Gene editing of deep intronic mutations

Abstract

To provide compositions, methods, kits and virus particles for treating disorders linked to deep intronic mutations, such as CEP290 mutation-caused LCA10.SOLUTION: A composition for treating a disease or disorder associated with a deep intronic mutation in a gene of an individual is provided, the composition comprising an engineered, non-naturally occurring CRISPR-Cas system comprising a) first and second guide RNAs which hybridize to the opposite strands of the target DNA sequences flanking the deep intronic mutation, and b) a Cas protein, wherein the Cas protein cleaves the target DNA molecule at sites flanking the deep intronic mutation, thereby excising a portion of the target DNA comprising the deep intronic mutation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 162,720, filed May 16, 2015, which is incorporated herein by reference in its entirety for all purposes.

[0002] Submitting a sequence listing as an ASCII text file The following submission, in an ASCII text file, is incorporated herein by reference in its entirety: Sequence Listing Computer Readable Form (CRF) (Filename: 159792013440SEQLIST.TXT, Date Recorded: April 14, 2016, Size: 49KB).

[0003] The present invention provides genetically engineered, non-naturally occurring clustered regularly interspaced short palindromic repeats (CIRs). The present invention relates to methods for treating diseases or disorders associated with deep intronic mutations using a Crispridine-CRISPR (CRISPR-Cas) system, as well as related compositions, kits, and viral particles. [Background technology]

[0004] Mutations in non-coding sequences, such as introns, are associated with a wide range of diseases. The human genome contains a higher proportion of long introns than other organisms, such as worms and flies; more than 90% of human introns are longer than 100 nucleotides, and more than one-third of all introns are 2,000 nucleotides or longer (Non-Patent Document 1). Furthermore, deep intron mutations have been identified as an important, potentially neglected, cause of human disease, and many attempts to identify disease-associated mutations have focused on coding sequences (Non-Patent Document 2). Due to the complexity of mRNA splicing in humans, these deep intron mutations can potentially cause a variety of pathologies due to mechanisms including, inter alia, mRNA destabilization, degradation, and mis-splicing (e.g., generating cryptic splice sites). In fact, some have estimated that up to 5% of Mendelian diseases in humans may be associated with deep intron mutations (Non-Patent Document 3).

[0005] As an illustrative example of a disorder that has been associated with deep intronic mutations in some cases, Leber congenital amaurosis (LCA) is the most severe form of hereditary retinal dystrophy, with symptoms occurring in the first year of life (Non-Patent Document 4). Visual acuity in LCA patients is rarely better than 20 / 400 (Non-Patent Document 5). LCA affects approximately 1 in 30,000 individuals in the general population, accounting for 5% of all hereditary retinal dystrophies (Non-Patent Document 6). The most frequent genetic cause of LCA, accounting for approximately 15% of all LCA cases in European countries and the United States, is the deep intronic mutation c.2991+1655A>G in intron 26 of the CEP290 gene, which generates a cryptic splice donor site and results in the inclusion of an aberrant exon containing a premature stop codon (p.C998X) in CEP290 mRNA (Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 10). LCA disease caused by CEP290 mutations is known as LCA10. In homozygous patients, some, but not all, mRNA transcripts show alternative splicing of the cryptic exon into CEP290 mRNA (Non-Patent Document 11), which suggests that this mutation may be involved in the development of LCA10. The hypomorphic nature of the thoron mutation is emphasized.

[0006] The human CEP290 gene contains 54 exons encoding a 2479-amino acid protein. CEP290 is a centrosomal protein that plays an important role in both cilium assembly and ciliary protein transport (Non-Patent Document 12; Non-Patent Document 13). In photoreceptors, the retinal cells most affected by CEP290 mutations, CEP290 is localized to the connecting cilium (Non-Patent Document 14), which connects the inner and outer segments of the photoreceptor.

