and inducing skipping and/or refraining of an exon of a DMD gene, therefore modifying a DMD gene in a cell or a subject.
  • compositions and method of the disclosure may be used to treat muscular dystrophy.
  • a composition comprising a first vector comprising a sequence encoding a DMD guide RNA and a second vector comprising a sequence encoding a Cas9 protein or a nuclease domain thereof, wherein the ratio of the first vector to the second vector is at least 1.5: 1, may be used for the treatment of muscular dystrophy.
  • the first vector and/or the second vector is a viral vector.
  • the first vector and/or the second vector is an adeno-associated viral (AAV) vector.
  • AAV adeno-associated viral
  • the disclosure provides a composition
  • a composition comprising (i) a first 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 sequence encoding a first promoter, wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding a second promoter, wherein the second promoter drives expression of the sequence encoding the second DMD guide RNA, and (ii) a second vector comprising a nucleic acid comprising a sequence encoding a Cas9 or a nuclease domain thereof; a sequence encoding a muscle-specific promoter, wherein the muscle-specific promoter drives expression of the sequence encoding a Cas9 or a nuclease domain thereof; wherein a ratio of the first vector to the second vector in the composition is between about 30: 1 to about
  • the ratio of the first vector to the second vector is about 1 : 1. In some embodiments, the ratio of the first vector to the second vector is about 1.7: 1. In some embodiments, the ratio of the first vector to the second vector is about 2: 1. In some embodiments, the ratio of the first vector to the second vector is about 2.5: 1. In some embodiments, the ratio of the first vector to the second vector is about 3: 1. In some embodiments, the ratio of the first vector to the second vector is about 4: 1. In some embodiments, the ratio of the first vector to the second vector is about 5: 1. In some embodiments, the ratio of the first vector to the second vector is about 6: 1. In some embodiments, the ratio of the first vector to the second vector is about 7: 1.
  • the ratio of the first vector to the second vector is about 8: 1. In some embodiments, the ratio of the first vector to the second vector is about 9: 1. In some embodiments, the ratio of the first vector to the second vector is about 10: 1. In some embodiments, the ratio of the first vector to the second vector is about 1 :2. In some embodiments, the ratio of the first vector to the second vector is about 1 :3. In some embodiments, the ratio of the first vector to the second vector is about 1 :4. In some embodiments, the ratio of the first vector to the second vector is about 1 :5. In some embodiments, the ratio of the first vector to the second vector is about 1 :6. In some embodiments, the ratio of the first vector to the second vector is about 1 :7.
  • the ratio of the first vector to the second vector is about 1 :8. In some embodiments, the ratio of the first vector to the second vector is about 1 :9. In some embodiments, the ratio of the first vector to the second vector is about 1 : 10. In some embodiments, the ratio of the first vector to the second vector is about 1 : 15. In some embodiments, the ratio of the first vector to the second vector is about 1 :20. In some embodiments, the ratio of the first vector to the second vector is about 1 :25. In some embodiments, the ratio of the first vector to the second vector is about 1 :30.
  • the first vector comprises at least one sequence encoding an additional DMD guide RNA targeting a genomic target sequence; and at least one sequence encoding an additional promoter, wherein the at least one additional promoter drives expression of the at least one sequence encoding an additional DMD guide RNA.
  • the first vector or the second vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).
  • the first vector and the second vector comprise a sequence isolated or derived from an adeno-associated virus (AAV).
  • the sequence encoding the muscle-specific promoter comprises or consists of a sequence encoding a muscle-specific creatine kinase 8 (CK8) promoter. In some embodiments, the sequence encoding the muscle-specific promoter comprises or consists of a sequence encoding a CK8e promoter.
  • the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 60-371, SEQ ID NO: 383-694, SEQ ID NO: 682-697, SEQ ID NO: 715-717, SEQ ID NO: 790-862, SEQ ID NO: 1036-1051, SEQ ID NO: 1066-1380, SEQ ID NO: 1392-1467, or SEQ ID NO: 1484-1491.
  • the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714, SEQ ID NO: 762, SEQ ID NO: 1039.
  • the sequence encoding the first DMD guide RNA and the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714, SEQ ID NO: 762, SEQ ID NO: 1039.
  • the sequence encoding the additional DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 60-371, SEQ ID NO: 383- 694, SEQ ID NO: 682-697, SEQ ID NO: 715-717, SEQ ID NO: 790-862, SEQ ID NO: 1036- 1051, SEQ ID NO: 1066-1380, SEQ ID NO: 1392-1467, or SEQ ID NO: 1484-1491.
  • the sequence encoding the additional DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714, SEQ ID NO: 762, SEQ ID NO: 1039.
  • the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714. In some embodiments, the sequence encoding the first DMD guide RNA and the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714. In some embodiments, the sequence encoding the additional DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714.
  • the composition comprises between 5 x 10 11 viral genomes (vg)/kilogram (kg) and 1 x 10 15 vg/kg, inclusive of the endpoints, of the first vector. In some embodiments, including those wherein the composition is administered locally or by an intramuscular delivery route, the composition comprises between 5 x 10 11 viral genomes (vg)/kilogram (kg) and 1 x 10 15 vg/kg, inclusive of the endpoints, of the first vector.
  • the composition comprises between 5 x 10 12 viral genomes (vg)/kilogram (kg) and 1 x 10 15 vg/kg, inclusive of the endpoints, of the first vector.
  • the composition comprises at least 5 x 10 11 viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 1 x 10 12 viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 5 x 10 12 viral genomes (vg)/kilogram (kg) of the first vector.
  • the composition comprises at least 1 x 10 13 viral genomes
  • the composition comprises at least 5 x 10 13 viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 1 x 10 14 viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 5 x 10 14 viral genomes
  • the composition comprises at least 1 x 10 15 viral genomes (vg)/kilogram (kg) of the first vector.
  • the composition comprises at least 4 x 10 12 viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 5 x 10 12 viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 6 x 10 12 viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 1 x 10 13 viral genomes
  • the composition comprises at least 2 x 10 13 viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 3 x 10 13 viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 5 x 10 13 viral genomes
  • the composition comprises at least 1 x 10 14 viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 2 x 10 14 viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 3 x 10 14 viral genomes
  • the composition comprises at least 4 x 10 14 viral genomes (vg)/kilogram (kg) of the first vector.
  • the ratio of the first vector to the second vector is 1 : 1 and the composition comprises at least 6 x 10 12 vg/kg of the first vector and at least 6 x 10 12 vg/kg of the second AAV vector.
  • the ratio of the first vector to the second vector is 1 :1 and the composition comprises at least 2.6 x 10 13 vg/kg of the first vector and at least 2.6 x 10 13 vg/kg of the second AAV vector.
  • the ratio of the first vector to the second vector is 1 : 1 and the composition comprises at least 1 x 10 14 vg/kg of the first vector and at least 1 x 10 14 vg/kg of the second AAV vector. In some embodiments, the ratio of the first vector to the second vector is 2: 1 and the composition comprises at least 2 x 10 14 vg/kg of the first vector and at least 1 x 10 14 vg/kg of the second AAV vector. In some embodiments, the composition further comprises a pharmaceutically-acceptable carrier.
  • the composition comprises between 5 x 10 11 viral genomes (vg)/kilogram (kg) and 1 x 10 15 vg/kg, inclusive of the endpoints, of the first vector. In some embodiments, the composition comprises between 5 x 10 11 viral genomes (vg)/kilogram (kg) and 1 x 10 15 vg/kg, inclusive of the endpoints, of the second vector.
  • a method of treating muscular dystrophy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition
  • a composition comprising (i) a first 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 sequence encoding a first promoter, wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding a second promoter, wherein the second promoter drives expression of the sequence encoding the second DMD guide RNA, and (ii) a second vector comprising a nucleic acid comprising a sequence encoding a Cas9 or a nuclease domain thereof; a sequence encoding a muscle-specific promoter, wherein the muscle- specific promoter drives expression of the sequence encoding a Cas9 or a nucleas
  • 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 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, the subject is a neonate, an infant, a child, a young adult, or an adult. In some embodiments, the subject has muscular dystrophy.
  • the subject is a genetic carrier for muscular dystrophy.
  • the subject is male.
  • the subject is female.
  • the subject is an adult.
  • the adult is at least 18 years old.
  • the targeted muscle cell type is a cardiac muscle cell
  • the adult is at least 25 years old.
  • the adult is at least 20 kg.
  • the subject is a child.
  • the child is less than 18 years of age.
  • the child is 20 kg or less.
  • the subject is an infant. In some embodiments, the subject is less than 2 years old.
  • the subject upon administering the therapeutically effective amount of the composition, the subject produces a minimal immune response to the composition.
  • the minimal immune response to the composition is reduced or eliminated ytreatment with an anti-inflammatory agent or an immune suppressive agent.
  • the composition does not induce breaks in a predicted alternative targeting site.
  • the predicted alternative targeting site comprises a coding sequence of the human genome and wherein the coding sequence comprises at least 2 mismatches with respect to the first genomic target sequence or the second genomic target sequence.
  • the predicted alternative targeting site comprises a coding sequence of the human genome and wherein the coding sequence comprises at least 3, 4, 5, 6, 7,8, 9, or 10 mismatches with respect to the first genomic target sequence or the second genomic target sequence.
  • the predicted alternative targeting site is identified using an algorithm (for example, those publicly available at Based on CRISPR design tools (crispr.mit.edu/ and benchling.com/).
  • confirmation that the composition does not induce breaks at predicted alternative targeting sites comprises DNA amplification, isolation of genomic PCR amplification products and sequencing of the isolated of genomic PCR amplification products spanning the predicted alternative targeting sites.
  • the administration of the therapeutically effective amount of the composition is provided as a single dose or provided within a single medical procedure.
  • the administration of the therapeutically effective amount of the composition is provided as multiple doses or provided over multiple medical procedures.
  • “a” or“an” may mean one or more.
  • the words“a” or“an” when used in conjunction with the word“comprising”, may mean one or more than one.
  • 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.
  • FIG. 1A-F.“Humanized’’-DEc50 mouse model [027] FIG. 1A-F.“Humanized’’-DEc50 mouse model.
  • FIG. 1A Strategy showing CRISPR/Cas9-mediated genome editing approach to generate a humanized mouse model.
  • FIG. IB RT-PCR analysis to validate deletion of exon 50 (DEc50).
  • FIG. ID Histochemistry of cardiac and skeletal muscle by hematoxylin and eosin (H&E) staining, and immunohistochemistry using dystrophin antibody. Scale bar: 50 mm.
  • FIG. IE Western blot analysis of dystrophin and vinculin expression in tibialis anterior and heart tissues.
  • FIG. IF Levels of serum CK, a marker of muscle dystrophy that reflects muscle damage and membrane leakage were measured in wildtype (WT), DEc50 and mdx mice.
  • FIG. 2A-B Exon 51 skipping.
  • FIG. 2A RT-PCR of RNA from DEc50 mice 3 weeks after intramuscular injection indicates deletion of exon 51 (termed DEc50-51, lower band).
  • FIG. 3A-G Correction of dystrophin expression using triplicate gRNA strategy 3 weeks after intra-muscular injection.
  • FIG. 3A Strategy showing CRISPR/Cas9-mediated genome editing approach to correct the reading frame in DEc50 mouse model.
  • FIG. 3B sgRNA targeting the splice acceptor site (sgRNA-ex5l-SA) sequence and schematic illustration of sgRNA binding position.
  • Fig. 3B discloses SEQ ID NOS: 954-957, respectively, in order of appearance.
  • FIG. 3C Schematic illustration of AAV injection plasmids and strategy.
  • FIG. 3D Dystrophin immunohistochemistry staining of tibialis anterior muscle.
  • FIG. 3E
  • FIG. 3F Western blot analysis of dystrophin (DMD) and vincubn (VCL) expression 3 weeks after intramuscular injection.
  • FIG. 3G Quantification of dystrophin expression after normalization to vinculin. Data are represented as mean ⁇ SEM. **P ⁇ 0.005. Scale bar: 50 mm
  • FIG. 4A-B Histological improvement of injected muscle after 3 weeks.
  • FIG. 4B Quantification of fiber size and percentage of frequency. Data are represented as mean ⁇ SEM. Scale bar: 50 mm.
  • FIG. 5 Screen of sgRNA in human 293 cells and mouse 10T cells. Undigested PCR products (upper panel) and T7E1 digestion (lower panel) on a 2% agarose gel. M denotes size marker lane bp indicates the length of the marker bands.
  • FIG. 6 Dystrophin immunohistochemistry staining of entire tibialis anterior muscle. Immunohistochemistry analysis demonstrates that dystrophin levels were restored in mice treated using the double guide constructs and the triple guide constructs. Mice treated using the triple guide constructs had higher levels of dystrophin expression.
  • FIG. 7A-B Strategy for CRISPR/Cas9-mediated genome editing in AEx50 mice.
  • FIG. 7A Strategy showing CRISPR/Cas9-mediated genome editing approach to correct the reading frame in AEx50 mouse model.
  • FIG. 7B sgRNA targeting the splice acceptor site (sgRNA-ex5l-SA2) sequence (SEQ ID NO: 708) and schematic illustration of sgRNA binding position.
  • Fig. 7B discloses SEQ ID NOS: 958-959, respectively, in order of appearance.
  • FIG. 8A-F sgRNA genomic analysis in mouse and human cells.
  • Cas9 was expressed in the presence or absence of mouse sgRNA-sgRNA-5l-SA2 in 10T1/2 cells and gene editing was monitored by T7E1 assay in fluorescence-based cell sorted (FACS) (+) and non-sorted cells (-). GFP was used as a control.
  • Undigested PCR products (upper panel) and T7E1 digestion (lower panel) are shown on a 2% agarose gel. Black arrowhead indicates the undigested 77lbp PCR band.
  • Green arrowheads in the lower panel indicate the cut bands by T7E1 assay.
  • M denotes size marker lane bp indicates the length of the marker bands.
  • FIG. 8B Genomic deep sequencing analysis of PCR amplicons generated across the exon 51 target site in 10T1/2 cells. Sequence of representative indels aligned with sgRNA sequence (indicated in blue) revealing insertions (highlighted in green) and deletions (highlighted in red) (SEQ ID NOS: 960-966, respectively, in order of appearance). The line indicates the predicted exon splicing enhancers (ESEs) sequence located at the site of sgRNA. Black arrow indicates the cleavage site.
  • FIG. 8C Mouse Exon 51 sequence (SEQ ID NO: 967) with the predicted exon splicing enhancers (ESEs) located at the site of sgRNA is indicated in red.
  • FIG. 8D Mouse and human ESE sites of exon 51 predicted using ESEfmder3.
  • FIG. 8E Cas9 was expressed in the presence or absence of mouse sgRNA-sgRNA-5l-SA2 in 293 T human cells and gene editing was monitored by T7E1 assay in fluorescence-based cell sorted (FACS) (+) and non- sorted cells (-). GFP was used as a control. Undigested PCR products (upper panel) and T7E1 digestion (lower panel) are shown on a 2% agarose gel.
  • Black arrowhead indicates the undigested 77lbp PCR band. Green arrowheads in the lower panel indicate the cut bands by T7E1 assay. M denotes size marker lane bp indicates the length of the marker bands.
  • FIG. 8F Sequence of representative indels aligned with sgRNA sequence (indicated in blue) revealing deletions and insertions (SEQ ID NOS: 969-978, respectively, in order of appearance). Black arrowhead indicates the cleavage site.
  • FIG. 9A-B Schematic illustration of AAV injection plasmids and strategy.
  • FIG. 9 A Muscle creatine kinase 8 (CK8) promoter to express SpCas9.
  • FIG. 9B Triplicate using rAAV9-sgRNA plasmid containing 3 copies of sgRNA-ex5l-SA2.
  • U6, Hl and 7SK promoter for RNA polymerase III were used to drive expression of sgRNA.
  • FIG. 10A-B In vivo Dmd gene editing.
  • FIG. 10A Undigested PCR products (upper panel) and T7E1 digestion (lower panel) are shown on a 2% agarose gel of TA (tibialis anterior) muscle samples from WT and AEx50 mice 3 weeks after intramuscular injection with AAV9-sgRNA-SA2 and AAV9-Cas9 expression vectors. Controls were injected with only AAV9-Cas9 not AAV9-sgRNA-SA2. Black arrowhead in the upper panel indicates the 77lbp PCR band. Green arrowheads in the lower panel indicate the cut bands by T7E1 assay.
  • M denotes size marker lane bp indicates the length of the marker bands.
  • N 4.
  • FIG. 11A-D RT-PCR analysis of correction of reading frame.
  • FIG. 11A RT-PCR of RNA from tibialis anterior muscles of wildtype (WT) and AEx50 mice 3 weeks after intramuscular injection of the sgRNA-5l-SA2 and Cas9 expression vectors. Lower dystrophin bands indicate deletion of exon 51. Primer positions in exons 48 and 53 are indicated (Fw, Rv).
  • FIG. 11B Percentage of events detected at exon 51 after AAV9- sgRNA-5l-SA2 treatment using RT-PCR sequence analysis of TOPO-TA generated clones. For each of 4 different samples, 40 clones were generated and sequenced.
  • RT-PCR products were divided into 4 groups: not-edited (NE), exon51 -skipped (SK), refrained (RF) and out of frame (OF).
  • NE not-edited
  • SK exon51 -skipped
  • RF refrained
  • OF out of frame
  • FIG. 11C Sequence of the RT-PCR products of the DEc50-51 mouse dystrophin lower band confirmed that exon 49 spliced directly to exon 52, excluding exon 51. Sequence of RT-PCR products of DEc50 refrained (AEx50-RF).
  • Fig. 11C discloses SEQ ID NOS: 1015-1022, respectively, in order of appearance.
  • FIG. 11D Deep sequencing analysis of RT-PCR products from the upper band containing DEc50 not-edited (NE) and ⁇ Ex50-RF.
  • FIG. 12A-D Intramuscular injection of AAV9-Cas9 and AAV9-sgRNA-51-SA2 corrects dystrophin expression.
  • FIG. 12A Tibialis anterior muscles of DEc50 mice were injected with AAV9 vector encoding sgRNA and Cas9 and analyzed 3 weeks later by immunostaining for dystrophin.
  • Wild type control (WT-CTL) mice and DEc50 mice control ( ⁇ Ex50-CTL) were injected with AAV9-Cas9 alone without sgRNAs.
  • FIG. 12B Hematoxylin and eosin (H&E) staining of tibialis anterior muscles.
  • FIG. 12C Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in tibialis anterior muscles 3 weeks after intramuscular injection.
  • FIG. 14 Histological improvement of injected muscle after 3 weeks.
  • H&E hematoxylin and eosin
  • FIG. 15A-B Rescue of dystrophin expression following intramuscular injections of AAV9-Cas9 combined with different AAV9s expressing single copy or triple copy of sgRNA in AEx50 mouse model.