[0007] Currently, there is no cure for LCA caused by CEP290 mutations. Two preclinical approaches to combat this disease are gene augmentation and antisense oligonucleotides (AONs). The size of human CEP290 complementary DNA (cDNA) exceeds the cargo size (approximately 4.8 kb) of recombinant adeno-associated virus (rAAV). Lentiviral vector systems can accommodate full-length CEP290 cDNA; however, the expression level of CEP290 may not be precisely controlled. Previous reports have demonstrated that photoreceptors are sensitive to the level of transgene expression and that overexpression of CEP290 is cytotoxic (Non-Patent Document 15; Non-Patent Document 16). An alternative strategy is to use AONs to interfere with the aberrant splicing of CEP290 (Non-Patent Document 17; Non-Patent Document 18). However, this approach requires weekly or monthly subretinal injections by a retinal specialist for several years. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Molecular Biology of the Cell, 6th edition (Alberts, B. et al., 2014) [Non-patent document 2] Homolova, K. et al. (2010) Hum. Mutat. 31:437-444 [Non-patent document 3] Cooper, DN et al. (2010) Hum. Mutat. 31:631-655 [Non-patent document 4] Leber, T.K.G.v (1869) Graefe's Archive for Clinical and Experimental Ophthalmology, Vol. 15, pp. 1-25 [Non-patent document 5] Cremers, FP et al. (2002) Human molecular genetics 11:1169-1176 [Non-patent document 6] Koenekoop, RK (2004) Survey of Ophthalmology 49:379-398 [Non-Patent Document 7] den Hollander, AI et al. (2006) Am. J. Hum. Genet. 79:556-561 [Non-patent document 8] Perrault, I. et al. (2007) Hum. Mutat. 28:416 [Non-Patent Document 9] Stone, EM (2007) Am. J. Ophthalmol. Vol. 144: pp. 791-811. [Non-Patent Document 10] Wiszniewski, W. et al. (2011) Hum. Genet. 129:319-327 [Non-Patent Document 11] den Hollander, AI et al. (2006) Am. J. Hum. Genet. 79:556-561 [Non-Patent Document 12] Barbelanne, M. et al. (2013) Hum. Mol. Genet. 22:2482-2494 [Non-Patent Document 13] Craige, B. et al. (2010) J. Cell Biol. 190:927-940 [Non-Patent Document 14] Chang, B. et al. (2006) Hum. Mol. Genet. 15:1847-1857 [Non-Patent Document 15] Burnight, E.R. et al. (2014) Gene Ther. 21:662-672 [Non-Patent Document 16] Tan, E. et al. (2001) Invest. Ophthalmol. Vis. Sci. Vol. 42: pp. 589-600 [Non-Patent Document 17] Collin, RW et al. (2012) Mol. Ther. Nucleic Acids Vol. 1: e14 [Non-Patent Document 18] Gerard, X. et al. (2012) Mol. Ther. Nucleic Acids Vol. 1: e29 Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, improved therapeutic approaches to treat disorders associated with deep intronic mutations, such as LCA10 caused by CEP290 mutations, are urgently needed. [Means for solving the problem]

[0010] The present invention provides a composition for treating a disease or disorder associated with a deep intronic mutation in a gene in an individual, the composition comprising an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking the deep intronic mutation; and b) a Cas protein, wherein the Cas protein cleaves the target DNA molecule at sites flanking the deep intronic mutation, thereby excising a portion of the target DNA containing the deep intronic mutation. In some embodiments, the invention provides compositions for treating a disease or disorder associated with a deep intronic mutation in a gene in an individual, the composition comprising a nucleic acid encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking the deep intronic mutation; and b) a nucleotide sequence encoding a Cas protein, wherein the Cas protein cleaves the target DNA molecule at sites flanking the deep intronic mutation, thereby excising a portion of the target DNA comprising the deep intronic mutation.In some embodiments, the disease or disorder associated with a deep intronic mutation is afibrinogenemia, Alport syndrome, amyotrophic lateral sclerosis, ataxia-telangiectasia, autosomal recessive polycystic kidney disease, Barth syndrome, beta thalassemia, congenital afibrinogenemia, congenital cataract, dysfacial and neuropathy syndrome, congenital glycosylation disorder type Ia, congenital glycosylation disorder type II, cystic fibrosis, dihydropteridine reductase deficiency, Fabry disease, familial platelet disorder with acute myeloid leukemia, Fanconi anemia, Gitelman syndrome, growth hormone insensitivity syndrome, Friedreich's ataxia, hemophilia A, genetic Megaloblastic anemia 1, Hermansky-Pudlak syndrome, homocystinuria, maple syrup urine disease, Marfan syndrome, methionine synthase deficiency, methylmalonic acidemia, mitochondrial trifunctional protein deficiency, mucopolysaccharidosis type II, multiminicore disease, muscular dystrophy, neurofibromatosis type I, Niemann-Pick disease type C, ocular albinism type I, ornithine delta-aminotransferase deficiency, predisposition to systemic lupus erythematosus, propionic acidemia, rhabdoid tumor, Schwartz-Jampel syndrome, Stickler syndrome, systemic lupus erythematosus, tuberous sclerosis complex, Werner syndrome, and X-linked hyperimmunoglobulin M syndrome. or X-linked hypophosphatemia. In some embodiments, the deep intronic mutation is a deep intronic mutation shown in Table 1.

[0011] In some aspects, the invention provides compositions for treating an ocular disease or disorder associated with a deep intronic mutation in a gene in an individual, the composition comprising a nucleic acid encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking the deep intronic mutation; and b) a nucleotide sequence encoding a Cas protein, wherein the Cas protein cleaves the target DNA molecule at sites flanking the deep intronic mutation, thereby excising a portion of the target DNA comprising the deep intronic mutation. In some embodiments, the present invention provides a composition for treating an ocular disease or disorder associated with a deep intronic mutation in an individual's gene, the composition comprising an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking the deep intronic mutation; and b) a Cas protein, wherein the Cas protein cleaves the target DNA molecule at sites flanking the deep intronic mutation, thereby excising the portion of the target DNA containing the deep intronic mutation. In some embodiments, the ocular disease is Leber's congenital amaurosis, optic atrophy, retinitis pigmentosa, retinoblastoma, Stargardt disease, Usher syndrome, or X-linked retinitis pigmentosa. In some embodiments, the deep intronic mutation is a deep intronic mutation listed in Table 2.

[0012] In some of the above embodiments, the ocular disease is Leber's congenital amaurosis. In some embodiments, the first guide RNA and second guide RNA guide sequences hybridize to opposite strands of a target DNA sequence located on either side of a deep intronic mutation in the Centrosomal Protein 290 kDa (CEP290) gene. In some embodiments, the deep intronic mutation is the c.2991+1655A>G mutation. In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:41 (for SpCas9), SEQ ID NO:45 (for SaCas9), SEQ ID NO:46 (for SaCas9), or SEQ ID NO:47 (for SaCas9). In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:19 (for SpCas9), SEQ ID NO:50 (for SaCas9), SEQ ID NO:51 (for SaCas9), or SEQ ID NO:52 (for SaCas9). In some embodiments, the second guide RNA is encoded by DNA comprising the sequence of SEQ ID NO: 42 (for SpCas9), SEQ ID NO: 43 (for SpCas9), SEQ ID NO: 44 (for SpCas9), SEQ ID NO: 48 (for SaCas9), or SEQ ID NO: 49 (for SaCas9). In some embodiments, the second guide RNA is encoded by DNA comprising the sequence of SEQ ID NO: 20 (for SpCas9), SEQ ID NO: 21 (for SpCas9), SEQ ID NO: 22 (for SpCas9), SEQ ID NO: 53 (for SaCas9), or SEQ ID NO: 54 (for SaCas9). In some embodiments, CEP290 is human CEP290. In some embodiments, CEP290 comprises a deep intron mutation of the sequence set forth in SEQ ID NO: 23.

[0013] In some of the above embodiments, the deep intronic mutation is located about 1-10,000 nucleotides, about 1-1000 nucleotides, or about 100-1000 nucleotides downstream of the 5' splice donor site of the gene. In some embodiments, the deep intronic mutation is located about 1-10,000 nucleotides downstream of the 3' splice acceptor site of the gene. In some embodiments, the deep intron mutation introduces a splice donor or splice acceptor site into the gene.

[0014] In some of the above embodiments, the Cas protein is a Cas9 protein. In some embodiments, the Cas9 protein is a Streptococcus pyogenes Cas9 protein (SEQ ID NO: 40), a Staphylococcus aureus Cas9 protein (SEQ ID NO: 55), a Streptococcus thermophilus Cas9 protein, a Neisseria meningitidis Cas9 protein, or a Treponema denticola Cas9 protein. In some embodiments, the Cas9 is codon-optimized for expression in a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell.

[0015] In some of the above embodiments, the CRISPR-Cas system further comprises one or more nuclear localization signals (NLS). In some embodiments, the Cas protein comprises one or more NLS. In some embodiments, the NLS is a C-terminal sequence in the SV40 large T antigen. In some embodiments, the NLS comprises the sequence PKKKRKV (SEQ ID NO: 26) or PKKKRKVEDPKKKRKVD (SEQ ID NO: 27).