  • FIG. 15A The U6, Hl and 7SK promoters for RNA polymerase III were each individually used to express sgRNA in a single copy (AAV9-U6- sgRNA-5l-SA2; AAV9-Hl-sgRNA-5l-SA2; AAV9-7SK-sgRNA-5l-SA2) or triple copy.
  • FIG. 15B Dystrophin immunohistochemistry of entire tibialis anterior muscle. Control (CTL) mice were injected with AAV9-Cas9 alone without AAV9-sgRNA-5l-SA2.
  • FIG. 16A-B Rescue of dystrophin expression 4 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2 in AEx50 mice.
  • FIG. 16A Dystrophin immunostaining of tibialis anterior (TA), triceps, diaphragm and cardiac muscles 4 weeks after systemic injection of AAV9-sgRNA-5l.
  • FIG. 17A-B Rescue of dystrophin expression 8 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2 in AEx50 mice.
  • FIG. 17A Dystrophin immunostaining of tibialis anterior (TA), triceps, diaphragm and cardiac muscles 8 weeks after systemic injection of AAV9-sgRNA-5l.
  • FIG. 18A-B Functional improvement 4 weeks after systemic delivery of AAV9- Cas9 and AAV9-sgRNA-51-SA2 in AEx50 mice.
  • FIG. 18A Wild type (WT) mice, control AEx50 mice and AEx50 mice treated with AAV9-sgRNA-5l-SA2 (AEx50-AAV9-sgRNA- 51-SA2) were subjected to grip strength testing to measure muscle performance (grams of force).
  • FIG. 19 Correction of dystrophin expression 6 weeks after intra-muscular injection in AEx50-MD Dog.
  • FIG. 20A-B Correction of dystrophin expression 6 weeks after intra-muscular injection in AEx50-MD Dog.
  • FIG. 20A Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in cranial tibialis muscles 6 weeks after intramuscular injection in 2 dogs (Newton (#lA) and Norman (#lB)).
  • FIG. 20B Quantification of dystrophin expression from blots after normalization to vinculin.
  • FIG. 21 Histological improvement of injected muscle after 6 weeks in AEx50-MD Dog. Histochemistry by hematoxylin and eosin (H&E) staining of cranial tibialis muscle from a wild type dog, AEx50 dog untreated, AEx50 contralateral uninjected and AEx50 dogs injected intramuscularly with AAV9-Cas9 and AAV9-sgRNA-5l (referred as AEx50-Dog- #lA-AAV9s (Newton) and AEx50-Dog-#lB-AAV9s (Norman)). Scale bar: 50mm.
  • H&E hematoxylin and eosin
  • FIG. 22A-B Rescue of dystrophin expression 4 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2 (referred as AAV9-sgRNA-51) in AEx50 mice.
  • FIG. 22A Dystrophin immunostaining of tibialis anterior (TA), triceps, diaphragm and cardiac muscles 4 weeks after systemic injection of different doses of AAV9-Cas9 and AAV9-sgRNA-5l-SA2 (referred as AAV9-sgRNA-5l.
  • FIG. 22B Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in triceps, gastrocnemius/plantaris (G/P) diaphragm (Dia) muscles and heart.
  • DMD dystrophin
  • FIG. 23A-C Correction of dystrophin expression 4 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2 (referred as AAV9-sgRNA-51) in 1 month old AEx50 mice.
  • FIG. 23A Dystrophin immunostaining of tibialis anterior, triceps and cardiac muscles 4 weeks after systemic injection of AAV9-Cas9 and AAV9-sgRNA-5l-SA2 (referred as AAV9-sgRNA-5l at a dose of 2.6xl0 13 vg/kg of each AAV9.
  • FIG. 23B Dystrophin immunostaining of tibialis anterior, triceps and cardiac muscles 4 weeks after systemic injection of AAV9-Cas9 and AAV9-sgRNA-5l-SA2 (referred as AAV9-sgRNA-5l at a dose of 2.6xl0 13 vg/kg of each AAV9.
  • FIG. 23C Wild type mice, control AEx50 mice and AEx50 mice treated with AAV9-Cas9 and AAV9-sgRNA-5l (AEx50-AAV9) were subjected to grip strength testing to measure muscle performance (grams of force).
  • FIG. 24A-C Correction of dystrophin expression 8 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2 (referred as AAV9-sgRNA-51) in 1 month old AEx50 mice.
  • FIG. 24A Dystrophin immunostaining of tibialis anterior, triceps, gastrocnemius, quadriceps, diaphragm and cardiac muscles 8 weeks after systemic injection of AAV9-Cas9 and AAV9-sgRNA-51 with 2.6 x 10 13 vg/kg of each AAV9.
  • FIG. 24B Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in various tissues.
  • DMD dystrophin
  • VCL vinculin
  • FIG. 24C Wildtype mice, control AEx50 mice and AEx50 mice treated with AAV9- Cas9 and AAV9-sgRNA-5l (AEx50-AAV9) were subjected to grip strength testing to measure muscle performance (grams of force).
  • FIG. 25A-B In vivo investigation of correction of dystrophin expression using different AAV9-Cas9 and AAV9-sgRNA-51-SA2 (referred as AAV9-sgRNA-51) ratios by intravenous injection of AAV9s. (FIG.
  • AEx50-KI-luciferase mice were injected with an AAV9 encoding Cas9 and an AAV9 encoding a sgRNA at a 1 : 1 ratio (1xl0 14 vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l) and at a 1 :2 ratio (1xl0 14 vg/kg of AAV9-Cas9 and 2xl0 14 vg/kg AAV9-sgRNA-5l).
  • AEx50-KI-luciferase mice were analyzed weekly by bioluminescence. (FIG.
  • FIG. 26A-G.“Humanized”-AEx44 mouse model (FIG. 26A) Outline of the CRISPR/Cas9 strategy used for generation of the AEx44 mice.
  • FIG. 26B Outline of the CRISPR/Cas9 strategy to deplete exon 44.
  • T7E1 assay using 10T1/2 mouse cells transfected with spCas9 with different sgRNAs targeting 5’ end (In44-l, In44-2 or In44-3) and 3’ end (In44-4, In44-5, In44-6) of exon 44 shows different cleavage efficiency at the Dmd exon 44. Red arrowheads show cleavage products of genome editing.
  • FIG. 26C PCR genotyping of 10 Fl pups shows efficient exon 44 depletion by CRISPR/Cas9-mediated genome editing.
  • FIG. 26E Western blot analysis shows loss of dystrophin expression in heart, TA muscle, and gastrocnemius/plantaris (G/P) muscle of AEx44 mice. Vinculin was used as a loading control.
  • FIG. 26F Dystrophin staining of TA, diaphragm and cardiac muscle.
  • FIG. 27A-E Characterization of DEc44 mice.
  • FIG. 28A-G Correction of Dmd exon 44 deletion in mice by intramuscular AAV9 delivery of gene editing components.
  • A RT-PCR analysis of TA muscles from WT and DEc44 mice 3 weeks after intramuscular injection of gene editing components carried by AAV9. Lower dystrophin bands (179 bp) indicate skipping of exon 45.
  • B Pie chart showing percentage of events detected at exon 45 after AAV-Cas9 and AAV-G6 treatment using RT- PCR sequence analysis of TOPO-TA (topoisomerase-based thymidine to adenosine) generated clones.
  • TOPO-TA topoisomerase-based thymidine to adenosine
  • C Sequences of RT-PCR products of WT, DEc44 and corrected DEc44 mice. In-frame sequences are shown in blue, including WT and exon 45-skipped sequences. Refrained sequence is shown in green, and out of frame sequence is shown in red. Figure discloses SEQ ID NOS 2305-2312, respectively, in order of appearance.
  • D Western blot analysis shows restoration of dystrophin expression in TA muscle and heart of DEc44 mice. Vinculin is loading control.
  • FIG. 29A-E Systemic AAV9 delivery of gene editing components to D44 mice rescues dystrophin expression.
  • Different AAV9-Cas9 and AAV9-exon45-sgRNA-G6 ratios were injected into D44 mice: 1.7: 1 (8.5xl0 13 vg/kg of AAV9-exon45-sgRNA to 5xl0 13 vg/kg of AAV9-Cas9); 2: 1 (1xl0 14 vg/kg of AAV9-exon45-sgRNA to 5xl0 13 vg/kg of AAV9-Cas9); 2.5: 1 (l.25xl0 13 vg/kg of AAV9-exon45-sgRNA-G6 to 5xl0 13 vg/kg of AAV9-Cas9), 5: 1 (2xl0 14 vg/kg of AAV9-exon45-sgRNA-G6 to 5xl0 13 vg/kg of AAV9-C
  • FIG. 29A Western blot analysis shows restoration of dystrophin expression in TA, diaphragm, triceps and cardiac muscles of D44 mice 4 weeks after systemic delivery of AAV9-Cas9 or AAV9- Cas9/exon45-sgRNA4. Vinculin was used as a loading control.
  • FIG. 29B Immunostaining shows restoration of dystrophin in TA, diaphragm, triceps and cardiac muscles of D44 mice 4 weeks after systemic delivery of AAV9-Cas9 or AAV9-Cas9/exon45-sgRNA4. Dystrophin stains in red. Nucleus marks by DAPI stains in blue.
  • FIG. 29C Reduction of serum creatine kinase activity in D44 mice 4 weeks after systemic delivery of AAV9-Cas9 or AAV9- Cas9/exon45-sgRNA4.
  • FIG. 30 Immunostaining of dystrophin following intravenous delivery of AAV9- encoded gene editing components in AEx50-MD Dogs.
  • FIG. 31A-D Western blot of dystrophin following intravenous delivery of AAV9- encoded gene editing components.
  • FIG. 31A Western blot analysis of dystrophin (DMD) and vinculin (VCL) of cranial tibialis, triceps, biceps muscles of wild type, untreated AEx50, and AEx50 injected with AAV9-Cas9 and AAV9-sgRNA at 2xl0 13 vg/kg for each virus (referred as AEx50-Dog #2A-AAV9s).
  • FIG. 31B Quantification of dystrophin expression from blots after normalization to vinculin.
  • FIG. 31C Western blot analysis of dystrophin (DMD) and vinculin (VCL) of cranial tibialis, triceps, biceps, diaphragm, heart, tongue muscles of wild type, untreated AEx50, and AEx50 injected with AAV9-Cas9 and AAV9- sgRNA at 1x10 14 vg/kg of each virus (referred as AEx50-Dog #2B-AAV9s).
  • FIG. 32 Muscle histology following intravenous delivery of AAV9-encoded gene editing.
  • H&E hematoxylin and eosin staining of cranial tibialis, diaphragm and biceps muscles of wild type dog untreated, AEx50-MD Dogs untreated and AAV9-Cas9-sgRNA injected with AAV9-Cas9/AAV9-sgRNA at 2xl0 13 vg/kg (Dog #2A) and 1xl0 14 vg/kg (Dog #2B) for each virus.
  • Scale bar 50mm.
  • FIG. 33 Blood analysis 8 weeks after intra-venous injection. Creatine kinase (CK) activities in untreated wild type dog, untreated DEc50 dog, AEx50-Dog-#2A injected with 2xl0 13 vg/kg and AEx50-Dog-#2B injected with1xl0 14 vg/kg of each of AAV9-Cas9 and AAV9-sgRNA.
  • CK Creatine kinase
  • FIG. 34A-F AEx50-Dmd-Luc mouse model.
  • FIG. 34A Strategy for creation of dystrophin reporter mice. Dystrophin ( Dmd) gene with exons is indicated in blue. Using CRISPR/Cas9 mutagenesis, the inventors inserted a DNA cassette encoding the Luciferase reporter with the protease 2A cleavage site at the 3’ end of the dystrophin coding region.
  • FIG. 34B Bioluminescence imaging of wild-type (WT) and Dmd knock-in luciferase reporter (referred as WT-Dmd-Luc) mice.
  • FIG. 34C Strategy for creation of AEx50-Dmd- Luc reporter mice. Dystrophin ( Dmd) gene with exons is indicated in blue. Using CRISPR/Cas9 mutagenesis, the inventors inserted a DNA cassette encoding the Luciferase reporter with the protease 2A cleavage site at the 3’
  • FIG. 34D Genotyping results of AEx50-Dmd-Luc reporter mice. Schematic of genotyping strategy and forward (Fw) and reverse (Rv) primers.
  • FIG. 34E Bioluminescence imaging of wild-type (WT), WT-Dmd-Luc and AEx50-Dmd-Luc reporter mice.
  • FIG. 34F Western blot analysis of dystrophin (DMD), Luciferase and vinculin (VCL) expression in skeletal muscle and heart tissues.
  • DMD dystrophin
  • VCL vinculin
  • FIG. 35A-E Correction of dystrophin expression by intra-muscular injection of AAV9-encoded gene editing components.
  • FIG. 35A The left tibialis anterior muscle of AEx50-Dmd-Luc mice were injected with AAV9-Cas9 and AAV9-sgRNA-5l-SA2 (referred as AAV9-sgRNA) SEQ. NO: 708.
  • AEx50-Dmd-Luc mice were analyzed weekly by bioluminescence. Control mice were injected with saline.
  • FIG. 35B Bioluminescence imaging of wild-type (WT), WT-Dmd-Luc and AEx50-Dmd-Luc mice injected with AAV9- Cas9 and AAV9-sgRNA 1 week and 4 weeks after injection.
  • FIG. 35C Dystrophin immunohistochemistry of entire tibialis anterior muscle of WT-Dmd-Luc mice, control AEx50-Dmd-Luc mice and AEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9- sgRNA, 4 weeks after injection.
  • 35D Dystrophin immunohistochemistry of tibialis anterior muscle of WT-Dmd-Luc mice, control AEx50-Dmd-Luc mice and AEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA, 4 weeks after injection.
  • FIG. 36A-D Correction of dystrophin expression by systemic delivery of AAV9- encoded gene editing components.
  • FIG. 36A AEx50-Dmd-Luc mice were injected intraperitoneally with AAV9-Cas9 and AAV9-sgRNA-5l-SA2 (referred as AAV9-sgRNA) SEQ. NO: 708 and analyzed by bioluminescence. Control mice were injected with saline.
  • FIG. 36B Bioluminescence imaging of WT-Dmd-Luc mice and AEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA.
  • FIG. 37A-B Western blot of dystrophin and luciferase following systemic delivery of AAV9-encoded gene editing components.
  • FIG. 37A Western blot analysis of dystrophin (DMD), Luciferase (Luc), Cas9 and vinculin (VCL) in diaphragm, heart, triceps muscles and tibialis anterior of WT-Dmd-Luc mice, control AEx50-Dmd-Luc mice and AEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA-5l-SA2 (referred as AAV9-sgRNA) SEQ. NO: 708.
  • FIG. 38 AEx50-Dmd-Luciferase mouse model analysis. Genomic sequence of targeted locus of WT-Dmd-Luc (top line) (SEQ ID NO: 2313) and AEx50-Dmd-Luciferase founder (bottom line) (SEQ ID NO: 2314) with a 215 base pair deletion that eliminated exon 50 (indicated in color green). sgRNA-#l and #2 are indicated in blue.
  • FIG. 39 AEx50-Dmd-Luciferase mouse model muscle histological analysis.
  • Hematoxylin and eosin (H&E) staining of tibialis anterior, quadriceps and diaphragm muscles of 6 weeks old WT-Dmd-Luc and AEx50-Dmd-Luciferase mice. n 5. Scale bar: 50mm.
  • FIG. 41A-D Single cut CRISPR editing of canine exon 50 in vivo and in vitro.
  • FIG. 41A Scheme showing the CRISPR/Cas9-mediated genome editing approach to correct the reading frame in AEx50 dogs by refraining and skipping of exon 51. Gray exons are out of frame.
  • FIG. 4 IB Illustration of sgRNA binding position and sequence for sgRNA-ex5l. PAM sequence for sgRNA is indicated in red. Black arrow indicates the cleavage site. Figure discloses SEQ ID NOS 2315-2316, respectively, in order of appearance.
  • FIG. 41C shows SEQ ID NOS 2315-2316, respectively, in order of appearance.
  • Dystrophin immunohistochemistry staining of cranial tibialis muscle of wild type dog untreated, AEx50 dog untreated, AEx50 dogs contralateral (uninjected) muscle and AEx50 dogs injected with AAV9-Cas9 and AAV9-sgRNA-5l referred as AEx50-#lA AAV9s and AEx50-#lB-AAV9s). Scale bar: 50mm.
  • FIG. 42A-D Rescue of dystrophin expression in human DMD iPSC-derived cardiomyocytes.
  • FIG. 42A Genomic deep sequencing analysis of PCR amplicons generated across the exon 51 target site in iPS cells. Sequence of representative indels aligned with sgRNA sequence (indicated in blue) revealing insertions (highlighted in green) and deletions (highlighted in red). Black arrowhead indicates the cleavage site. Figure discloses SEQ ID NOS 2325-2336, respectively, in order of appearance.
  • FIG. 42B Genomic deep sequencing analysis of PCR amplicons generated across the exon 51 target site in iPS cells. Sequence of representative indels aligned with sgRNA sequence (indicated in blue) revealing insertions (highlighted in green) and deletions (highlighted in red). Black arrowhead indicates the cleavage site. Figure discloses SEQ ID NOS 2325-2336, respectively, in order of appearance.
  • FIG. 42B shows SEQ ID NOS 2325-
  • Immunocytochemistry of dystrophin expression shows DMD iCMs lacking dystrophin expression. Following successful gene editing, the corrected DMD iCMs express dystrophin. Immunofluorescence (green) detects cardiac marker troponin-I. Nuclei are labeled by Hoechst dye (blue).
  • FIG. 42C Western blot analysis of dystrophin (DMD) and vinculin (VCL) of WT untreated and GFP treated, iCM DMD carrying deletion from exon 48 to exon 50 untreated and GFP treated, iCM DMD carrying deletion from exon 48 to exon 50 from mixed clone treated with high and low concentration of Cas9 and human sgRNA-5l SEQ.
  • FIG. 43A-B Validation of sgRNA-51 in dog and human 293T cells.
  • Undigested PCR products (upper panel) and T7E1 digestion (lower panel) are shown on a 2% agarose gel.
  • Black arrowhead indicates the undigested 574bp PCR band amplified from human genomic samples.
  • Grey arrowhead indicates the undigested 748bp PCR band amplified from dog genomic samples.
  • Green arrowheads in the lower panel indicate the cut bands by T7E1 assay from PCR bands amplified from human genomic DNA (H).
  • Red arrowheads in the lower panel indicate the cut bands by T7E1 assay from PCR bands amplified from dog genomic DNA (D).
  • M denotes size marker lane bp indicates the length of the marker bands.
  • FIG. 44A-C In vivo Dmd gene editing after intramuscular injection.