[0016] In some of the above embodiments, the first guide RNA and / or the second guide RNA is fused to a transactivating cr (tracr) sequence. In some embodiments, the tracr sequence comprises a nucleotide sequence encoded by SEQ ID NO:25.

[0017] In some of the above embodiments, the CRISPR-Cas system (e.g., the first guide RNA, the second guide RNA, and the Cas protein) is complexed with a lipid, a cationic lipid, a liposome, a polycation, or a substance that enhances cellular uptake of nucleic acids and / or proteins.

[0018] In some of the above embodiments, the nucleic acid encoding the first guide RNA, the second guide RNA, and the Cas protein is expressed in a eukaryotic cell. In some embodiments, the nucleic acid encoding the first guide RNA, the second guide RNA, and / or the Cas protein is operably linked to one or more regulatory control elements. In some embodiments, the first guide RNA and / or the second guide RNA is operably linked to an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6, 7SK, or H1 promoter. In some embodiments, the nucleic acid encoding the Cas protein is operably linked to an RNA polymerase II promoter. In some embodiments, the RNA polymerase II promoter is selected from the group consisting of a cytomegalovirus (CMV) immediate early promoter, a minimal promoter fragment derived from the CMV promoter (minCMV promoter), an RSV LTR, an MoMLV LTR, a phosphoglycerate kinase-1 (PGK) promoter, a simian virus 40 (SV40) promoter, a CK6 promoter, a transthyretin promoter (TTR), a TK promoter, a tetracycline-responsive promoter (TRE), an HBV promoter, a hAAT promoter, an LSP promoter, a chimeric liver-specific promoter (LSP), an E2F promoter, an EF1α promoter, a telomerase (hTERT) promoter, a cytomegalovirus enhancer / The promoters are the chicken β-actin / rabbit β-globin promoter (CAG) promoter, the rod opsin promoter, the cone opsin promoter, the β-phosphodiesterase (PDE) promoter, the retinitis pigmentosa (RP1) promoter, or the interphotoreceptor retinoid-binding protein gene (IRBP) promoter.

[0019] In some of the above embodiments, the nucleic acids encoding one or more of the first guide RNA, the second guide RNA, or the Cas protein are located on the same or different vectors of the system. In some embodiments, the vector is a plasmid. In some embodiments, the vector is complexed with a delivery system. In some embodiments, the vector is complexed with a lipid, a cationic lipid, a liposome, a polycation, or a substance that enhances cellular uptake of the nucleic acid.

[0020] In some of the above embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector, a recombinant adenovirus vector, a recombinant lentivirus vector, or a recombinant herpes simplex virus (HSV) vector. In some embodiments, the vector is a recombinant adenovirus vector. In some embodiments, the recombinant adenoviral vector is derived from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3. In some embodiments, the recombinant adenoviral vector is derived from a variant of adenovirus serotype 2 or adenovirus serotype 5.

[0021] In some embodiments, the vector is a recombinant lentiviral vector, hi some embodiments, the recombinant lentiviral vector is derived from a lentivirus pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD114 or a mutant thereof.

[0022] In some embodiments, the vector is an rHSV vector. In some embodiments, the rHSV vector is derived from rHSV-1 or rHSV-2.

[0023] In some embodiments, the vector is a recombinant AAV (rAAV) vector. In some embodiments, the nucleic acid encoding one or more of the first guide RNA, second guide RNA, or Cas protein is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the nucleic acid encoding one or more of the first guide RNA, second guide RNA, or Cas protein is flanked by two AAV ITRs. In some embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV capsid serotype ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the vector is a self-complementary vector.

[0024] In some embodiments, the vector is encapsidated in a viral particle. In some embodiments, the viral particle is a recombinant adenoviral particle that encapsidates a recombinant adenoviral vector. In some embodiments, the recombinant adenoviral particle is a recombinant adenoviral particle that encapsidates a recombinant adenoviral vector. The recombinant adenovirus particles comprise capsids derived from adenovirus serotypes 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3. In some embodiments, the recombinant adenovirus particles comprise a mutant of an adenovirus serotype 2 capsid or an adenovirus serotype 5 capsid.

[0025] In some embodiments, the viral particle is a recombinant lentiviral particle that encapsidates a recombinant lentiviral vector. In some embodiments, the recombinant lentiviral particle comprises a capsid pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD114, or a mutant thereof.

[0026] In some embodiments, the viral particle is a recombinant HSV particle that encapsidates a recombinant HSV vector. In some embodiments, the recombinant HSV particle is a rHSV-1 or rHSV-2 viral particle.

[0027] In some embodiments, the viral particle is a recombinant AAV viral particle comprising a recombinant AAV vector. In some embodiments, the recombinant AAV viral particle comprises an AAV serotype capsid from clades A-F. In some embodiments, the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or murine AAV capsid rAAV2 / HBoV1 serotype capsid. In some embodiments, the ITRs and capsid of the rAAV viral particle are derived from the same AAV serotype. In some embodiments, the ITRs and capsid of the rAAV viral particle are derived from different AAV serotypes. In some embodiments, the recombinant AAV viral particle comprises an AAV1, AAV2, AAV8, AAVrh8R, AAV9, and / or AAVrh10 capsid. In some embodiments, the AAV1, AAV2, AAV8, AAVrh8R, AAV9, and / or AAVrh10 capsid comprises a tyrosine mutation or a heparan binding mutation. In some embodiments, the rAAV vector comprises an AAV2 ITR.

[0028] In some aspects, the invention provides methods for treating a disease or disorder associated with a deep intron mutation in an individual's gene, the method comprising administering to the individual a therapeutically effective amount of a composition comprising an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking the deep intron mutation; and b) a Cas protein, wherein the Cas protein cleaves the target DNA molecule at sites flanking the deep intron mutation, thereby excising a portion of the target DNA comprising the deep intron mutation. In some embodiments, the invention provides methods for treating a disease or disorder associated with a deep intron mutation in an individual's gene, the method comprising: a) hybridizing to opposite strands of a target DNA sequence flanking the deep intron mutation; and a) a nucleotide sequence encoding a Cas protein, wherein the Cas protein cleaves a target DNA molecule at sites flanking the deep intron mutation, thereby excising a portion of the target DNA containing the deep intron mutation. In some embodiments, the disease or disorder associated with a deep intronic mutation is afibrinogenemia, Alport syndrome, amyotrophic lateral sclerosis, ataxia-telangiectasia, autosomal recessive polycystic kidney disease, Barth syndrome, beta-thalassemia, congenital afibrinogenemia, congenital cataract, dysfacial, and neuropathy syndrome, congenital glycosylation disorder type Ia, congenital glycosylation disorder type II, cystic fibrosis, dihydropteridine reductase deficiency, Fabry disease, familial platelet disorder with acute myeloid leukemia, Fanconi anemia, Gitelman syndrome, growth hormone insensitivity syndrome, Friedreich's ataxia, hemophilia A, hereditary megaloblastic anemia. The deep intronic mutation is selected from the group consisting of methionine synthase deficiency, methylmalonic acidemia, mitochondrial triprotein deficiency, mucopolysaccharidosis type II, multiminicore disease, muscular dystrophy, neurofibromatosis type I, Niemann-Pick disease type C, ocular albinism type I, ornithine delta aminotransferase deficiency, predisposition to systemic lupus erythematosus, propionic acidemia, rhabdoid tumor, Schwartz-Jampel syndrome, Stickler syndrome, systemic lupus erythematosus, tuberous sclerosis complex, Werner syndrome, X-linked hyperimmunoglobulin M syndrome, and X-linked hypophosphatemia. In some embodiments, the deep intronic mutation is a deep intronic mutation set forth in Table 1.

[0029] In aspects, the invention provides methods for treating an ocular disease or disorder associated with a deep intronic mutation in a gene in an individual, the method comprising administering to the individual a therapeutically effective amount of a composition comprising an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking the deep intronic mutation; and b) a nucleotide sequence encoding a Cas protein, wherein the Cas protein cleaves the target DNA molecule at sites flanking the deep intronic mutation, thereby excising a portion of the target DNA comprising the deep intronic mutation. In some embodiments, the present invention provides a method for treating an ocular disease or disorder associated with a deep intronic mutation in a gene in an individual, the method comprising: using an engineered, non-naturally occurring Clustered Regularly Interspaced DNA sequence (CRISPR) comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking the deep intronic mutation; and b) a nucleotide sequence encoding a Cas protein. The present invention provides a method for treating a deep intronic mutation in a subject, comprising administering to an individual a therapeutically effective amount of a composition comprising a nucleic acid encoding a Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system, wherein the Cas protein cleaves a target DNA molecule at sites flanking a deep intronic mutation, thereby excising the portion of the target DNA comprising the deep intronic mutation. In some embodiments, the eye disease is Leber's congenital amaurosis, optic atrophy, retinitis pigmentosa, retinoblastoma, Stargardt disease, Usher syndrome, or X-linked retinitis pigmentosa. In some embodiments, the deep intronic mutation is a deep intronic mutation listed in Table 2.