  • FIG. 44A Genomic deep sequencing analysis of PCR amplicons generated across the exon 51 target site in DEc50 dogs injected with AAV9-sgRNA-5l and AAV9-Cas9. Sequences of representative indels are aligned with the sgRNA sequence (indicated in blue), revealing insertions
  • FIG. 44B RT-PCR of RNA from cranial tibialis muscles of wild type dog untreated, DEc50 dog untreated, DEc50 dogs contralateral uninjected and DEc50 dogs injected with AAV9-Cas9 and AAV9-sgRNA-5l (referred as AEx50-# 1 A-AAV9s and AEx50-#lB-AAV9s) 6 weeks after intramuscular injection. Lower bands indicate deletion of exon 51. Primer positions in exons 48 and 53 are indicated (Fw, Rv). (FIG.
  • FIG. 45 List of potential off-target sites in the dog genome for sgRNA-51.
  • OT1 to OT3 Three potential genome-wide off- target sites (OT1 to OT3) were predicted in the coding regions of mitochondrial pyruvate carrier 2 (MCP2), microphthalmia-associated transcription factor (MITF) and prolyl 4- hydroxylase transmembrane (P4HTM).
  • MCP2 mitochondrial pyruvate carrier 2
  • MITF microphthalmia-associated transcription factor
  • P4HTM prolyl 4- hydroxylase transmembrane
  • FIG. 46 Deep sequencing analyses of off-target sites for sgRNA-51. Genomic deep sequencing analysis of PCR amplicons generated across the exonic off-target sites in cranial tibialis muscles. Mismatches in the target sequence are highlighted in red. Muscle samples from untreated wild type dog, untreated DEc50 dog and contralateral uninjected DEc50 dogs were used for analysis to determine the background of the sequencing analysis. Figure discloses SEQ ID NOS 2372-2425, respectively, in order of appearance.
  • FIG. 47A-B Intramuscular delivery of AAV9-Cas8 and AAV9-sgRNA-51 in AEx50 dogs reduces expression and numbers of develommental myosin (dMHC)-positive fibers.
  • dMHC develommental myosin
  • FIG. 47A Perlecan and develommental myosin (dMHC) immunohistochemistry of cranial tibialis muscles 6 weeks after intramuscular injection of AAV9s.
  • FIG. 47B Western blot analysis of dMHC and vinculin (VCL) in cranial tibialis muscles 6 weeks after intramuscular injection. Scale bar: 50mm.
  • FIG. 48 Intramuscular delivery of AAV9-Cas8 and AAV9-sgRNA-51 in DEc50 dogs restores dystroglycan complex protein expression b-dystroglycan
  • FIG. 49 Lack of immune infiltration in muscles after intramuscular injection of AAV9-Cas9 and AAV9-sgRNA-51.
  • Scale bar 50mm.
  • FIG. 50 Blood analysis 6 weeks after intramuscular injection of AAV9-Cas9 and AAV9-sgRNA-51. Hematology analysis in wild type dog and DEc50 dogs injected with AAV9-Cas9 and AAV9-sgRNA-5l (referred as AEx50-#lA-AAV9s and AEx50-#lb- AAV9s).
  • White blood cells (WBC) red blood cells (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and platelet count (PLT) were evaluated.
  • FIG. 51A-B AAV9-Cas9 expression after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in dogs.
  • FIG. 51A qRT-PCR analysis of Cas9 expression of cranial tibialis, heart left ventricle, heart right ventricle, heart septum, biceps, diaphragm, triceps, temporalis and masseter of untreated wild type dog, untreated AEx50 dog, AEx50-dog-#2A injected with 2xl0 13 vg/kg of AAV9-Cas9 and AAV9-sgRNA-5l.
  • FIG. 51A qRT-PCR analysis of Cas9 expression of cranial tibialis, heart left ventricle, heart right ventricle, heart septum, biceps, diaphragm, triceps, temporalis and masseter of untreated wild type dog, untreated AEx50 dog, AEx50-dog-#
  • FIG. 52A-D In vivo DMD gene editing after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in dogs.
  • FIG. 52A Percentage of indels detected at genomic locus of exon 51 after AAV9-Cas9 and AAV9-sgRNA-5l treatment using tracking indels by decomposition (TIDE) analysis of PCR products amplifying the targeted region from cranial tibialis, heart left ventricle, heart right ventricle, heart septum, diaphragm, biceps and triceps samples of untreated wild type dog, untreated AEx50 dog, AEx50-Dog-#2A injected with 2xl0 13 vg/kg and AEx50-Dog-#2B injected with 1xl0 14 vg/kg of AAV9-Cas9 and AAV9- sgRNA.
  • TIDE tracking indels by decomposition
  • FIG. 52B RT-PCR of RNA from cranial tibialis, heart left ventricle, heart right ventricle, heart septum, diaphragm, biceps and triceps samples of untreated wild type dog, untreated AEx50 dog, AEx50-Dog-#2A injected with 2xl0 13 vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l (referred as AAV9s).
  • Black arrowhead indicates the 822bp WT PCR.
  • Red arrowhead indicates the 7l3bp AEx50 PCR. Lower bands indicate deletion of exon 51.
  • Black arrowhead indicates the 822bp WT PCR band.
  • Red arrowhead indicates the 7l3bp AEx50 PCR band.
  • Grey arrowhead indicates the deletion of exon 51 and 480bp AEx50-5l PCR band.
  • 52D Percentage of indels detected at exon 51 after AAV9-Cas9 and AAV9-sgRNA-5l treatment using tracking indels by decomposition (TIDE) analysis of RT-PCR products from cranial tibialis, heart left ventricle, heart right ventricle, heart septum, diaphragm, biceps and triceps samples of untreated wild type dog, untreated AEx50 dog, AEx50-Dog-#2A injected with 2xl0 13 vg/kg and AEx50-Dog-#2B injected with 1xl0 14 vg/kg of AAV9-Cas9 and AAV9-sgRNA.
  • TIDE tracking indels by decomposition
  • FIG. 53 Correction of dystroglycan complex protein expression after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in AEx50 dogs, b-dystroglycan
  • FIG. 54A-B Decrease of regeneration markers after systemic delivery of AAV9- Cas9 and AAV9-sgRNA-51 in DEc50 dogs.
  • FIG. 54A Perlecan and develomment myosin (dMHC) immunohistochemistry of triceps, diaphragm and semitendinosus muscles 8 weeks after systemic injection of AAV9-Cas9 and AAV9-sgRNA-5l (referred as AAV9s).
  • FIG. 54B Western blot analysis of dMHC and vinculin (VCL) in cranial tibialis and diaphragm muscles of wild type, DEc50 injected with AAV9-Cas9 at 1x10 14 vg/kg and untreated DEc50. Scale bar: 50mm.
  • FIG. 55 ELISpot analysis before and after systemic injection of AAV9-Cas9 and AAV9-sgRNA-51. T-cell reactivity to Cas9 measured using ELISpot analysis of peripheral blood mononuclear cells (PBMCs) of AEx50-Dog-#2A injected with 2xl0 13 vg/kg and AEx50-Dog-#2B injected with 1xl0 14 vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l. PBMCs were isolated from blood samples before injection (0), 1, 2, 4, 6 and 8 weeks after injection.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • PMA phorbol l2-myristate 13- acetate
  • SFU Spot forming units
  • FIG. 56A-B Hematology and biochemistry analyses before and after systemic injection of AAV9-Cas9 and AAV9-sgRNA-51. Blood samples collected before injection (0), 1, 2, 4, 6 and 8 weeks after injection were used for the hematology and biochemistry analysis.
  • FIG. 56A Hematology analyses in wild type dog untreated, DEc50 dog untreated, DEc50 dogs injected with AAV9-Cas9 and AAV9-sgRNA-5l.
  • WBC white blood cells
  • RBC red blood cells
  • HGB Hemoglobin
  • HCT Hematocrit
  • MCV Mean corpuscular volume
  • MH mean corpuscular hemoglobin
  • MCHC mean corpuscular hemoglobin concentration
  • PTT platelet count
  • Total protein, albumin, globulin, sodium, potassium, chloride, calcium, inorganic phosphate, urea, creatinine, cholesterol, total bilirubin, amylase, lipase, alanine transaminase (ALT) and alkaline phosphatase (ALP) were measured in serum samples from untreated wild type dog, untreated DEc50 dog, AEx50-Dog-#2A injected with 2xl0 13 vg/kg and AEx50-Dog-#2B injected with 1x10 14 vg/kg of each of AAV9-Cas9 and AAV9-sgRNA.
  • FIG. 57A-B Validation of sgRNA-51 in human DMD iPS cells.
  • Cas9 was expressed in the presence or absence of human sgRNA-5l (hsgR) in human iPS cells and gene editing was monitored by T7E1 assay. High and low concentration of Cas9 and human sgRNA (hsgR) were tested. Undigested PCR products (upper panel) and T7E1 digestion (lower panel) are shown on a 2% agarose gel. Black arrowhead indicates the undigested 574bp PCR band. Green arrowheads in the lower panel indicate the cut bands by T7E1 assay. M denotes size marker lane bp indicates the length of the marker bands.
  • FIG. 57B shows that
  • FIG. 58 Deep sequencing analysis of genomic DNA after systemic delivery of AAV9-Cas9 and AAV9 sgRNA-51 in dogs. Genomic deep sequencing analysis of PCR amplicons generated across the exon 51 target site in heart, biceps and triceps of DEx50 dogs systemically injected with AAV9-sgRNA-5l and AAV9-Cas9. Sequences of representative indels are aligned with the sgRNA sequence (indicated in blue), revealing insertions
  • FIG. 59A-B Testes analyses after systemic delivery of AAV9-Cas9 and AAV9- sgRNA-51 in dogs.
  • FIG. 59A Percentage of indels detected at genomic locus of exon 51 after AAV9-Cas9 and AAV9-sgRNA-5l treatment using tracking indels by decomposition (TIDE) analysis of PCR products amplifying the targeted region from testes, biceps and diaphragm samples of AEx50-Dog-#2B injected with 1xl0 14 vg/kg of each virus (total 2xl0 14 vg/kg).
  • TIDE tracking indels by decomposition
  • FIG. 60 Western blot titration of dystrophin following intravenous delivery of AAV9-encoded gene editing components.
  • DMD dystrophin
  • VCL vinculin
  • FIG. 61A-F Exon 44-deleted DMD patient iPSC-derived cardiomyocytes express dystrophin after CRISPR/Cas9 mediated genome editing.
  • FIG. 61A Schematic of the procedure for derivation and editing of DMD patient-derived iPSCs and iPSC-CMs.
  • the protospacer adjacent motif (PAM) (denoted as red nucleotides) of the sgRNAs is located near the exon 43 splice junctions. Exon sequence is bold upper case. Intron sequence is lower case. Arrowheads show sites of Cas9 DNA cutting with each sgRNA. Splice acceptor and donor sites are shaded in yellow. Figure discloses SEQ ID NOS 2486-2489, respectively, in order of appearance.
  • FIG. 61D Sequence of sgRNAs targeting exon 45 splice acceptor site in the human DMD gene.
  • the PAM (denoted as red nucleotides) of the sgRNAs is located near the exon 45 splice acceptor site.
  • FIG. 61E Western blot analysis shows restoration of dystrophin expression in exon 43-edited (E43) and exon 45-edited (E45) AEx44 patient iPSCCMs with sgRNAs (G) 3, 4 and 6, as indicated. Vinculin is loading control. HC, iPSC-CMs from a healthy control. The second lane is unedited AEx44 patient iPSC-CMs.
  • FIG. 62A-B Analysis of mice with a DMD exon 44 deletion.
  • FIG. 62A RT-PCR analysis of TA muscles to validate deletion of exon 44.
  • RT-PCR primers were in exons 43 and 46, and the amplicon size is 503 base pairs (bp) for WT mice and 355 bp for AEx44 DMD mice.
  • FIG. 62B Sequencing of RT-PCR products from AEx44 DMD mouse muscle confirmed deletion of exon 44 and generation of a premature stop codon in exon 45, indicated by red asterisk.
  • Figure discloses SEQ ID NOS 2492-2943, respectively, in order of appearance.
  • FIG. 63A-B Rescue of dystrophin expression after systemic AAV9 delivery of gene editing components to AEx44 mice.
  • FIG. 64A-E Analysis of sgRNAs that target the splice acceptor or donor sites for exon 43 and 45.
  • FIG. 64A Alignment of human and mouse DNA sequence at the intron-exon junction of exon 45. The conserved region is shaded in light blue. Exon sequence is in bold upper case and intron sequence is in lower case.
  • FIG. 64B T7E1 assay using human 293 cells transfected with plasmids that express SpCas9 and exon 43 sgRNAl (Gl), sgRNA2 (G2), sgRNA3 (G3) or sgRNA4 (G4) shows cleavage of the DMD locus at the intron-exon junctions of exon 43.
  • FIG. 64C T7E1 assay using mouse 10T1/2 and human 293 cells transfected with plasmids that express 5/;Cas9 and exon 45 sgRNA5 (G5), sgRNA6 (G6), sgRNA7 (G7) or sgRNA8 (G8) shows cleavage of the Dmd locus at the intron-exon junction of exon 45. Red arrowheads denote cleavage products. PCR indicates the undigested PCR product.
  • FIG. 64D Sequences of the G6 edited 34 single clones. HC is sequence of the healthy human control.
  • FIG. 64E Western blot analysis showing restoration of dystrophin expression in three exon 45-skipped single iPSC clones (clones #3, #11 and #13). Clone #3 and #11 were corrected through exon 45 skipping, and clone #9 was corrected through exon 45 reframing.
  • HC iPSC-derived CM from a healthy human control.
  • NE non-edited. Vinculin is loading control.
  • FIG. 65A-B Characterization of AEx44 mouse line.
  • sgRNA sequences are indicated in blue, protospacer adjacent motifs (PAMs) are indicated in red, and genotyping primers are highlighted in yellow. Exon 44 sequence is in bold upper case and intron sequence is in lower case.
  • Figure discloses SEQ ID NO: 2529.
  • FIG. 65B Picrosirius red staining of TA, diaphragm, and heart of WT and AEx44 mice. Scale bar is 50 mm.
  • FIG. 66A-D Intramuscular AAV9 delivery of gene editing components rescues dystrophin expression.
  • T7E1 assay shows cleavage of the Dmd locus at the intron-exon junction of exon 45 in mouse C2C12 cells with electroporation of G5 or G6 in PX458 or Trispr backbone. Red arrowheads show cleavage products of genome editing. PCR indicates the undigested PCR product.
  • FIG. 66B T7E1 assay shows cleavage of the Dmd locus at the intron-exon junction of exon 45 in TA muscle of corrected AEx44 mice. Red arrowheads show cleavage products of genome editing.
  • PCR indicates the undigested PCR product.
  • Bold represents substitutions, red square is insertions, is deletion.
  • Vertical pink line indicates intron-exon junction in (FIG. 66C) and exon-exon junction in (FIG. 66D).
  • Black arrowhead points to dotted vertical line representing the predicted cleavage site.
  • Figures 66C-D disclose SEQ ID NOS 2530-2566, respectively, in order of appearance.
  • FIG. 67A-C Analysis of top ten potential off-target sites.
  • FIG. 67A T7E1 analysis of the top 10 predicted off-target (OT) sites of sgRNA-G6 assayed in TA muscle 3 weeks following intramuscular injection of 2.5 c 10 10 vg AAV9-Cas9 and 2.5 c 10 10 vg AAV-G6. Red arrowheads denote on-target cleavage products. No off-target cleavage products were detected. PCR indicates the undigested PCR product.
  • FIG. 67B Amplicon genomic deep sequencing analysis on the top 10 predicted off-target sites of G6.
  • FIG. 67C Percentage of NHEJ in amplicon genomic deep sequencing analysis on the top 10 predicted off-target sites of G6. Blue indicates AAV-Cas9 only control, and red indicates AAV-Cas9/AAV-G6 injected TA muscle.
  • FIG. 68 Correction of AEx44 mice by systemic delivery of AAV9 expressing gene editing components.
  • AAV-Cas9 was administered at 5 c 10 13 vg/kg.
  • Dystrophin is shown in red. Nuclei are marked by DAPI stain in blue. 10X tile scan of the entire TA muscle. Scale bar is 500 um.
  • FIG. 69A-G Western blot analysis of corrected AEx44 mice by systemic delivery of AAV9 expressing gene editing components.
  • FIG. 69A-F Western blot analysis of dystrophin, Cas9, and GFP protein expression in TA, triceps, diaphragm, and heart of AEx44 mice 4 weeks after systemic delivery of AAVCas9 and AAV-G6 at the indicated ratios.
  • FIG. 69G Quantification of the Western blot analysis in TA, triceps, diaphragm, and heart.
  • FIG. 70 Histology of AEx44 mice after systemic delivery of AAV9 expressing gene editing components. H&E staining of TA, triceps, diaphragm and heart of AEx44 mice 4 weeks after systemic delivery of AAV-Cas9 and AAV-G6 at the indicated ratios. AAV- Cas9 was administered at 5 c 10 13 vg/kg.
  • FIG. 71 Whole muscle scanning of TA, triceps, diaphragm and heart of corrected AEx44 DMD mice. H&E staining of WT, AEx44 DMD and corrected AEx44 DMD 4 weeks after systemic injection of a 1 :5 ratio and 1: 10 ratio of AAV-Cas9 to AAV-G6. AAV-Cas9 was administered at 5 c 10 13 vg/kg. 4X tile scan of the entire muscle. Scale bar in TA, triceps, diaphragm is 500um, in heart is l.5mm.
  • FIG. 72A-B qPCR analysis of the skeletal and cardiac muscle groups comparing low and high doses of AAV-G6.
  • FIG. 72A qPCR analysis of Cas9 mRNA expression in TA, triceps, diaphragm, and heart of AEx44 mice 4 weeks after systemic delivery of AAV- Cas9 and AAVG6 at the indicated ratios.
  • FIG. 73A-B Histological analysis showing dystrophin restoration in EDL muscle of corrected AEx44 DMD mice.
  • FIG. 73A Dystrophin immunostaining of EDL muscle in AEx44 DMD and corrected AEx44 DMD 4 weeks after systemic injection of a 1:5 ratio and 1: 10 ratio of AAV-Cas9 to AAV-G6. AAV-Cas9 was administered at 5 c 10 13 vg/kg.
  • Dystrophin is shown in red. Nuclei are marked by DAPI stain in blue. (FIG. 73B) H&E staining of EDL muscle in AEx44 DMD and corrected AEx44 DMD 4 weeks after systemic injection of a 1:5 ratio and 1: 10 ratio of AAV-Cas9 to AAV-G6. AAV-Cas9 was
  • FIG. 74 Picrosirius red staining of TA, triceps, diaphragm and heart of corrected AEx44 DMD mice. Picrosirius red staining of TA, triceps, diaphragm and heart of AEx44 mice 4 weeks after systemic delivery of AAV-Cas9 and AAV-G6 at the indicated ratios. AAV-Cas9 was administered at 5 c 10 13 vg/kg. Scale bar is 50 mm.