[0030] In some embodiments of the above methods, the individual is a mammal. In some embodiments, the mammal is a human. In some embodiments, the composition is administered to the eye of the individual. In some embodiments, administration is subretinal or intravitreal.

[0031] In some embodiments of the above method, the eye disease is Leber's congenital amaurosis. In some embodiments, the first guide RNA and second guide RNA guide sequences hybridize to opposite strands of a target DNA sequence located on either side of a deep intronic mutation in the Centrosomal Protein 290 kDa (CEP290) gene. In some embodiments, the deep intronic mutation is the c.2991+1655A>G mutation. In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:41 (for SpCas9), SEQ ID NO:45 (for SaCas9), SEQ ID NO:46 (for SaCas9), or SEQ ID NO:47 (for SaCas9). In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:19 (for SpCas9), SEQ ID NO:50 (for SaCas9), SEQ ID NO:51 (for SaCas9), or SEQ ID NO:52 (for SaCas9). In some embodiments, the second guide RNA is encoded by DNA comprising the sequence of SEQ ID NO: 42 (for SpCas9), SEQ ID NO: 43 (for SpCas9), SEQ ID NO: 44 (for SpCas9), SEQ ID NO: 48 (for SaCas9), or SEQ ID NO: 49 (for SaCas9). In some embodiments, the second guide RNA is encoded by DNA comprising the sequence of SEQ ID NO: 20 (for SpCas9), SEQ ID NO: 21 (for SpCas9), SEQ ID NO: 22 (for SpCas9), SEQ ID NO: 53 (for SaCas9), or SEQ ID NO: 54 (for SaCas9). In some embodiments, CEP290 is human CEP290. In some embodiments, CEP290 comprises a deep intron mutation of the sequence set forth in SEQ ID NO: 23.

[0032] In some embodiments of the above methods, the deep intron mutation is located about 1 to 10,000 nucleotides, about 1 to 1,000 nucleotides, or about 100 to 1,000 nucleotides downstream of the 5' splice donor site of the gene. In some embodiments, the deep intron mutation is located about 1 to 10,000 nucleotides, about 1 to 1,000 nucleotides, or about 100 to 1,000 nucleotides upstream of the 3' splice acceptor site of the gene. In some embodiments, the deep intron mutation introduces a splice donor site or a splice acceptor site into the gene.

[0033] In some embodiments of the above methods, the Cas protein is a Cas9 protein. In some embodiments, the Cas9 protein is a Streptococcus pyogenes Cas9 protein, a Staphylococcus aureus Cas protein, a Streptococcus thermophilus Cas9 protein, a Neisseria meningitidis Cas9 protein, or a Treponema denticola Cas9 protein. In some embodiments, the Cas9 is codon-optimized for expression in a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell.

[0034] In some embodiments of the above methods, the CRISPR-Cas system further comprises one or more nuclear localization signals (NLS). In some embodiments, the Cas protein comprises one or more NLS. In some embodiments, the NLS is a C-terminal sequence in the SV40 large T antigen. In some embodiments, the NLS comprises the sequence PKKKRKV (SEQ ID NO: 26) or PKKKRKVEDPKKKRKVD (SEQ ID NO: 27).

[0035] In some embodiments of the above methods, the first guide RNA and / or the second guide RNA is fused to a transactivating cr (tracr) sequence. In one embodiment, the tracr sequence comprises the nucleotide sequence encoded by SEQ ID NO:25.

[0036] In some embodiments of the above methods, the CRISPR-Cas system (e.g., the first guide RNA, the second guide RNA, and the Cas protein) is complexed with a lipid, a cationic lipid, a liposome, a polycation, or a substance that enhances cellular uptake of nucleic acids and / or proteins.