  • FIG. 75 Correction of dystrophin expression 4 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in AEx50 mice using different AAV9-Cas9 and AAV9-sgRNA-51-SA2 ratios.
  • DMD is a new mutation syndrome with more than 4,000 independent mutations that have been identified in humans.
  • 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.
  • BMD Becker muscular dystrophy
  • the disclosure provides Clustered Regularly Interspaced Short Palindromic
  • CRISPR/Cas9 Repeat/Cas9-mediated genome editing compositions for correcting a dystrophin gene (DMD) mutation or for use in a method of correcting a dystrophin gene (DMD) mutation, a mutation which left untreated, results in the onset of DMD.
  • the data presented herein show that in vivo AAV-mediated delivery of gene-editing components successfully remove the mutant genomic sequence by reframing and/or exon skipping in muscle cells of mice, dogs and humans. Using different modes of AAV9 delivery, dystrophin protein expression was restored in muscle cells of DMD mouse and dog models,
  • compositions and methods for treating DMD are provided herein.
  • an AAV construct is provided, wherein the AAV construct comprises a nucleic acid encoding three promoters that each drive expression of a DMD guide RNA.
  • a more robust and safe form of genome editing was achieved in a humanized mouse model for DMD with a deletion in exon 50, in a AEx50- MD Dog and in human IPS cells.
  • “refraining” is used to refer to a genome editing strategy in which small INDELs restore the protein reading frame.
  • the term“skipping”or“exon skipping” is used to refer to a genom editing strategy wherein a splice donor or splice acceptor site is disrupted, which results in exon skipping and restoration of the protein reading frame.
  • 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.
  • gRNA Guide RN A
  • 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 here without a protospacer target. However, the Cas9-gRNA complex 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 10Obp 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.
  • the gRNA 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 gRNA targets a site within a mutant dystrophin gene. In some embodiments, the gRNA targets a dystrophin intron. In some embodiments, the gRNA targets a dystrophin exon. In some embodiments, the gRNA targets a site in a dystrophin exon that is expressed and is present in one or more of the dystrophin isoforms shown in
  • the gRNA targets a dystrophin splice site. In some embodiments, the gRNA targets a splice donor site on the dystrophin gene. In embodiments, the gRNA targets a splice acceptor site on the dystrophin gene.
  • the guide RNA targets a mutant DMD exon.
  • the mutant exon is exon 23 or 51.
  • the guide RNA targets at least one of exons 1, 23, 41, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 of the dystrophin gene.
  • the guide RNA targets at least one of introns 44, 45, 50, 51, 52, 53, 54, or 55 of the dystrophin gene.
  • the guide RNAs are designed to induce skipping and/or reframing of exon 51 or exon 23.
  • the gRNA is targeted to a splice acceptor site of exon 51 or exon 23.
  • Suitable gRNAs for use in various compositions and methods disclosed herein are provided as SEQ ID NOs: 383-705, 709-711, 715-717, 790-862, 864 (Tables 7, 9, 11, 13, and 15).
  • the gRNA is selected from any one of SEQ ID No: 790 to SEQ ID No: 862.
  • gRNAs 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.
  • gRNAs for Cpfl comprise a single crRNA containing a direct repeat scaffold sequence followed by 24 nucleotides of guide sequence.
  • a“guide” sequence of the crRNA comprises a sequence of the gRNA that is complementary to a target sequence.
  • crRNA of the disclosure comprises a sequence of the gRNA that is not complementary to a target sequence.“Scaffold” sequences of the disclosure link the gRNA to the Cpfl polypeptide.“Scaffold” sequences of the disclosure are not equivalent to a tracrRNA sequence of a gRNA-Cas9 construct.
  • 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.
  • all of the sequences encode different gRNAs.
  • 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.
  • 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, Apem, 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.
  • RAMPs repeat-associated mysterious proteins
  • 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. The team demonstrated that they could disable one or both sites while preserving Cas9’s ability to locate its target DNA. Jinek et al. (2012) combined tracrRNA and spacer RNA 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.
  • scaRNA CRISPR/Cas-associated RNA
  • CRISPR/Cas are separated into three classes.
  • Class 1 uses several Cas proteins together with the CRISPR RNAs (crRNA) to build a functional endonuclease.
  • Class 2 CRISPR systems use a single Cas protein with a crRNA.
  • Cpfl has been recently identified as a Class II, Type V CRISPR/Cas systems containing a 1,300 amino acid protein. See also U.S. Patent Publication 2014/0068797, which is incorporated by reference in its entirety.
  • 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 (AddGene).
  • CRISPR/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
  • 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,
  • 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 fmger-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 (proximately 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 consist 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.
  • Cas9 requires two RNA molecules to cut DNA while Cpfl needs one.
  • the proteins also cut DNA at different places, offering researchers more options when selecting an editing site.
  • Cas9 cuts both strands in a DNA molecule at the same position, leaving behind‘blunt’ ends.
  • Cpfl leaves one strand longer than the other, creating 'sticky' ends that are easier to work with.
  • Cpfl appears to be more able to insert new sequences at the cut site, compared to Cas9.
  • the CRISPR/Cas9 system can efficiently disable genes, it is challenging to insert genes or generate a knock-in.
  • Cpfl lacks tracrRNA, utilizes a T-rich PAM and cleaves DNA via a staggered DNA DSB.
  • Cpfl recognizes different PAMs, enabling new targeting possibilities, creates 4-5 nt long sticky ends, instead of blunt ends produced by Cas9, enhancing the efficiency of genetic insertions and specificity during NHEJ or HDR, and cuts target DNA further away from PAM, further away from the Cas9 cutting site, enabling new possibilities for cleaving the DNA.
  • 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. Exemplary genomic target sequences can be found in Tables 6,
  • 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.
  • 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.
  • 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. U2UMQ6; SEQ ID NO: 870.
  • 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 Cas 9 or Cpfl -expressing cells to be sorted using fluorescence activated cell sorting (FACS).
  • a fluorescent protein such as GFP
  • 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 (“refraining” 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.
  • 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 is 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 Cpfl 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.
  • 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.
  • 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 Mafl 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 muscle-cell specific promoter.
  • This muscle-cell specific promoter may be constitutively active or may be an inducible 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 virus 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 virus 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 purpose.
  • a promoter with well
  • 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 5, 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.v, 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),
  • inducible elements may be used.
  • the inducible element is, for example, MTII, MMTV (mouse mammary tumor virus), b- interferon, adenovirus 5 E2, collagenase, stromelysin, SV40, murine MX gene, GRP78 gene, a-2-macroglobulin, vimentin, MHC class I gene H-2k ⁇ >, 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 (TP A), interferon, Newcastle Disease Virus, A23187, IL-6, serum, interferon, SV40 large T antigen, PMA, and/or thyroid hormone.
  • TFA phorbol ester
  • TP A phorbol ester
  • 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. 875):
  • 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 EGRGSLLTCGDVEENPGP
  • 2A-like peptides include, but are not limited to, equine rhinitis A virus (ERAV) 2A peptide (SEQ ID NO: 877; QCTNYALLKLAGDVESNPGP), porcine teschovirus-l (PTV1) 2A peptide (SEQ ID NO: 878; ATNFSLLKQAGDVEENPGP) and foot and mouth disease virus (FMDV) 2A peptide (SEQ ID NO: 879; PVKQLLNFDLLKLAGDVESNPGP) or modified versions thereof.
  • EAV equine rhinitis A virus
  • PTV1 porcine teschovirus-l
  • FMDV foot and mouth disease virus
  • the 2A peptide is used to express a reporter and a Cas9 or a Cpfl simultaneously.
  • the reporter may be, for example, GFP.
  • peptides that may be used include, but are not limited to nuclear inclusion protein a (Nia) protease, a Pl protease, a 3C protease, an L protease, a 3C-like protease, or modified versions thereof.
  • Nia nuclear inclusion protein a
  • Pl protease a protease
  • 3C protease an L protease
  • L protease a 3C-like protease
  • modified versions thereof include, but are not limited to nuclear inclusion protein a (Nia) protease, a Pl protease, a 3C protease, an L protease, a 3C-like protease, or modified versions thereof.
  • 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, AAVrh74, AAVrh10 or any combination thereof.
  • Exemplary 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, AAV 8, AAV9, AAV 10, AAV11 AAVrh74, AAVrh10 or any combination thereof.
  • the ITRs comprise or consist of full-length and/or wildtype sequences for an AAV serotype.
  • the ITRs comprise or consist of truncated sequences for an AAV serotype.
  • 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,
  • 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,
  • 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:
  • the AAV-Cas9 vector may contain one or more nuclear localization signals (NLS).
  • NLS nuclear localization signals
  • the AAV-Cas9 vector contains 1, 2, 3, 4, or 5 nuclear localization signals.
  • Exemplary NLS include the c-myc NLS (SEQ ID NO:
  • 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
  • KRKGDEVDGVDEVAKKKSKK (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 poly A sequence.
  • the polyA sequence may be a mini-poly A 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 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.
  • 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 selected from
  • SEQ ID NO: 899 SEQ ID NO: 900, SEQ ID NO: 901, or SEQ ID NO: 902, as shown in Table 4.
  • Table 4 Exemplary gene editing constructs (from ITR to ITR for delivery via AAV vector)
  • the construct comprises or consists of a promoter and a nuclease.
  • the construct comprises or consists of a CK8e promoter and a Cas9 nuclease.
  • the construct comprises or consists of a CK8e promoter and a Cas9 nuclease isolated or derived from Staphylococcus pyogenes (“SpCas9”).
  • the CK8e promoter comprises or consists of a nucleotide sequence of
  • the construct comprising a promoter and a nuclease further comprises at least two inverted terminal repeat (ITR) sequences.
  • the construct comprising a promoter and a nuclease further comprises at least two ITR sequences from isolated or derived from an AAV of serotype 2 (AAV2).
  • the construct comprising a promoter and a nuclease further comprises at least two ITR sequences each comprising or consisting of a nucleotide sequence of
  • the construct comprising a promoter and a nuclease further comprises at least two ITR sequences, wherein the first ITR sequence comprises or consists of a nucleotide sequence of
  • 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 sequence encoding 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 sequence encoding a CK8e promoter, a sequence encoding a SpCas9 nuclease and a second AAV2 ITR.
  • the construct comprising or consisting of, from 5’ to 3’ a first ITR, a sequence encoding 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
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding 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 sequence encoding a promoter, a sequence encoding a nuclease, a minipoly A sequence and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a sequence encoding a SpCas9 nuclease, a minipoly A sequence and a second AAV2 ITR.
  • the construct comprising, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR, further comprises at least one nuclear localization signal.
  • the construct comprising, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR, further comprises at least two nuclear localization signals.
  • Exemplary nuclear localization signals of the disclosure comprise or consist of a nucleotide
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a poly A sequence and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding 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 sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 885, 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 sequence encoding a CK8e promoter, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a minipoly A sequence and a second AAV2 ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 885, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a minipoly A sequence and a second AAV2 ITR.
  • the construct comprising, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a poly A sequence and a second ITR, 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, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding 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 sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 885, 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 sequence encoding a CK8e promoter, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, 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 sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 885, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a stop codon a minipoly A sequence and a second AAV2 ITR.
  • the construct comprising or consisting of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence and a second ITR, further comprises transposable element inverted repeats.
  • exemplary transposable element inverted repeats of the disclosure comprise or consist of a nucleotide sequence of
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat having a sequence of SEQ ID NO: 907, a first AAV2 ITR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 885, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a stop codon a minipoly A sequence, a second AAV2 ITR, and a second transposable element inverted repeat having a sequence of SEQ ID NO: 908.
  • the construct comprising or consisting of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, and a second transposable element inverted repeat, further comprises a regulatory sequence.
  • Exemplary regulatory sequences of the disclosure comprise or consist of a nucleotide sequence of
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding 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 comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat having a sequence of SEQ ID NO: 907, a first AAV2 ITR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 885, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a stop codon, a minipoly A sequence, a second AAV2 ITR, a regulatory sequence having a sequence of SEQ ID NO: 909, and a second transposable element inverted repeat having a sequence of SEQ ID NO: 908.
  • 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, or SEQ ID NO: 902.
  • At least a first sequence encoding a gRNA and a second sequence encoding a gRNA may be packaged into an AAV vector.
  • at least a first sequence encoding a gRNA, a second sequence encoding a gRNA, and a third sequence encoding a gRNA may be packaged into an AAV vector.
  • at least 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 may be packaged into an AAV vector.
  • At least 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 may be packaged into an AAV vector.
  • a plurality of sequences encoding a gRNA are packaged into an AAV vector. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 sequences encoding a gRNA may be packaged into an AAV vector.
  • each sequence encoding a gRNA is different. In some embodiments, at least 1, 2, 3, 4, 5, 6,
  • 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the sequences encoding a gRNA are the same. In some embodiments, all of the sequence encoding a gRNA are the same.
  • 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, AAV 10, AAV11, AAVrh74, AAVrh10 or any combination thereof.
  • Exemplary 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, AAV 3, AAV4, AAV 5, AAV6, AAV7, AAV 8, AAV9, AAV 10, AAV11, AAVrh74, AAVrh10 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.
  • 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, AAVrh74, or AAVrh10. In some embodiments, the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAVrh74, or AAVrh10.
  • the first serotype is AAV2 and the second serotype is AAV1, AAV2, AAV 3, AAV4, AAV5, AAV6, AAV7, AAV 8, AAV9, AAV10, AAV11, AAVrh74, or AAVrh10.
  • the first serotype is AAV2 and the second serotype is AAV9.
  • Exemplary AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the gRNA sequences.
  • the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV 3, AAV4, AAV 5, AAV6, AAV7, AAV 8, AAV9, AAV 10, AAV11, AAVrh74, AAVrh10 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, AAVrh74, or AAVrh10.
  • the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAVrh74, or AAVrh10.
  • the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV 7, AAV8, AAV9, AAV 10, AAV11, AAVrh74, or AAVrh10.
  • the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV1, AAV2, AAV 3, AAV4, AAV5, AAV6, AAV7, AAV 8, AAV9, AAV10, AAV11, AAVrh74, or AAVrh10.
  • the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV9.
  • Exemplary 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, AAV 10, AAV11, AAVrh74, or AAVrh10 or any combination thereof.
  • the ITRs comprise or consist of full-length and/or wildtype sequences for an AAV serotype.
  • the ITRs comprise or consist of truncated sequences for an AAV serotype.
  • the ITRs comprise or consist of elongated sequences for an AAV serotype.
  • the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype.
  • 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,
  • 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,
  • 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: 880, SEQ ID NO: 881, SEQ ID NO: 882, or SEQ ID NO: 883.
  • 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.
  • Exemplary 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 5, 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.v 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
  • CMV cytomegalovirus
  • gibbon ape leukemia virus cytomegalovirus
  • Further exemplary promoters include the U6 promoter, the Hl promoter, and the 7SK promoter.
  • the sequence encoding the gRNA comprises a sequence selected from SEQ ID Nos: 383-705, 709-711, 715-717, 790-862, and 864.
  • the AAV 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 Hl 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 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
  • 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 Hl promoter, the U6 promoter, and the 7SK promoter.
  • the first promoter is the U6 promoter.
  • the second promoter is the Hl promoter.
  • the third promoter is the 7SK promoter.
  • the first promoter is the U6 promoter, the second promoter is the Hl 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 Hl 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 Hl 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 selected from SEQ ID NO: 910, SEQ ID NO: 911, SEQ ID NO: 912, or SEQ ID NO: 913. In some embodiments, the AAV-sgRNA vector comprises a sequence selected from SEQ ID NO: 914, SEQ ID NO: 915, SEQ ID NO: 916, or SEQ ID NO: 917. In some embodiments, the AAV- sgRNA vector comprises a sequence selected from SEQ ID NO: 918, SEQ ID NO: 919, SEQ ID NO: 920, or SEQ ID NO: 921. Exemplary AAV-sgRNA vectors are provided in Table 5. Table 5. Exemplary AAV-sgRNA vectors.
  • the construct comprises or consists of 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.
  • Exemplary sequences encoding gRNAs of the disclosure are SEQ ID NO: 383-705, 709-711, 715-717, 790-862, 864.
  • the sequence encoding the gRNA is
  • the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA of SEQ ID NO.: 708, a second promoter, a second sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, and a third sequence encoding a gRNA of SEQ ID NO: 708.
  • the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, a second sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, and a third sequence encoding a gRNA of SEQ ID NO: 714.
  • the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, a second sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, and a third sequence encoding a gRNA of SEQ ID NO: 863.
  • 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: 922), the Hl promoter (SEQ ID NO: 923), and the 7SK promoter (SEQ ID NO: 924). In some embodiments, the first, second, and third promoter are each individually selected from the U6 promoter (SEQ ID NO: 922), and the Hl promoter (SEQ ID NO: 923). In some embodiments,
  • the construct comprises, from 5’ to 3’, a U6 promoter, a first sequence encoding a gRNA, a Hl 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 of SEQ ID NO: 708, the Hl promoter, a second sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, and a third sequence encoding a gRNA of SEQ ID NO: 708.
  • the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the Hl promoter, a second sequence encoding a gRNA of 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 of SEQ ID NO: 863, the Hl promoter, a second sequence encoding a gRNA of SEQ ID NO: 863, the 7SK promoter, and a third sequence encoding a gRNA 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.
  • ITR inverted terminal repeat
  • 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).
  • AAV2 serotype 2
  • 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, a second promoter, and a second sequence encoding a gRNA, a third 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 U6 promoter, a first sequence encoding a gRNA, a Hl 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 of SEQ ID NO: 708, a second promoter, and the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA 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 of SEQ ID NO: 714, a second promoter, and the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA 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 of SEQ ID NO: 863, a second promoter, and the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6, the sequence encoding a gRNA of SEQ ID NO: 708, a Hl promoter, and the sequence encoding a gRNA of SEQ ID NO: 708, a 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6, the sequence encoding a gRNA of SEQ ID NO: 714, a Hl promoter, and the sequence encoding a gRNA of SEQ ID NO: 714, a 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 714, 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 polyA sequence comprises or consists of a minipolyA sequence.
  • Exemplary minipolyA sequences of the disclosure comprise or consist of a nucleotide sequence of
  • the construct 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 s gRNA, 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 Hl 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 of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA 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 of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO.: 714, a third promoter, the sequence encoding a gRNA 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 of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA 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 of SEQ ID NO: 708, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA 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 of SEQ ID NO: 714, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, 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, a minipolyA sequence, and a second ITR further comprises transposable element inverted repeats.
  • exemplary transposable element inverted repeats of the disclosure comprise or consist of a nucleotide sequence of
  • the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, the U6 promoter, a first sequence encoding a gRNA, the Hl promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat.
  • the construct comprising a first transposable element inverted repeat, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, a second transposable element inverted repeat, further comprises a regulatory sequence.
  • Exemplary regulatory sequences of the disclosure comprise or consist of a nucleotide sequence of
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, a first sequence encoding a gRNA, the Hl promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, a sequence encoding a gRNA of SEQ ID NO: 714, the Hl promoter, a sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, a sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • the construct comprising 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, a minipolyA sequence, and a second ITR, further comprises a stuffer sequence.
  • the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, a first sequence encoding a gRNA, the Hl promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a stuffer sequence, 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 of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a stuffer sequence 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 of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO. 714, a stuffer sequence, 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 of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a stuffer sequence, 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 of SEQ ID NO: 708, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a stuffer sequence, 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 of SEQ ID NO: 714, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a stuffer sequence, a minipolyA sequence, and a second ITR.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • a first transposable element inverted repeat a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, a first sequence encoding a gRNA, the Hl promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat.
  • a first transposable element inverted repeat a first ITR
  • the U6 promoter the sequence encoding a gRNA of SEQ ID NO: 708, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence
  • the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, a sequence encoding a gRNA of SEQ ID NO: 714, the Hl promoter, a sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, a sequence encoding a gRNA of SEQ ID NO: 714, a stuffer sequence, a minipolyA 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 at a position that is 5’ to or 3’ to an ITR, a promoter, a sequence encoding a gRNA, a polyA sequence, a transposable element inverted repeat, a stuffer sequence, and/or a regulator element.
  • 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 sequence encoding a first promoter wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding 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 sequence encoding the first promoter and the sequence encoding the second promoter are identical. In some embodiments, the sequence encoding the first promoter and the sequence encoding the second promoter not identical. In some embodiments, the sequence encoding the first promoter and the sequence encoding 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 sequence encoding 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 sequences encoding the first promoter, the sequence encoding the second promoter, and the sequence encoding the third promoter are identical.
  • at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, and the sequence encoding the third promoter are not identical.
  • At least two of the sequences encoding the first promoter, the sequence encoding the second promoter, and the sequence encoding the third promoter 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 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 sequence encoding 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 fourth genomic target sequence comprises a dystrophin splice acceptor site.
  • at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, and the sequence encoding the fourth promoter are identical.
  • At least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, and the sequence encoding the fourth promoter are not identical. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, and the sequence encoding the fourth promoter 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, the third genomic target sequence, and the fourth genomic target sequence are 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 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%, 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, 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 sequence encoding 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 sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter are identical.
  • At least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter are not identical. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter 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, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence are 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 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%, 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, 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 sequence encoding 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 sequence encoding the first promoter or the sequence encoding the second promoter comprises a sequence encoding a constitutive promoter.
  • the first promoter or the second promoter comprises a constitutive promoter.
  • At least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding 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 sequence encoding the first promoter or the sequence encoding the second promoter comprises a sequence encoding an inducible promoter.
  • At least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises an inducible promoter.
  • the sequence encoding the first promoter or the sequence encoding the second promoter comprises a sequence encoding a cell-type specific promoter.
  • at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises a cell-type specific promoter.
  • the cell type specific promoter comprises a muscle-specific promoter.
  • the sequence encoding the first promoter or the sequence encoding the second promoter comprises a sequence encoding a U6 promoter, an Hl promoter, or a 7SK promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises a U6 promoter, an Hl promoter, or a 7SK promoter.
  • At least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises a U6 promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises an Hl promoter.
  • At least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding 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 sequence encoding the first promoter comprises a sequence encoding a U6 promoter
  • the sequence encoding the second promoter comprises a sequence encoding an Hl promoter
  • the sequence encoding 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 virus (AAV).
  • AAV adeno- associated virus
  • 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).
  • AAV adeno-associated virus
  • 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.
  • 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. In some embodiments, 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 and 712-789. 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.
  • 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 sequence encoding a first promoter wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding 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 vector further comprises a sequence encoding an inverted terminal repeat of a transposable element.
  • 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),
  • the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 9 (AAV 9). 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.
  • the vector is optimized for expression in mammalian cells. In some embodiments, the vector is optimized for expression in human cells. In some embodiments, the vector comprises the sequence of SEQ ID NO: 914, SEQ ID NO: 915, SEQ ID NO: 916, or SEQ ID NO: 917.
  • nucleic acid comprising a sequence encoding a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the sequence encoding the promoter comprises a sequence encoding a muscle-specific promoter.
  • the sequence encoding the muscle-specific promoter comprises a sequence encoding a CK8 promoter.
  • sequence encoding the muscle-specific promoter comprises a sequence encoding a CK8e promoter.
  • the sequence encoding the Cas9 or the nuclease domain thereof is isolated or derived from a sequence encoding an S.
  • 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.
  • the nucleic acid comprising a sequence encoding 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 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.
  • 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.
  • the nucleic acid is optimized for expression in mammalian cells. In some embodiments, the nucleic acid is optimized for expression in human cells.
  • a vector comprising a nucleic acid comprising a sequence encoding a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the sequence encoding the promoter comprises a sequence encoding a muscle-specific promoter such as the CK8 or CK8e promoter.
  • the vector further comprises a sequence encoding an inverted terminal repeat (ITR) of a transposable element.
  • 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 (AAV 6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11), AAVrh74, AAVrh10 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. In some embodiments, the vector comprises the nucleic acid sequence of SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, or SEQ ID NO: 902.
  • compositions comprising one or more vectors 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.
  • Aqueous compositions of the present disclosure comprise an effective amount of the drug, vector or proteins, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • pharmaceutically acceptable carrier or aqueous medium refers 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, drug 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
  • 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 sequence encoding a first promoter, wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA; and a sequence encoding a second promoter, wherein the second promoter drives expression of the sequence encoding the second DMD guide RNA.
  • the second vector comprise a nucleic acid comprising a sequence encoding a Cas9 or a nuclease domain thereof; a sequence encoding a muscle- specific promoter, wherein the muscle-specific promoter drives expression of the sequence encoding a Cas9 or a nuclease domain thereof.
  • a first vector and a second vector are administered to a patient in a therapeutically effective ratio.
  • the term“ratio” may refer to a ratio of the amount of vector in a composition (concentration), amount delivered to a patient (dosage), amount available to a therapeutic site (bioavailability), amount expressed by a target cell (copy number), amount of modifications made (efficacy), amount of DNA, or 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
  • 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 the“Ratio Table” below: [0216] 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 the“Ratio Table.”
  • 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 the “Ratio Table.”
  • the ratio of the first vector to the second vector is greater than 10: 1.
  • 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. In some embodiments, 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 sequence encoding a first promoter wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding 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 and (ii) a second nucleic acid sequence comprising a sequence encoding a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the sequence encoding the promoter comprises a sequence encoding a muscle-specific promoter such as the CK8 or CK8e promoter.
  • the sequence encoding the promoter comprises a sequence
  • 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 sequence encoding a first promoter wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding 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 and (ii) a second vector comprising a nucleic acid sequence comprising a sequence encoding a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the sequence encoding the promoter comprises a sequence encoding a muscle-specific promoter such as the CK8 or CK8
  • 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.
  • 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 reframing.
  • the at least one DMD guide RNA- Cas9 complex disrupts a dystrophin splice site and induces a refraining of a dystrophin reading frame.
  • 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.
  • the insertion comprises an insertion of a single adenosine.
  • Also provided is a method for inducing selective skipping and/or refraining 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 refraining 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. 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.
  • 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 serum 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 develonce, curvature of the spine, loss of movement, and paralysis.
  • the subject presents a neurological 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 develonce, curvature of the spine, loss of movement,
  • 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 6-25 provide exemplary primer and genomic targeting 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.
  • adenovirus a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB.
  • 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 mid sized 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 (El A 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 5‘- tripartite leader
  • recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus 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. Combined with the approximately 5.5 kb of DNA that is replaceable in the El 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.
  • 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 adenovirus 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 develonce. 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.
  • 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.
  • 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 sialogly coprotein 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 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 (Roux el al, 1989).
  • 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.
  • retrovirus vectors 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.
  • 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, AAV 5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV 11 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.
  • Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. 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, /. 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 virus (HVJ) to facilitate fusion with the cell membrane and promote cell entry of liposome- encapsulated DNA.
  • HVJ hemagglutinating virus
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-l).
  • HMG-l nuclear non-histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-L
  • 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.
  • a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
  • 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.
  • a cell receptor-specific ligand Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are
  • ASOR asialoorosomucoid
  • transferrin A synthetic neoglycoprotein, which recognizes the same receptor as ASOR, 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.l 1), 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
  • 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
  • Duchenne muscular dystrophy a progressive neuromuscular disorder
  • Muscle weakness also occurs later, in the arms, neck, and other areas. Calves are often enlarged. Symptoms usually appear before age 6 and may appear in early infancy. Other physical symptoms are:
  • Lumbar hyperlordosis possibly leading to shortening of the hip-flexor muscles. This has an effect on overall posture and a manner of walking, stepping, or running.
  • Muscle contractures of Achilles tendon and hamstrings impair functionality because the muscle fibers shorten and fibrose in connective tissue.
  • a positive Gowers' sign reflects the more severe impairment of the lower extremities muscles. The child helps himself to get up with upper extremities: first by rising to stand on his arms and knees, and then "walking" his hands up his legs to stand upright. Affected children usually tire more easily and have less overall strength than their peers. Creatine kinase (CPK-MM) levels in the bloodstream are extremely high.
  • An electromyography CPK-MM
  • EMG shows that weakness is caused by destruction of muscle tissue rather than by damage to nerves. Genetic testing can reveal genetic errors in the Xp2l gene. A muscle biopsy (immunohistochemistry or immunoblotting) or genetic test (blood test) confirms the absence of dystrophin, although improvements in genetic testing often make this unnecessary.
  • DMD patients may suffer from:
  • Respiratory disorders including pneumonia and swallowing with food or fluid passing into the lungs (in late stages of the disease).
  • DMD Duchenne muscular dystrophy
  • Xp2l located on the short arm of the X chromosome.
  • Dystrophin is responsible for connecting the cytoskeleton of each muscle fiber to the underlying basal lamina (extracellular matrix), through a protein complex containing many subunits. The absence of dystrophin permits excess calcium to penetrate the sarcolemma (the cell membrane). Alterations in calcium and signaling pathways cause water to enter into the mitochondria, which then burst.
  • mitochondrial dysfunction gives rise to an amplification of stress-induced cytosolic calcium signals and an amplification of stress-induced reactive- oxygen species (ROS) production.
  • ROS reactive- oxygen species
  • DMD is inherited in an X-linked recessive pattern.
  • Females will typically be carriers for the disease while males will be affected.
  • a female carrier will be unaware they carry a mutation until they have an affected son.
  • the son of a carrier mother has a 50% chance of inheriting the defective gene from his mother.
  • the daughter of a carrier mother has a 50% chance of being a carrier and a 50% chance of having two normal copies of the gene.
  • an unaffected father will either pass a normal Y to his son or a normal X to his daughter.
  • Female carriers of an X-linked recessive condition such as DMD, can show symptoms depending on their pattern of X-inactivation.
  • Duchenne muscular dystrophy has an incidence of 1 in 5000 male infants. Mutations within the dystrophin gene can either be inherited or occur spontaneously during germline transmission. A table of exemplary but non-limiting mutations and corresponding models are set forth below:
  • DMD Diagnosis Genetic counseling is advised for people with a family history of the disorder. Duchenne muscular dystrophy can be detected with about 95% accuracy by genetic studies performed during pregnancy.
  • DNA test The muscle-specific isoform of the dystrophin gene is composed of 79 exons, and DNA testing and analysis can usually identify the specific type of mutation of the exon or exons that are affected. DNA testing confirms the diagnosis in most cases.
  • Muscle biopsy If DNA testing fails to find the mutation, a muscle biopsy test may be performed. A small sample of muscle tissue is extracted (usually with a scalpel instead of a needle) and a dye is applied that reveals the presence of dystrophin. Complete absence of the protein indicates the condition. [0279] Over the past several years DNA tests have been developed that detect more of the many mutations that cause the condition, and muscle biopsy is not required as often to confirm the presence of Duchenne's.
  • DMD is carried by an X-linked recessive gene. Males have only one X chromosome, so one copy of the mutated gene will cause DMD. Fathers cannot pass X- linked traits on to their sons, so the mutation is transmitted by the mother.
  • Prenatal tests can tell whether their unborn child has the most common mutations. There are many mutations responsible for DMD, and some have not been identified, so genetic testing only works when family members with DMD have a mutation that has been identified.
  • Amniocentesis can be done after 15 weeks, and has a 0.5% risk of miscarriage. Fetal blood sampling can be done at about 18 weeks. Another option in the case of unclear genetic test results is fetal muscle biopsy.
  • 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.
  • DMD generally progresses through five stages, as outlined in Bushby et al, Lancet Neurol., 9(1): 77-93 (2010) and Bushby et al, Lancet Neurol., 9(2): 177-198 (2010), incorporated by reference in their entireties.
  • patients typically show develommental delay, but no gait disturbance.
  • patients typically show the Gowers’ sign, waddling gait, and toe walking.
  • patients typically exhibit an increasingly labored gait and begin to lose the ability to climb stairs and rise from the floor.
  • patients are typically able to self-propel for some time, are able to maintain posture, and may develop scoliosis.
  • upper limb function and postural maintenance is increasingly limited.
  • treatment is initiated in the presymptomatic stage of the disease. In some embodiments, treatment is initiated in the early ambulatory stage. In some embodiments, treatment is initiated in the late ambulatory stage. In embodiments, treatment is initiated during the early non-ambulatory stage. In embodiments, treatment is initiated during the late non-ambulatory stage.
  • the ventilator may require an invasive endotracheal or tracheotomy tube through which air is directly delivered, but, for some people non-invasive delivery through a face mask or mouthpiece is sufficient. Positive airway pressure machines, particularly bi-level ones, are sometimes used in this latter way.
  • the respiratory equince may easily fit on a ventilator tray on the bottom or back of a power wheelchair with an external battery for portability.
  • Ventilator treatment may start in the mid to late teens when the respiratory muscles can begin to collapse. If the vital capacity has dropped below 40% of normal, a volume ventilator/respirator may be used during sleeping hours, a time when the person is most likely to be under ventilating (“hypoventilating”). Hypoventilation during sleep is determined by a thorough history of sleep disorder with an oximetry study and a capillary blood gas (See Pulmonary Function Testing). A cough assist device can help with excess mucus in lungs by hyperinflation of the lungs with positive air pressure, then negative pressure to get the mucus up. If the vital capacity continues to decline to less than 30 percent of normal, a volume ventilator/respirator may also be needed during the day for more assistance. The person gradually will increase the amount of time using the ventilator/respirator during the day as needed. Prognosis
  • Duchenne muscular dystrophy is a progressive disease which, left untreated, eventually affects all voluntary muscles and involves the heart and breathing muscles in later stages. The life expectancy is currently estimated to be around 25, but this varies from patient to patient. Recent advancements in medicine are extending the lives of those afflicted.
  • the Muscular Dystrophy Campaign which is a leading UK charity focusing on all muscle disease, states that“with high standards of medical care young men with Duchenne muscular dystrophy are often living well into their 30s.”
  • ILM intrinsic laryngeal muscles
  • the disclosure provides mouse and canine models of DMD to provide not only proof of concept data but also to demonstrate the safety and efficacy of the pharmaceutical compositions of the disclosure when used in vivo.
  • the most common hot spot mutation region in DMD patients is the region between exon 45 to 51. Reframing and/or skipping of exon 51 may be used to treat the largest group (13-14%) of patients.
  • FIG. 1A CRISPR/Cas9 system directed by 2 sgRNAs
  • FIG. 1B Deletion of exon 50 placed the dystrophin gene out of frame leading to the absence of dystrophin protein in skeletal muscle and heart (FIGS. 1C-1E). Mice lacking exon 50 showed pronounced dystrophic muscle changes by 2 months of age (FIG. 1E). Serum analysis of delta-exon 50 mice showed a significant increase in creatine kinase (CK) levels, indicative of muscle damage (FIG. 1F). Taken together, dystrophin protein expression, muscle histology and serum CK levels validated the dystrophic phenotype of the DEc50 mouse model.
  • CK creatine kinase
  • the sgRNA-ex51 -S A2 was delivered to mice in triple copy (AAV-Tri-SA2), along with a Cas9 (AAV-Cas9), by intra-muscular (IM) injection. Following the injection, muscle tissues were analyzed. In vivo targeting efficiency was estimated by RT-PCR with primers for sequences in exons 48 and 53 and the T7E1 assay for the targeted genomic regions. To investigate whether efficient target cleavage was achieved, the inventors amplified a 771 bp region spanning the target site and analyzed it using the T7EI assay (FIG. 10A). The activity of SpCas9 with the corresponding sgRNA was analyzed on the target site.
  • T7EI assays revealed mutagenesis of the Dmd locus after delivery of AAV-Cas9 and AAV9-sgRNA-5l- SA2 (FIG. 10A).
  • genomic PCR amplification products spanning the target site were analyzed by amplicon deep-sequencing analysis. Deep sequencing of the targeted region indicated that 27.9% of total reads contained changes at the targeted genomic site (FIG. 10B).
  • 15% of the identified mutations contained the same A insertion seen in mouse 10T1/2 and human 293 cells in vitro.
  • the deletions identified using this method encompassed a highly -predicted exonic splicing enhancer site for exon 51 (FIG.
  • RT-PCR products of RNA from muscle of AEx50 mice injected intramuscularly with AAV9-Cas9 and AAV9-sgRNA-5l showed that deletion of exon 51 (DEc50-51) allowed splicing from exon 49 to 52 (FIG. 11 A, lower band).
  • DEc50-51 adenosine
  • RT-PCR amplification products from 4 samples were directly subjected to topoisomerase-based thymidine to adenosine (TOPO-TA) cloning without gel purification, then sequenced.
  • TOPO-TA adenosine
  • sequence analysis of 40 clones from each sample showed that in addition to exon 5l-skipped cDNA products (DEc50-51) identified in 15% of sequenced clones, DEc50 mice injected with AAV9-Cas9 and AAV9- sgRNA-5l showed a high frequency of reframing events.
  • DEc50 mice injected with AAV9-Cas9 and AAV9- sgRNA-5l showed a high frequency of reframing events.
  • 63% contained a single nucleotide insertion in the sequence of exon 51 (FIGS. 11 B-C). The most dominant insertion mutation seen was an A insertion.
  • dystrophin immunostaining of muscle cryosections from AEx50 mice injected with AAV-Tri-SA2 revealed significantly higher numbers of dystrophin-positive fibers with an average of 99% restoration of normal fibers (FIG. 12A, FIG. 13).