[0037] In some embodiments of the above method, the nucleic acid encoding the first guide RNA, the second guide RNA, and the Cas protein is expressed in a eukaryotic cell. In some embodiments, the first guide RNA, the second guide RNA, and / or the nucleic acid encoding the Cas protein are operably linked to one or more regulatory control elements. In some embodiments, the first guide RNA and / or the second guide RNA are operably linked to an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6, 7SK, or H1 promoter. In some embodiments, the nucleic acid encoding the Cas protein is operably linked to an RNA polymerase II promoter. In some embodiments, the RNA polymerase II promoter is a cytomegalovirus (CMV) immediate early promoter, a minimal promoter fragment derived from the CMV promoter (minCMV promoter), an RSV LTR, an MoMLV LTR, a phosphoglycerate kinase-1 (PGK) promoter, a simian virus 40 (SV40) promoter, a CK6 promoter, a transthyretin promoter (TTR), a TK promoter, a tetracycline-responsive promoter (TRE), an HBV promoter, a hAAT promoter, an LSP promoter, a chimeric liver-specific promoter (LSP), an E2F promoter, an EF1α promoter, a telomerase (hTERT) promoter, a cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG) promoter, a rod opsin promoter, a cone opsin promoter, a β-phosphodiesterase (PDE) promoter, a retinitis pigmentosa (RP1) promoter, or an interphotoreceptor retinoid-binding protein gene (IRBP) promoter.

[0038] In some embodiments of the above method, the nucleic acids encoding one or more of the first guide RNA, the second guide RNA, or the Cas protein are located on the same or different vectors of the system. In some embodiments, the vector is a plasmid. In some embodiments, the vector is complexed with a delivery system. In some embodiments, the vector is complexed with a lipid, a cationic lipid, a liposome, a polycation, or a substance that enhances cellular uptake of the nucleic acid.

[0039] In some of the above embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector, a recombinant adenovirus vector, a recombinant lentivirus vector, or a recombinant herpes simplex virus (HSV) vector. In some embodiments, the vector is a recombinant adenovirus vector. In some embodiments, the recombinant adenoviral vector is derived from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3. In some embodiments, the recombinant adenoviral vector is derived from a variant of adenovirus serotype 2 or adenovirus serotype 5.

[0040] In some embodiments, the vector is a recombinant lentiviral vector, hi some embodiments, the recombinant lentiviral vector is derived from a lentivirus pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD114 or a mutant thereof.

[0041] In some embodiments, the vector is an rHSV vector. In some embodiments, the rHSV vector is derived from rHSV-1 or rHSV-2.

[0042] In some embodiments, the vector is a recombinant AAV (rAAV) vector. In some embodiments, the nucleic acid encoding one or more of the first guide RNA, second guide RNA, or Cas protein is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the nucleic acid encoding one or more of the first guide RNA, second guide RNA, or Cas protein is flanked by two AAV ITRs. In some embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV capsid serotype ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the vector is a self-complementary vector.

[0043] In some embodiments, the vector is encapsidated in a viral particle, hi some embodiments, the viral particle is a recombinant adenoviral particle that encapsidates a recombinant adenoviral vector. In some embodiments, the recombinant adenoviral particle comprises a capsid derived from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3. In some embodiments, the recombinant adenovirus particle comprises a mutant of an adenovirus serotype 2 capsid or an adenovirus serotype 5 capsid.

[0044] In some embodiments, the viral particle is a recombinant lentiviral particle that encapsidates a recombinant lentiviral vector. In some embodiments, the recombinant lentiviral particle comprises a capsid pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD114, or a mutant thereof.

[0045] In some embodiments, the viral particle is a recombinant HSV particle that encapsidates a recombinant HSV vector. In some embodiments, the recombinant HSV particle is a rHSV-1 or rHSV-2 viral particle.

[0046] In some embodiments, the viral particle is a recombinant AAV viral particle comprising a recombinant AAV vector. In some embodiments, the recombinant AAV viral particle comprises an AAV serotype capsid from clades A-F. In some embodiments, the AAV viral particle is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, The capsids include AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or murine AAV capsids, and rAAV2 / HBoV1 serotype capsids. In some embodiments, the ITRs and capsid of the rAAV viral particle are derived from the same AAV serotype. In some embodiments, the ITRs and capsid of the rAAV viral particle are derived from different AAV serotypes. In some embodiments, the recombinant AAV viral particle comprises an AAV1, AAV2, AAV8, AAVrh8R, AAV9, and / or AAVrh10 capsid. In some embodiments, the AAV1, AAV2, AAV8, AAVrh8R, AAV9 and / or AAVrh10 capsid comprises a tyrosine mutation or a heparan-binding mutation. In some embodiments, the rAAV vector comprises an AAV2 ITR.