  • Western blot analysis confirmed the restoration of dystrophin expression in skeletal muscle. (FIG. 12C, FIG. 12D).
  • Hematoxylin and eosin (H&E) staining of muscle showed that histopathologic hallmarks of muscular dystrophy, such as necrotic myofibers, were corrected in TA muscle at 3-weeks post-AAV delivery (FIG. 12B and FIG. 14).
  • This method using a distinct sgRNA design, represents a major advance in efficiency of DMD correction with direct applicability to the patients with the most common dystrophin mutations.
  • RNA polymerase III promoter U6 or Hl or 7SK
  • U6, Hl and 7SK three RNA polymerase III promoters
  • U6 promoter AAV9-U6-sgRNA-5l-SA2
  • Hl promoter AAV9-Hl-sgRNA-5l-SA2
  • the 7SK promoter AAV9-7SK-sgRNA-5l-SA2
  • Triple-sgRNA-5l-SA2 triple copy
  • IM intra-muscular
  • dystrophin immunostaining of muscle cryosections from AEx50 mice injected with AAV9-Triple-sgRNA-5l-SA2 revealed significantly higher numbers of dystrophin-positive fibers with an average of 95% restoration of normal fibers compared to AEx50 mice injected with AAV9-U6-sgRNA-5l- SA2, AAV -H 1 -sgRNA-51 -S A2 and AAV-7SK-sgRNA-5l-SA2 with an average of 70%; 40% and 50% restoration of normal fibers respectively (FIGS. 15B).
  • the triple gRNA provides superior and unexpected restoration of dystrophin-positive fibers.
  • AEx50 mice Grip strength testing also showed a significant increase in muscle strength of AEx50 mice at 4 weeks post-intraperitoneal AAV9 injection compared to AEx50 control mice (wildtype control 92.6 ⁇ l.63; AEx50 control 50.5 ⁇ 1.85; AEx50-AAV9-sgRNA-5l 79.7 ⁇ 2.63) (FIG. 18A). Consistently, AAV9-sgRNA- 51-SA2 gene-edited AEx50 mice also showed significant reductions in serum CK
  • AAV9-Cas9 and AAV9-sgRNA-5l were tested different doses and delivery strategies for AAV9-Cas9 and AAV9-sgRNA-5l.
  • the inventors systemically delivered by intraperitoneal injection the AAV9-Cas9 and AAV9-sgRNA-5l to P4 AEx50 mice using 2 different doses (2.6xl0 13 vg/kg of each AAV9 referred as Dose 1 and 6xl0 12 vg/kg of each AAV9 referred as Dose 2).
  • Dose 1 and 6xl0 12 vg/kg of each AAV9 referred as Dose 2 The systemic delivery yielded widespread dystrophin expression in the heart, triceps, tibialis anterior muscle, and diaphragm in gene- edited AEx50 mice at 4 weeks post-injection (FIG.
  • AAV9-Cas9 and AAV9-sgRNA-5l were tested different ratios of AAV9-Cas9 and AAV9-sgRNA-5l.
  • the inventors delivered the AAV9- Cas9 and AAV9-sgRNA-ex5l in different doses and ratios (1: 1 and 1:2) using IP injection P4 DEc50 mice (FIG. 75) using the following conditions: 5xl0 13 vg/kg AAV9-Cas9 and
  • dystrophin immunostaining of muscle cryosections from AEx50 mice injected mice injected with 5xl0 13 vg/kg AAV9-Cas9 and 1xl0 14 vg/kg (1:2) of AAV9- Cas9 to AAV9-sgRNA-ex5l displayed better dystrophin correction then 5xl0 13 vg/kg AAV9- Cas9 and 5xl0 13 vg/kg AAV9-sgRNA-5l.
  • dystrophin immunostaining of muscle cryosections from AEx50 mice injected mice injected with 5xl0 13 vg/kg AAV9-Cas9 and 1xl0 14 vg/kg showed comparable correction to the mice injected with 1x10 14 vg/kg AAV9- sgRNA-51 and 1x10 14 vg/kg AAV9-Cas9 and 1xl 0 14 vg/kg AAV9-sgRNA-5l (FIG. 75).
  • the inventors treated 1 month old AEx50 mice with AAV9-Cas9 and AAV9-sgRNA-5l.
  • the inventors systemically delivered 2.6xl0 13 vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l via a tail vein injection to 1 month old AEx50 mice.
  • the systemic delivery resulted in widespread dystrophin expression in the heart, triceps, tibialis anterior muscle, and diaphragm in gene-edited AEx50 mice at 4 weeks and 8 weeks post-injection (FIG. 23 A and FIG. 24 A).
  • the therapeutic compositions of the disclosure restore dystrophin expression following administration to these subjects.
  • the administration is systemic as opposed to local. Local administration would be expected to provide even greater efficacy because the AAV constructs would not have to be delivered from the injection point to the treatment site, which is likely a farther distance and which involves traversing the subject’s blood stream, where the AAV constructs are exposed to the immune system.
  • a luciferase reporter was introduced in-frame with the C-terminus of the dystrophin gene in mice. Expression of this reporter mimics endogenous dystrophin expression and DMD mutations that disrupt the dystrophin open reading frame extinguish luciferase expression. The inventors evaluated the correction of the dystrophin reading frame coupled to luciferase in mice lacking exon 50, a common mutational hotspot, after delivery of
  • sgRNA-5l an sgRNA targeting a region adjacent to the exon 51 splice acceptor site
  • CK8e muscle creatine kinase
  • RNA polymerase III promoters U6, Hl and 7SK.
  • FIG. 36A To further evaluate the sensitivity of the Luciferase reporter to in vivo, the inventors administered AAV9-Cas9 and AAV9-sgRNA-5l intraperitoneally to ⁇ Ex50-Dmd-Luc mice at P4 and monitored the signal over time (FIG. 36A). Widespread bioluminescence was observed 3 weeks after injection and continued to increase to a level -70% of wild-type by 10 weeks. (Fig. 36B). Histological analysis revealed widespread dystrophin expression in the diaphragm, heart, TA and triceps muscles of gene-edited AEx50-Dmd-Luc mice at 10 weeks post-injection (FIG. 36C).
  • a 1:2 ratio of AAV9-Cas9 to AAV9- sgRNA may provide superior efficacy to when compared with a 1:1 ratio of AAV9-Cas9 to AAV9-sgRNA.
  • AAV9-Cas9 and AAV9-sgRNA-5l were tested different ratios of AAV9-Cas9 and AAV9-sgRNA-5l.
  • the inventors delivered the AAV9- Cas9 and AAV9-sgRNA-ex5l in different ratios (1 :1 and 1 :2) using intravenous injection in the tail vein of 1 month old ⁇ Ex50-/9m/-l uci ferase mice (FIG. 25 A).
  • injection is at least 30% when all muscle types are considered but over 60% for core/essential body muscles of the diagraphram and heart.
  • TIDE decomposition
  • DMD Duchenne muscular dystrophy
  • AEx44 DMD mice were generated in the C57/BL6J background using the CRISPR/Cas9 system.
  • Two sgRNAs specific to the intronic regions surrounding exon 44 of the mouse Dmd locus were cloned into vector PX458 (Addgene plasmid #48138) using the primers from Table 16.
  • AAV9 was utilized to package the gene editing components.
  • AAV9 is a single-stranded DNA virus that displays tropism to both skeletal muscle and heart and has been used in numerous clinical trials.
  • the CK8e regulatory cassette was used, which combines key elements of the enhancer and promoter regions of the muscle creatine kinase gene to drive SpCas9 expression in skeletal muscle and heart.
  • three RNA polymerase III promoters U6, Hl, and 7SK
  • Fig. 9A three RNA polymerase III promoters
  • TA muscles were collected for analysis.
  • In vivo gene editing by AAV-G5 and AAV-G6 was compared by the T7E1 assay and RT-PCR of the targeted region (Fig. 28E and Fig. 66B).
  • Gene editing with AAV-G6 showed higher efficiency based on DNA cutting in vivo (Fig. 66B).
  • RT-PCR with primers that amplify the region from exon 43 to exon 46 revealed deletion of exon 45 in TA muscle injected with AAV-Cas9 and AAV- G6 (Fig. 28E). This allows exon 43 to skip exon 45 and directly splice to exon 46 when processing the pre-mRNA.
  • the alternate mRNA enables the production of a truncated dystrophin protein in corrected TA muscle of AEx44 DMD mice.
  • topoisomerase-based thymidine to adenosine (TOPO-TA) cloning was performed using the RT-PCR amplification products and sequenced the cDNA products. Sequencing results demonstrated that 7% of sequenced clones represented exon 45-skipped cDNA products, and 42% of sequenced clones contained a single adenosine (A) insertion in exon 45 that resulted in reframing of dystrophin protein (Fig. 28F, Fig. 28G, and Fig. 66B).
  • the predominance of reframing explains the high abundance of the RT-PCR band at 355 bp and the lower abundance of the smaller RT-PCR product of 179 bp that reflects exon skipping (Fig. 28E).
  • Genomic and cDNA amplicon deep sequencing on the target region of the TA muscles with AAV-G6 IM injection also confirmed that 9.8% of mutations at the genomic level and 35.7% of mutations at the mRNA level contain a single A insertion at the cutting site after gene editing with AAV-G6 (Fig. 66C and 66D). This single A insertion leads to refraining of exon 45, and restores the dystrophin protein reading frame. Minor AAV integration events were also observed at the cutting site, with 0.2% at the genomic level (Fig. 66C), and 1.2% at the mRNA level (Fig. 66D). The integrated sequence is from the ITR region of AAV and prevents production of functional dystrophin protein from those transcripts and, thus, has neither positive nor negative effects on the dystrophic muscle phenotype.
  • the top 10 potential off-target sites were determined and, based on sequencing analysis, no off-target effects were detected at these sites (Fig. 67A-C).
  • T7E1 analysis confirmed the absence of off-target cutting in the top 10 potential off target sites, and DNA sequencing of the isolated genomic PCR amplification products spanning the potential off-target sites confirmed the absence of sgRNA/Cas9-mediated mutations at the predicted sites (Fig. 67 A).
  • genomic amplicon deep sequencing of the top 10 predicted off-target sites within protein coding exons was performed. None of these sites showed significant sequence alterations (Fig. 67B and 67C).
  • AAV- Cas9 and AAV-G6 was delivered systemically by intraperitoneal (IP) injection.
  • IP intraperitoneal
  • AAV-Cas9 was injected at a dosage of 5 c 10 13 vg/kg.
  • Multiple ratios of AAV-G6 to AAV-Cas9 were tested to determine whether there might be an optimal ratio of the viruses for maximal systemic editing efficiency.
  • dystrophin protein expression in several muscle tissues was assessed, including TA muscle of the hindlimb, triceps of the forelimb, diaphragm, and cardiac muscle.
  • dystrophin expression was observed in 94%, 90% and 95% of myofibers in the TA, triceps, and diaphragm, respectively, and in 94% of cardiomyocytes when AEx44 mice were injected with a 1 : 10 ratio of AAV- Cas9:AAVG6 (Fig. 29B and Fig. 68).
  • the restoration of dystrophin protein in skeletal muscles correlated with the dosage of AAV-G6 delivered through IP injection.
  • dystrophin positive cardiomyocytes were seen at a low dosage of AAV-G6 and remained consistent at higher dosages.
  • Western blot analysis of the same muscle groups after systemic delivery showed similar trends of dystrophin correction (Fig. 29 A, and Fig. 69).
  • cardiac muscle showed higher dystrophin restoration than skeletal muscle. Correction of cardiac muscle reached 82% when injected at a 1 : 1 ratio of AAV-Cas9:AAV-G6 and increased an additional 12% at a 1 : 10 ratio.
  • an increase of dystrophin-expressing hallmarks of muscular dystrophy such as necrotic myofibers and regenerated fibers with central nuclei, were diminished in the TA, diaphragm, and triceps muscles at 4 weeks after AAV-Cas9/AAV-G6 delivery (Fig. 70 and Fig. 71).
  • AAV-nuclease e.g., Cas9
  • AAV-gRNA may be varied to achieve optimal therapeutic efficacy.
  • the required ratio may vary by route of administration, type of tissue targeted (skeletal muscle, cadiac muscle or smooth muscle), or type of genetic modification
  • the amount of the AAV-gRNA delivered to the subject, the amount of the AAV-gRNA delivered to a target cell, the amount of AAV- gRNA expressed (including copy number) and/or the amount of gRNA operably linked to a nuclease may affect the activity of the nuclease.
  • the amount of the AAV -nuclease delivered to the subject, the amount of the AAV-nuclease delivered to a target cell, the amount of AAV- nuclease expressed (including copy number) and/or the amount of nuclease operably linked to a gRNA may affect the ability of a gRNA to selectively and specifically bind to a target sequence.
  • the AE50-MD dog model harbors a missense mutation in the 5’ donor splice site of exon 50 that results in deletion of exon 50 ((Walmsley et al, 2010)).
  • this represents an ideal canine model for the investigation of gene-editing as an approach to permanently correct the most common DMD mutations in humans.
  • Fig. 41A To correct the dystrophin reading frame in the deltaE50-MD canine model (henceforth referred to as AEx50) (Fig. 41A), the inventors used S. pyogenes Cas9 coupled with a sgRNA to target a region adjacent to the exon 51 splice acceptor site (referred to as sgRNA-5l) (Fig. 41B).
  • the sgRNA-5l corresponded to a highly conserved sequence that differs by only one nucleotide between the human and dog genomes (Fig. 43 A, Table 4).
  • the inventors evaluated the specificity of Cas9 activity by testing the sgRNA-5l sequences in human and dog cell lines. Cas9 coupled with each of these sgRNA-5l sequences only introduced a genomic cut in each respective species’ DNA, highlighting the specificity of CRISPR cutting (Fig. 43B).
  • AAV9 AAV9, which displays preferential tropism for these tissues.
  • a muscle-specific creatine kinase (CK) regulatory cassette was used to drive expression of Cas9; three RNA polymerase III promoters (U6, Hl and 7SK) directed expression of the sgRNA, as described previously in mice (Fig. 3D).
  • AAV9-Cas9 and AAV9-sgRNA-51 were initially introduced into the cranial tibialis muscles of two 1 -month-old dogs by intramuscular (IM) injection with l.2xl0 13 AAV9 viral genomes (vg) of each virus. Muscles were analyzed 6 weeks after injection. To evaluate dystrophin correction at the protein level, the inventors performed histological analysis of AAV9-injected cranial tibialis muscles 6 weeks after viral injection.
  • Western blot analysis confirmed the restoration of dystrophin expression in skeletal muscle (Fig. 20A and 20B) to -60% of wildtype levels. Injected muscles also appeared markedly healthier via H&E staining, with fewer
  • dMHC develommental myosin heavy chain
  • Dystrophin nucleates a series of proteins into the dystrophin-associated glycoprotein complex (DCG) to link the cytoskeleton and extracellular matrix.
  • DCG dystrophin-associated glycoprotein complex
  • AEx50 mice, dogs, and DMD patients these proteins are destabilized and fail to appropriately localize to the sub- sarcolemmal region.
  • Muscles injected with AAV9-Cas9 and AAV9-sgRNA-5l showed recovery of the DCG protein beta-dystroglycan compared to contralateral uninjected muscles (Fig. 48).
  • single-cut genomic editing using AAV9-Cas9 and AAV9-sgRNA-5l is highly efficient in restoration of dystrophin expression and assembly of the DGC in dystrophic muscles.
  • Dystrophin and muscle structure correction in DEc50 dogs by systemic delivery of Cas9 and sgRNA Based on the high dystrophin-correction efficiency observed following IM injection of AAV9-Cas9 and AAV9-sgRNA-5l, the inventors tested for rescue of dystrophin expression in DEc50 dogs following systemic delivery of gene editing components. Dogs at 1 month of age were injected with the viruses and analyzed 8 weeks later. The inventors tested two doses (2xl0 13 vg/kg and 1x10 14 vg/kg) of each of the two viruses AAV9-Cas9 and AAV9-sgRNA-5l.
  • AAV9-Cas9 and AAV9-sgRNA-5l performed amplicon deep-sequencing analysis of the genomic DNA from heart, triceps and biceps muscles.
  • the genomic deep sequencing analysis revealed an increase of percentage of reads containing changes at the targeted genomic site, especially of the 1 A insertion mutation in the samples from Dog-#2B compared to Dog-#2A (FIG. 58). Proof of concept - efficacious gene editing in a variety of muscle cell types following systemic administration.
  • AEx50 dogs of this age typically exhibit prominent pelvic limb paresis, displaying a distinctive“bunny -hopping” phenotype when walking and trotting. Additionally, AEx50 dogs demonstrate marked reluctance to jump or rear up. The untreated AEx50 dog displayed all these clinical signs. The AEx50 dog that received 2xl0 13 vg/kg showed a mild
  • IL-2 was measured, as a marker of T-cell reactivity in peripheral blood mononuclear cells using a canine IFN-gamma/IL-2 Dual-Color ELISpot Kit. Blood samples were collected the day before injection and then at 1, 2, 4, 6 and 8 weeks post-injection for peripheral blood mononuclear cell (PBMC) isolation. No increase of immune response over time or compared to blood samples collected before the day of injection was seen (Fig. 55). Furthermore, hematological evaluation of treated dogs revealed no significant abnormalities in comparison with untreated controls or reference ranges (Fig. 56A).
  • Example 4 As shown in Example 4, to evaluate the efficiency of the equivalent single cut strategy using sgRNA-5l to correct human DMD mutations, a DMD iPSC line carrying a deletion from exon 48 to 50 was used. Deletion of exons 48 to 50 leads to a frameshift mutation and appearance of a premature stop codon in exon 51. To correct the dystrophin reading frame, the inventors introduced Cas9 and sgRNA-5l into cells using nucleofection. Two concentrations of Cas9 and sgRNA were tested (26 ng/pl, referred to as high, and l3ng/pl, referred to as low).
  • the inventors evaluated the DNA cutting activity of Cas9 coupled with the human sgRNA-5l sequence at different concentrations in DMD-iPSCs using the mismatch-specific T7 endonuclease I (T7E1) assay (Fig. 57A).
  • Indel analysis showed 55.8% and 31.9% of indels for the high and low concentrations, respectively (Fig. 57B).
  • Genomic deep-sequencing analysis revealed that 27.94% of mutations contained a single A insertion 3’ to the PAM sequence for the high concentration condition and 19.03% for the low concentration condition (Fig. 42 A), as observed in mouse and dog cells with a similar sgRNA directed against exon 51.
  • the inventors also observed genomic sequences that contained deletions covering the splice acceptor site and ESE site for exon 51 (Fig. 42A).
  • the inventors differentiated the mixed DMD iPSCs into induced cardiomyocytes (iCMs) to investigate the restoration of dystrophin protein by immunocytochemistry and Western blot analysis.
  • the DMD-iCMs treated with Cas9 and sgRNA-5l were dystrophin positive (Fig. 42B).
  • Dystrophin protein expression levels of the corrected DMD-iCMs were comparable to WT cardiomyocytes (67 to 100%) by Western blot analysis (Fig. 42C and 42D).
  • iPSCs patient-derived induced pluripotent stem cells
  • reframing of exon 43 or 45 can restore the protein reading frame by inserting one nucleotide (+3n+l insertion) or deleting two nucleotides (+3n-2 deletion).
  • sgRNAs that target the splice acceptor or donor sites for exon 43 and 45 were selected, thereby allowing splicing between surrounding exons to recreate in-frame dystrophin.
  • sgRNAs For editing exon 43, four sgRNAs (Gl, G2, G3 and G4) directed against sequences near the 5’ and 3’ boundaries of the splice junctions of exon 43 (Fig. 61 C) were designed.
  • bp 33-base pair
  • sgRNAs (G5, G6, G7, and G8) were generated to target the 5’ boundary of exon 45, within the conserved region of the human and mouse genomes (Fig. 61D).
  • T7E1 mismatch-specific T7 endonuclease I
  • the sgRNAs were compared for their ability to direct Cas9-mediated gene editing in human 293 cells (Fig. 64B).
  • Two out of four sgRNAs for exon 43 efficiently edited the targeted region, and all four sgRNAs for exon 45 generated precise cuts at the conserved region (Fig. 67C).
  • the editing activity of the same four sgRNAs for exon 45 in mouse 10T1/2 cells was concurrently tested, and confirmed the effectiveness of the four sgRNAs in both the human and mouse genomes (Fig. 67C).
  • sgRNAs with the highest gene editing activity were then tested for the ability to efficiently edit the corresponding exons in patient-derived iPSCs lacking exon 44 (referred to as AEx44).
  • a plasmid encoding optimized sgRNAs (G3 or G4 for exon 43, or G6 for exon 45) along with SpCas9 was introduced into AEx44 patient-derived iPSCs by electroporation, and the edited iPSCs were differentiated into cardiomyocytes (CMs).
  • Dystrophin expression was assessed by Western blot analysis and immunostaining, confirming restoration of dystrophin protein expression in edited AEx44 iPSC-derived CMs (Fig. 61E and 61F).
  • Levels of dystrophin protein expression in AEx44 iPSC-derived CMs edited with sgRNAs G4 and G6 were approximately comparable to those seen in healthy control iPSC-derived CMs (Fig. 61E).
  • sgRNA G6 was used to derive single clones of AEx44 iPSCs that were edited within exon 45. Thirty-four single clones were isolated by flow cytometry and expanded. Sequence analysis of the clones showed exon skipping events in 3 out of 34 clones, and dystrophin refraining by either +3n+l or +3n-2 in 13 out of 34 clones (Fig. 64D). Western blot analysis confirmed the restoration of dystrophin expression in the three CRISPR/Cas9 corrected clones (Fig. 64E). Proof of Concept— Correction of dystrophin expression in human iPSCs with a large deletion.
  • Example 4 As shown in Example 4, to evaluate the efficiency of the equivalent single cut strategy using sgRNA-5l to correct human DMD mutations, a DMD iPSC line carrying a deletion from exon 48 to 50 was used. Deletion of exons 48 to 50 leads to a frameshift mutation and appearance of a premature stop codon in exon 51. To correct the dystrophin reading frame, the inventors introduced Cas9 and sgRNA-5l into cells using nucleofection. Two concentrations of Cas9 and sgRNA were tested (26 ng/pl, referred to as high, and l3ng/pl, referred to as low).
  • the inventors evaluated the DNA cutting activity of Cas9 coupled with the human sgRNA-5l sequence at different concentrations in DMD-iPSCs using the mismatch-specific T7 endonuclease I (T7E1) assay (Fig. 57A).
  • Indel analysis showed 55.8% and 31.9% of indels for the high and low concentrations, respectively (Fig. 57B).
  • Genomic deep-sequencing analysis revealed that 27.94% of mutations contained a single A insertion 3’ to the PAM sequence for the high concentration condition and 19.03% for the low concentration condition (Fig. 42 A), as observed in mouse and dog cells with a similar sgRNA directed against exon 51.
  • the inventors also observed genomic sequences that contained deletions covering the splice acceptor site and ESE site for exon 51 (Fig. 42A).
  • the therapeutic or pharmaceutical compositions described herein comprising at least one gRNA and at least one nuclease specifically and selectively target mutations in the DMD gene, effectively induce breaks in the target sequence, and by a variety of mechanisms (including reframing and/or exon skipping depending on the DMD mutation targeted), restore DMD expression and function.
  • the therapeutic and/or pharmaceutical compositions described herein induce a reframing of the DMD gene and exon skipping to restore DMD gene expression in each of the mouse models, dog model and human cells lines. Accordingly, the proof of concept studies provided in this disclosure recapitulate the in vivo activity of these compositions when used as a human therapeutic.
  • compositions described herein are shown in vivo to have no detectable off-target activity, demonstrating that these compositions not only efficacious, but also safe. Moreover, the compositions described herein are shown in vivo to have no detectable immune response from a host with a functional immune system, demonstrating that these compositions not only efficacious, but also well-tolerated. Data presented herein demonstrate the composition of the disclosure are efficacious for treating and/or curing late-staged or advanced DMD in individuals with substantial muscle deterioration and functional losses.
  • Dosages provided herein may be scaled to human adults or to children of various ages using known equivalents, for example, as shown below in Table A or B (Reproduced from “Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers”, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), July 2005, Pharmacology and Toxicology):
  • CRISPR/Cas9-mediated exon 50 deletion in mice Two single-guide RNA (sgRNA) specific to the intronic region surrounding exon 50 sequence of the mouse Dmd locus were cloned into vector px330 using the following primers: Dmd exon 50 F1 : 5’- CACCGAAATGATGAGTGAAGTTATAT-3’ (SEQ ID NO: 926); Dmd exon 50 R1 : 5’- AAAC ATATAACTTCACTCATCATTTC-3’ (SEQ ID NO: 927); Dmd exon 50 F2: 5’
  • AAAC AGCC ACGCTTTTGAACAAAC-3’ (SEQ ID NO: 929).
  • T7 promoter sequence was added to the sgRNA template by PCR using the following primers: Dmd exon 50 T7-F1 :
  • AAV TRIPSR plasmids were obtained from Dr. Dirk Grimm (Heidelberg University Hospital). Cloning of sgRNA was done using a Bbsl site. pGL3-CK8e plasmid was obtained from Dr. Stephen Hauschka (Department of Biochemistry, University of Washington, Seattle, USA). AAV-miniCMV-Cas9-shortPolyA plasmid was obtained from Dr. Dirk Grimm (Heidelberg University Hospital).
  • AAV-miniCMV-Cas9-short-PolyA was digested with Pad and Nhel enzyme to remove the miniCMV promoter.
  • CK8 promoter was amplified from pGL3- CK8e plasmid using primers containing PacI and Nhel site sequence and cloned into digested vector to generate AAV-CK8-Cas9-shortPolyA plasmid.
  • Dmd exon 51 guide RNAs were defined using crispr.mit.edu. Guide sequences were cloned into Addgene plasmid #42230, a gift from Feng Zhang, using the following primers: Dmd exon 51 _F 1 : 5'- CACCGAGAGTAACAGTCTGACTGG -3’ (SEQ ID NO: 942); Dmd exon 51 R1 : 5'- AAACGTC AGACTGTTACTCTAGTGC-3 ' (SEQ ID NO: 943); Dmd exon 51 F2: 5'- CACCGCACTAGAGTAACAGTCTGAC -3' (SEQ ID NO: 944); Dmd exon 51 R2: 5'- AAACCC AGTC AGACTGTT ACTCTC -3' (SEQ ID NO: 945). Guide sequences were tested in culture using 10T1/2 cells before cloning into the AAV backbone.
  • Annealed oligonucleotides encoding for the sgRNA are cloned into donor plasmids that carry the negative selection marker ccdB (to reduce background during cloning) as well as the
  • Second step is that three of these donor plasmids driving expression of one sgRNA under transcriptional control of U6, Hl or 7SK promoter are pooled in a second Golden Gate assembly along with a recipient plasmid that carries AAV ITRs.
  • the assembly reaction will contain all four plasmids: donor plasmid-# l-U6-sgRNA, donor plasmid-#2-Hl -sgRNA, donor plasmid-#3-7SK-sgRNA and recipient plasmid containing the ITR.
  • Digest with Bbsl will generate unique overhangs for each fragment (U6, Hl, 7SK, recipient backbone). During the ligation procedure, these overhangs anneal a circularized plasmid is only obtained, when the three cassettes match each other.
  • Serum creatine kinase (CK) measurement Mouse serum CK was measured by the Metabolic Phenotyping Core at UT Southwestern Medical Center. Blood was collected from the submandibular vein and serum CK level was measured by VITROS Chemistry 7 Products CK Slides to quantitatively measure CK activity using VITROS 250 Chemistry System.
  • a humanized model of DMD The most common hot spot mutation region in DMD patients is the region between exon 45 to 51, and skipping of exon 51 could be used to treat the largest group (13-14%) of patients.
  • the inventors generated a mouse model that mimics the human“hot spot” region by deleting exon 50 using the CRISPR/Cas9 system directed by 2 sgRNAs (FIG. 1 A). The deletion of exon 50 was confirmed by DNA sequencing (FIG. 1B). Deletion of exon 50 placed the dystrophin gene out of frame leading to the absence of dystrophin protein in skeletal muscle and heart (FIGS. 1C-1E).
  • mice lacking exon 50 showed pronounced dystrophic muscle changes by 2 months of age (FIG. 1E).
  • Serum analysis of delta-exon 50 mice showed a significant increase in creatine kinase (CK) levels, indicative of muscle damage (FIG. 1F).
  • CK creatine kinase
  • the inventors compared 2 different strategies: (1) double-guide strategy in which one copy of a first sgRNA targeting splice acceptor site (sgRNA-SA) and one copy of a second sgRNA targeting donor acceptor site (sgRNA-SD), were cloned into the rAAV9-sgRNA vector; (2) triplicate strategy in which the inventors cloned 3 copies of the same sgRNA (sgRNA-SA) into the rAAV9-sgRNA vector (FIG. 3C). Expression of each copy of sgRNA-SA was driven by a different RNA promoter (U6, Hl and 7SK).
  • the inventors generated AAV-Cas9 using an AAV- Cas9 vector (CK8-Cas9-shortPolyA), which employs a CK8 promoter to drive expression of the humanized SpCas9 specifically in skeletal muscle and heart tissues.
  • AAV- Cas9 vector CK8-Cas9-shortPolyA
  • IM intra-muscular
  • muscle tissues were analyzed.
  • RT-PCR of RNA from DEc50 mice injected with AAV-Tri-SA and AAV-SA+SD showed that deletion of exon 51 (DEc50-51) allowed splicing from exon 49 to 52 (FIG. 2A, lower band). Sequencing of RT-PCR products of the DEc50-51 band confirmed that exon 49 spliced to exon 52 (FIG. 2B).
  • RT-PCR analysis showed that a single cut strategy using a triplicate version of sgRNA-SA (AAV-Tri-SA) is as efficient as using two sgRNAs - sgRNA-SA and sgRNA-SD (AAV-SA+SD).
  • Hematoxylin and eosin (H&E) staining of muscle showed that histopathologic hallmarks of muscular dystrophy, such as necrotic myofibers, were diminished in TA muscle at 3-weeks post- AAV delivery (FIG. 4A).
  • Quantitative analysis of the distribution of myofiber areas showed a clear increase in fiber size for both AAV-Tri-SA and AAV-SA-SD treated muscles compared to DEc50 muscles (FIG. 4B).
  • AAV-Tri-SA treated muscles revealed a higher decrease in the frequency of small fibers ( ⁇ 500 mm) compared to AAV-SA+SD treated muscles.
  • This gRNA uses a PAM sequence 3 nucleotides further into the exon in order to generate the DSB close to the splice acceptor site for exon 51 (FIG. 7A-FIG. 7B). Cutting in the vicinity of the splice acceptor region and within the exon sequence resulted in reframing events and exon skipping events. Moreover, designing the sgRNA in the exon sequence that shows higher conservation than intron sequence across species facilitates translation of the sgRNA to other species.
  • the sgRNA-ex5l- SA2 corresponds to a highly conserved region of the Dmd gene (FIGS. 8C-D), and the inventors tested the ability of Cas9 and human sgRNA-51 to cut the human Dmd locus in 293T cells.
  • the T7E1 assay revealed clear cleavage at the predicted site (FIG. 8E).
  • sequence analysis revealed that Cas9 coupled with human sgRNA-ex5l-SA2 generated the same adenosine (A) insertion and a different range of deletions around the cleavage site (FIG. 8F).
  • adeno-associated virus 9 (AAV9) was used, which displays preferential tropism for these tissues.
  • AAV9-Cas9 vector (CK8e-Cas9- shortPolyA) was employed, which contains a muscle-specific CK regulatory cassette, referred to as the CK8e promoter, which is highly specific for expression in muscle and heart (FIG. 9A).
  • CK8e-Cas9- shortPolyA a muscle-specific CK regulatory cassette, referred to as the CK8e promoter, which is highly specific for expression in muscle and heart
  • this 436 bp muscle-specific cassette and the 4101 bp Cas9 cDNA are within the packaging limit of AAV9.
  • Expression of each sgRNA was driven by one of three RNA polymerase III promoters (U6, Hl and 7SK) (FIG. 9B).
  • the sgRNA-ex5l-SA2 was delivered to mice in triple copy (AAV-Tri-SA2), along with a Cas9 (AAV-Cas9), by intra-muscular (IM) injection. Following the injection, muscle tissues were analyzed. In vivo targeting efficiency was estimated by RT-PCR with primers for sequences in exons 48 and 53 and the T7E1 assay for the targeted genomic regions. To investigate whether efficient target cleavage was achieved, the inventors amplified a 771 bp region spanning the target site and analyzed it using the T7EI assay (FIG. 10 A). The activity of SpCas9 with the corresponding sgRNA was analyzed on the target site.
  • T7EI assays revealed mutagenesis of the Dmd locus after delivery of AAV-Cas9 and AAV9-sgRNA-5l-SA2 (FIG. 10A).
  • genomic PCR amplification products spanning the target site were analyzed by amplicon deep-sequencing analysis. Deep sequencing of the targeted region indicated that 27.9% of total reads contained changes at the targeted genomic site (FIG. 10B).
  • 15% of the identified mutations contained the same A insertion seen in mouse 10T1/2 and human 293 cells in vitro.
  • the deletions identified using this method encompassed a highly-predicted exonic splicing enhancer site for exon 51 (FIG. 10B).
  • RT-PCR products of RNA from muscle of DEc50 mice injected intramuscularly with AAV9-Cas9 and AAV9-sgRNA-5l showed that deletion of exon 51 (DEc50-51) allowed splicing from exon 49 to 52 (FIG. 11 A, lower band).
  • exon 51 DEc50-51
  • RT-PCR amplification products from 4 samples were directly subjected to topoisomerase-based thymidine to adenosine (TOPO-TA) cloning without gel purification, then sequenced.
  • TOPO-TA adenosine
  • sequence analysis of 40 clones from each sample showed that in addition to exon 5 l-skipped cDNA products (DEc50-51) identified in 15% of sequenced clones, DEc50 mice injected with AAV9-Cas9 and AAV9- sgRNA-5l showed a high frequency of refraining events.
  • DEc50 mice injected with AAV9-Cas9 and AAV9- sgRNA-5l showed a high frequency of refraining events.
  • 63% contained a single nucleotide insertion in the sequence of exon 51 (FIGS. 11 B-C). The most dominant insertion mutation seen was an A insertion.
  • the inventors delivered the sgRNA-ex5l-SA2 in single copy driven separately by the U6 promoter (AAV9-U6-sgRNA-5l-SA2), the Hl promoter (AAV9-H1- sgRNA-5l-SA2), the 7SK promoter (AAV9-7SK-sgRNA-5l-SA2) and triple copy (AAV9- Triple-sgRNA-5l-SA2).
  • U6 promoter AAV9-U6-sgRNA-5l-SA2
  • Hl promoter AAV9-H1- sgRNA-5l-SA2
  • the 7SK promoter AAV9-7SK-sgRNA-5l-SA2
  • Triple-sgRNA-5l-SA2 triple copy
  • IM intra-muscular
  • dystrophin immunostaining of muscle cryosections from DEc50 mice injected with AAV9-Triple-sgRNA-5l-SA2 revealed significantly higher numbers of dystrophin-positive fibers with an average of 95% restoration of normal fibers compared to DEc50 mice injected with AAV9-U6-sgRNA-5l-SA2, AAV-Hl-sgRNA-5 l-SA2 and AAV-7SK-sgRNA-5l-SA2 with an average of 70%; 40% and 50% restoration of normal fibers respectively (FIGS. 15B).
  • AAV9-Cas9 and AAV9-sgRNA-5l were tested different doses and delivery strategies for AAV9-Cas9 and AAV9-sgRNA-5l.
  • Dose 2 The systemic delivery yielded widespread dystrophin expression in the heart, triceps, tibialis anterior muscle, and diaphragm in gene-edited DEc50 mice at 4 weeks post-injection (FIG. 22A). Western blot analysis confirmed the restoration of dystrophin expression in muscles (FIG. 22B).
  • the AAV dose 2 (6x10 12 vg/kg) lead to lower dystrophin correction in diaphragm and triceps muscle compared to AAV dose 1 (2.6xl0 13 vg/kg), suggesting that the efficient AAV dose should not be lower than 2.6xl0 13 vg/kg.
  • the inventors delivered the AAV9-Cas9 and AAV9- sgRNA-ex5l in different doses and ratios (1 : 1 and 1 :2) using IP injection P4 DEc50 mice (FIG. 75) using the following conditions: 5xl0 13 vg/kg AAV9-Cas9 and 5xl0 13 vg/kg AAV9-sgRNA-5l
  • dystrophin immunostaining of muscle cryosections from DEc50 mice injected mice injected with 5xl0 13 vg/kg AAV9-Cas9 and 1xl0 14 vg/kg showed comparable correction to the mice injected with 1x10 14 vg/kg AAV9-sgRNA-5l and 1x10 14 vg/kg AAV9- Cas9 and 1x10 14 vg/kg AAV9-sgRNA-51 (FIG. 75).
  • AAV9-Cas9 with AAV9-sgRNA-51 at later stages of DMD disease was treated 1 month old DEc50 mice with AAV9-Cas9 and AAV9- sgRNA-5l.
  • DMD Duchenne muscular dystrophy
  • Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), which is characterized by lethal degeneration of cardiac and skeletal muscles. Mutations that delete exon 44 of the dystrophin gene represent one of the most common causes of DMD and can be corrected in -12% of patients by skipping surrounding exons, which restores the dystrophin open reading frame.
  • CRISPR/Cas9 gene editing in cardiomyocytes obtained from patient-derived induced pluripotent stem cells and in a new mouse model harboring the same deletion mutation.
  • AAV9 encoding Cas9 and single guide RNAs the importance of the dosages of these gene editing components for optimal gene correction in vivo is also demonstrated.
  • PBMCs from healthy individuals and DMD patients were generated at the UT Southwestern Wellstone Myoediting Core. Male donors’ PBMCs were used in all experiments. PBMCs were collected based on the mutation of the patients; exclusion, randomization, or blinding approaches were not used to select the donors. Animal work was approved and conducted under the oversight of the UT Southwestern Institutional Animal Care and Use Committee. Animals were allocated to experimental groups based on genotype; exclusion, randomization, or blinding approaches were not used to assign the animals for the experiments. AAV injection and dissection experiments were conducted in a nonblinded fashion. Blinding approaches were used during grip strength tests, histology validation, immunostaining analysis, CK analysis, and muscle electrophysiology. For each experiment, sample size reflects the number of independent biological replicates and was provided in the figure legend.
  • Plasmids and cloning The pSpCas9(BB)-2A-GFP (PX458) plasmid contained the human codon optimized SpCas9 gene with 2A-EGFP.
  • pSpCas9(BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138). Cloning of sgRNA was done using Bbs I sites. The sgRNAs in this study, listed in table 21, were selected using prediction of crispr.mit.edu. sgRNA sequences were cloned into PX458, then tested in tissue culture using HEK 293 and 10T cells.
  • the AAV TRISPR-sgRNAs-CK8e-GFP plasmid contained three sgRNAs driven by the U6, Hl or 7SK promoters and GFP driven by the CK8e regulatory cassette.
  • TRISPR backbone cloning system relies on two consecutive steps of the Golden Gate Assembly (New England Biolabs).
  • Human iPSCs maintenance and nucleofection Human iPSCs were cultured in mTeSR TM1 media (STEMCELL Technologies) and passaged approximately every 4 days (1 : 12 to 1 : 18 split ratio depending on the cell lines). One hour before nucleofection, iPSCs were treated with 10 mM ROCK inhibitor (Y-27632) and dissociated into single cells using Accutase (Innovative Cell Technologies, Inc.). 1 x 10 6 iPSCs were mixed with 5 pg of PX458-sgRNA-2A-GFP plasmid and nucleofected using the P3 Primary Cell 4D-Nucleofector X kit (Lonza) according to manufacturer's protocol.
  • iPSCs were cultured in mTeSR TM1 media supplemented with 10 mM ROCK inhibitor and changed to mTeSR TM1 media the next day.
  • GFP(+) and (-) cells were sorted by FACS and subjected to T7E1 assay. Single clones derived from GFP(+) iPSCs were picked and sequenced.
  • mice were housed in a barrier facility with a l2-hour light/dark cycle and maintained on standard chow (2916 Teklad Global).
  • DEc44 DMD mice were generated in the C57/BL6J background using the CRISPR/Cas9 system.
  • Two sgRNAs specific to the intronic regions surrounding exon 44 of the mouse Dmd locus were cloned into vector PX458 (Addgene plasmid #48138) using the primers from Table 16.
  • PX458 Additional plasmid #48138
  • the gel purified PCR products were used as template for in vitro transcription using the MEGAshortscript T7 Kit (Life Technologies).
  • sgRNA were purified by MEGAclear kit (Life Technologies) and eluted with nuclease-free water (Ambion). The concentration of guide RNA was measured by a NanoDrop instrument (Thermo Scientific).
  • AEx44 DMD mice were backcrossed with C57/BL6J mice for more than three generations. DEc44 DMD mice and WT littermates were genotyped using primers encompassing the targeted region from Table 18.
  • Tail biopsies were digested in 100 pl of 25 mMNaOH, 0.2 mMEDTA (pH 12) for 20 min at 95 °C.
  • Tails were briefly centrifuged followed by addition of 100 pl of 40 mM Tris-HCl (pH 5) and mixed to homogenize. Two milliliters of this reaction was used for subsequent PCR reactions with the primers in Table 18, followed by gel electrophoresis.
  • Genomic DNA isolation, PCR amplification and T7E1 analysis of PCR products Genomic DNA of mouse 10T1/2 fibroblasts, mouse C2C12 myoblasts, human HEK 293 cells and human iPSCs was isolated using DirectPCR (cell) lysis reagent (VIAGEN) according to manufacturer's protocol. Genomic DNA of mouse muscle tissues was isolated using GeneJET genomic DNA purification kit (Thermo Fisher Scientific) according to manufacturer’s protocol. Genomic DNA was PCR-amplified using GoTaq DNA polymerase (Promega) or with primers. PCR products were gel purified and subcloned into pCRII-TOPO vector (Invitrogen) according to the manufacturer's protocol.
  • Mismatched duplex DNA was obtained by denaturing/renaturing of 25 m ⁇ of the genomic PCR product using the following conditions: 95 °C for 5 min, 95 °C to 85 °C (-2.0 °C /seconds), 85 °C to 25 °C (-0.1 °C /seconds), hold at 4 °C. Then 25 m ⁇ of the mismatched duplex DNA was incubated with 2.7 m ⁇ of 10X NEB buffer 2 and 0.3 m ⁇ of T7E1 (New England BioLabs) at 37 °C for 90 minutes. The T7E1 digested PCR product was analyzed by 2% agarose gel electrophoresis.
  • Human cardiomyocyte differentiation Human iPSCs were cultured in mTeSR TM1 media for 3 days until they reached 90-95% confluence. To differentiate the iPSCs into cardiomyocytes, the cells were cultured in CDM3-C media for 2 days, followed by CDM3-WNT media for 2 days, followed by BASAL media for 6 days, followed by SELECTIVE media for 10 days and lastly by BASAL media for 2 days. Then, the cardiomyocytes were dissociated using TrypLExpress media and re-plated at 2 x 10 6 cells per well in a 6-well dish. The contents of the differentiation medium can be found in the Table below. Media for iPSC-CMs differentiation
  • AAV9 delivery to DEc44 DMD mice Before AAV9 injections, the DEc44 DMD mice were anesthetized. For intramuscular injection, the TA muscle of P12 male DEc44 DMD mice was injected with 50 m ⁇ of 22 AAV9 (1 x 10 12 vg/ml) preparations or with saline solution. For intraperitoneal injection, the P4 DEc44 DMD mice were injected using an ultrafme needle (31 gauge) with 80 m ⁇ of AAV9 preparations with a dosage of 5 x 10 13 vg/kg of AAV-Cas9 and a corresponding ratio of AAV-G6 indicated in the figure legend or with saline solution.
  • H&E staining was performed according to established staining protocols and dystrophin immunohistochemistry was performed using MANDYS8 monoclonal antibody (Sigma-Aldrich) with modifications to manufacturer’s instructions.
  • MANDYS8 monoclonal antibody Sigma-Aldrich
  • cryostat sections were thawed and rehydrated/delipidated in 1% triton/phosphate-buffered-saline, pH 7.4 (PBS). Following delipidation, sections were washed free of Triton, incubated with mouse IgG blocking reagent (M.O.M. Kit, Vector Laboratories), washed, and sequentially equilibrated with MOM protein concentrate/PBS, and MANDYS8 diluted 1 : 1800 in MOM protein concentrate/PBS.
  • mouse IgG blocking reagent M.O.M. Kit, Vector Laboratories
  • iPSCs Patient-derived induced pluripotent stem cells
  • iPSCs were generated from a DMD patient lacking exon 44 of the dystrophin gene ⁇ DMD) and from the patient’s brother as a heathy control (Fig. 61A).
  • Deletion of exon 44 (DEc44) disrupts the open reading frame of dystrophin by causing splicing of exon 43 to exon 45 and introducing a premature termination codon (Fig. 61B).
  • the reading frame can be restored using CRISPR/Cas9 gene editing by skipping exon 43, which allows splicing between exons 42 and 45, or by skipping exon 45, which allows splicing between exons 43 and 46.
  • refraining of exon 43 or 45 can restore the protein reading frame by inserting one nucleotide (+3n+l insertion) or deleting two nucleotides (+3n-2 deletion).
  • sgRNAs that target the splice acceptor or donor sites for exon 43 and 45 were selected, thereby allowing splicing between surrounding exons to recreate in-frame dystrophin.
  • sgRNAs For editing exon 43, four sgRNAs (Gl, G2, G3 and G4) directed against sequences near the 5’ and 3’ boundaries of the splice junctions of exon 43 (Fig. 61C) were designed.
  • exon 45 it was observed that the intron-exon junction of the splice acceptor site is contained within a 33-base pair (bp) region that is identical in the human and mouse genomes, allowing exon skipping strategies to be interchanged between the two species (Fig. 64A).
  • sgRNAs (G5, G6, G7, and G8) were generated to target the 5’ boundary of exon 45, within the conserved region of the human and mouse genomes (Fig. 61D).
  • T7E1 mismatch-specific T7 endonuclease I
  • sgRNAs were compared for their ability to direct Cas9-mediated gene editing in human 293 cells (Fig. 64B).
  • Two out of four sgRNAs for exon 43 efficiently edited the targeted region, and all four sgRNAs for exon 45 generated precise cuts at the conserved region (Fig. 64B, C).
  • the editing activity of the same four sgRNAs for exon 45 in mouse 10T1/2 cells was concurrently tested, and confirmed the effectiveness of the four sgRNAs in both the human and mouse genomes (Fig. 64C).
  • sgRNAs with the highest gene editing activity were then tested for the ability to efficiently edit the corresponding exons in patient-derived iPSCs lacking exon 44 (referred to as DEc44).
  • a plasmid encoding optimized sgRNAs (G3 or G4 for exon 43, or G6 for exon 45) along with SpCas9 was introduced into DEc44 patient-derived iPSCs by electroporation, and the edited iPSCs were differentiated into cardiomyocytes (CMs).
  • Dystrophin expression was assessed by Western blot analysis and immunostaining, confirming restoration of dystrophin protein expression in edited DEc44 iPSC-derived CMs (Fig. 61E and 61F).
  • Levels of dystrophin protein expression in DEc44 iPSC-derived CMs edited with sgRNAs G4 and G6 were approximately comparable to those seen in healthy control iPSC-derived CMs (Fig. 61E).
  • sgRNA G6 was used to derive single clones of DEc44 iPSCs that were edited within exon 45. Thirty-four single clones were isolated by flow cytometry and expanded. Sequence analysis of the clones showed exon skipping events in 3 out of 34 clones, and dystrophin refraining by either +3n+l or +3n-2 in 13 out of 34 clones (Fig. 64D). Western blot analysis confirmed the restoration of dystrophin expression in the three CRISPR/Cas9 corrected clones (Fig. 64E).
  • mice with a DMD exon 44 deletion To optimize gene editing for correction of an exon 44 deletion in vivo , a mouse model bearing an exon 44 deletion in the Dmd gene was generated by CRISPR/Cas9 gene editing (Fig. 26A). Zygotes of C57BL/6 mice were injected with two sgRNAs that target the introns flanking exon 44, and the zygotes were injected into surrogate female mice (fig. 65). An F0 founder with a 957 bp deletion that eliminated exon 44 was chosen for further studies. These DEc44 DMD mice contain one of the most common deletions responsible for DMD in humans.
  • Necrotic fibers, inflammatory infiltration, and regenerative fibers with centralized nuclei were observed in 4-week old DEc44 DMD mice, indicative of a severe muscular dystrophy phenotype (Fig. 26G).
  • Serum creatine kinase levels in the DEc44 DMD mice were elevated 22-fold compared to WT littermates, similar to mdx mice, an established DMD mouse model (Fig. 26D).
  • Shear force generated during muscle contraction leads to muscle membrane tearing in muscle lacking dystrophin, eventually causing myofiber degeneration and muscle fibrosis. Fibrotic tissue increases muscle stiffness and compromises contractility of muscles.
  • DEc44 DMD mice To further analyze muscle function of DEc44 DMD mice, maximal tetanic force was measured in the extensor digitorum longus (EDL) muscle ex vivo. Compared with WT littermates at 4 weeks of age, DEc44 DMD mice showed an about 50% decrease in the specific and absolute tetanic force in the EDL muscle (Fig. 27, C and D). A similar decrease of muscle strength was observed by grip strength analysis in 8-week old DEc44 DMD mice (Fig. 27E).
  • AAV9 Correction of DMD exon 44 deletion in mice by intramuscular AAV9 delivery of gene editing components.
  • AAV9 was utilized to package the gene editing components.
  • AAV9 is a single-stranded DNA virus that displays tropism to both skeletal muscle and heart and has been used in numerous clinical trials.
  • the CK8e regulatory cassette was used, which combines key elements of the enhancer and promoter regions of the muscle creatine kinase gene to drive ripCas9 expression in skeletal muscle and heart.
  • three RNA polymerase III promoters U6, Hl, and 7SK
  • Fig. 9A three RNA polymerase III promoters
  • topoisomerase-based thymidine to adenosine (TOPO-TA) cloning was performed using the RT-PCR amplification products and sequenced the cDNA products. Sequencing results demonstrated that 7% of sequenced clones represented exon 45-skipped cDNA products, and 42% of sequenced clones contained a single adenosine (A) insertion in exon 45 that resulted in reframing of dystrophin protein (Fig. 28F, Fig. 28G, and Fig. 66B). The predominance of refraining explains the high abundance of the RT-PCR band at 355 bp and the lower abundance of the smaller RT-PCR product of 179 bp that reflects exon skipping (Fig. 28E).
  • Genomic and cDNA amplicon deep sequencing on the target region of the TA muscles with AAV-G6 IM injection also confirmed that 9.8% of mutations at the genomic level
  • H&E staining showed a pronounced reduction in fibrosis, necrotic myofibers and regenerating fibers with central nuclei, indicating amelioration of the abnormalities associated with muscular dystrophy in the TA muscle 3 weeks after AAV9-Cas9 and AAV-G6 injection (Fig. 28B).
  • AAV9 expressing gene editing components rescues dystrophin expression in DEc44 mice.
  • AAV-Cas9 and AAV-G6 was delivered systemically by intraperitoneal (IP) injection.
  • IP intraperitoneal
  • AAV- Cas9 was injected at a dosage of 5 c 10 13 vg/kg.
  • Multiple ratios of AAV-G6 to AAV-Cas9 were tested to determine whether there might be an optimal ratio of the viruses for maximal systemic editing efficiency.
  • dystrophin protein expression in several muscle tissues was assessed, including TA muscle of the hindlimb, triceps of the forelimb, diaphragm, and cardiac muscle.
  • dystrophin expression was observed in 94%, 90% and 95% of myofibers in the TA, triceps, and diaphragm, respectively, and in 94% of cardiomyocytes when DEc44 mice were injected with a 1 : 10 ratio of AAV-Cas9:AAVG6 (Fig. 29B and Fig. 68).
  • the restoration of dystrophin protein in skeletal muscles correlated with the dosage of AAV-G6 delivered through IP injection.
  • dystrophin positive cardiomyocytes were seen at a low dosage of AAV-G6 and remained consistent at higher dosages.
  • Western blot analysis of the same muscle groups after systemic delivery showed similar trends of dystrophin correction (Fig. 29A, and Fig.

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Abstract

L'invention concerne l'édition génomique médiée par CRISPR/Cas9 qui présente un potentiel clinique quant au traitement de maladies génétiques, telles que la Dystrophie Musculaire de Duchenne (DMD), qui est provoquée par des mutations dans le gène de la dystrophine. L'invention concerne des compositions et des méthodes pour le traitement de la DMD. Dans certains modes de réalisation, une composition fournissant des rapports thérapeutiquement efficaces d'un vecteur codant pour un ARN guide de l'invention et un vecteur codant pour une protéine Cas9 ou un domaine nucléase de celle-ci de l'invention sont fournis pour une utilisation dans le traitement de la DMD.
EP19705415.8A 2018-01-05 2019-01-04 Compositions crispr/cas9 thérapeutiques et méthodes d'utilisation Withdrawn EP3735462A1 (fr)

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WO2019246480A1 (fr) * 2018-06-21 2019-12-26 The Board Of Regents Of The University Of Texas System Correction des délétions de l'exon 43, de l'exon 45 ou de l'exon 52 de la dystrophine dans la dystrophie musculaire de duchenne
US11168141B2 (en) 2018-08-02 2021-11-09 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
AU2019316103A1 (en) 2018-08-02 2021-03-11 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
AU2019312692A1 (en) 2018-08-02 2021-03-11 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US20230024301A1 (en) * 2019-10-02 2023-01-26 Helmholtz Zentrum München Deutsches Forschungszentrum Für Gesundheit Und Umwelt (Gmbh) Treatment of diseases caused by frame shift mutations
CA3165316A1 (fr) * 2019-12-19 2021-06-24 Nippon Shinyaku Co., Ltd. Acide nucleique anti-sens permettant un saut d'exon
WO2021138286A1 (fr) * 2020-01-03 2021-07-08 The Board Of Regents Of The University Of Texas System Système d'administration d'aav auto-complémentaire pour crispr/cas9
JP2023515709A (ja) * 2020-04-27 2023-04-13 デューク ユニバーシティ 筋肉特異的プロモーターをコードするａａｖベクターを使用するインビボでの衛星細胞の遺伝子編集
TW202218686A (zh) * 2020-09-09 2022-05-16 美商維泰克斯製藥公司 用於治療杜興氏肌肉失養症(duchenne muscular dystrophy)之組合物及方法
WO2022204476A1 (fr) * 2021-03-26 2022-09-29 The Board Of Regents Of The University Of Texas System Édition de nucléotides pour remettre en phase des transcrits de la dmd par édition de base et édition génomique prémium (« prime editing »)
EP4323522A1 (fr) * 2021-04-16 2024-02-21 Editas Medicine, Inc. Méthodes et compositions associées à la nucléase guidée par crispr/arn pour le traitement de la rétinite pigmentaire autosomique dominante associée à rho (adrp)
US11771776B2 (en) 2021-07-09 2023-10-03 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11638761B2 (en) 2021-07-09 2023-05-02 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating Facioscapulohumeral muscular dystrophy
WO2023159103A1 (fr) * 2022-02-17 2023-08-24 The Board Of Regents Of The University Of Texas System Variant crispr/spcas9 et procédés pour une correction améliorée de mutations de dystrophie musculaire de duchenne
TW202345911A (zh) * 2022-03-08 2023-12-01 美商維泰克斯製藥公司 用於治療杜興氏肌肉失養症(duchenne muscular dystrophy)之部分外顯子44、50及53之精確切除

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