[0047] In some embodiments of the above methods, the composition is a pharmaceutical composition.

[0048] In some aspects, the invention provides the use of a composition of any one of the above embodiments for treating a disorder associated with a deep intronic mutation in a gene in an individual. In some aspects, the invention provides the use of a composition of any one of the above embodiments in the manufacture of a medicament for treating a disorder associated with a deep intronic mutation in a gene in an individual. In some embodiments, the disease or disorder associated with a deep intronic mutation is afibrinogenemia, Alport syndrome, amyotrophic lateral sclerosis, ataxia-telangiectasia, autosomal recessive polycystic kidney disease, Barth syndrome, beta-thalassemia, congenital afibrinogenemia, congenital cataract, dysfacial, and neuropathy syndrome, congenital glycosylation disorder type Ia, congenital glycosylation disorder type II, cystic fibrosis, dihydropteridine reductase deficiency, Fabry disease, familial platelet disorder with acute myeloid leukemia, Fanconi anemia, Gitelman syndrome, growth hormone insensitivity syndrome, Friedreich's ataxia, hemophilia A, hereditary megaloblastic anemia. The deep intronic mutation may be a deep intronic mutation listed in Table 1. In some embodiments, the deep intronic mutation is a deep intronic mutation listed in Table 1. In some embodiments, the disease or disorder associated with a deep intronic mutation is an ocular disease. In some embodiments, the ocular disease is Leber's congenital amaurosis, optic atrophy, retinitis pigmentosa, retinoblastoma, Stargardt disease, Usher syndrome, or X-linked retinitis pigmentosa. In some embodiments, the deep intronic mutation is a deep intronic mutation set forth in Table 2. In some embodiments, the individual is a mammal.In some embodiments, the mammal is a human. In some embodiments, the eye disease is Leber's congenital amaurosis.

[0049] In some embodiments of the above uses, the composition is formulated for administration to the eye of an individual, hi some embodiments, the administration is formulated for subretinal or intravitreal administration.

[0050] In some embodiments of the above uses, the first guide RNA and the second guide RNA guide sequences hybridize to opposite strands of a target DNA sequence located on either side of a deep intron mutation in the Centrosomal Protein 290 kDa (CEP290) gene. In some embodiments, the deep intronic mutation is a c.2991+1655A>G mutation. In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:41 (for SpCas9), SEQ ID NO:45 (for SaCas9), SEQ ID NO:46 (for SaCas9), or SEQ ID NO:47 (for SaCas9). In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:19 (for SpCas9), SEQ ID NO:50 (for SaCas9), SEQ ID NO:51 (for SaCas9), or SEQ ID NO:52 (for SaCas9). In some embodiments, the second guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:42 (for SpCas9), SEQ ID NO:43 (for SpCas9), SEQ ID NO:44 (for SpCas9), SEQ ID NO:48 (for SaCas9), or SEQ ID NO:49 (for SaCas9). In some embodiments, the second guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:20 (for SpCas9), SEQ ID NO:21 (for SpCas9), SEQ ID NO:22 (for SpCas9), SEQ ID NO:53 (for SaCas9), or SEQ ID NO:54 (for SaCas9). In some embodiments, CEP290 is human CEP290. In some embodiments, CEP290 comprises a deep intron mutation of the sequence set forth in SEQ ID NO:23.

[0051] In some embodiments of the above uses, the deep intron mutation is located about 1 to 10,000 nucleotides, about 1 to 1,000 nucleotides, or about 100 to 1,000 nucleotides downstream of the 5' splice donor site of the gene. In some embodiments, the deep intron mutation is located about 1 to 10,000 nucleotides, about 1 to 1,000 nucleotides, or about 100 to 1,000 nucleotides upstream of the 3' splice acceptor site of the gene. In some embodiments, the deep intron mutation introduces a splice donor site or a splice acceptor site.

[0052] In some aspects, the present invention provides a kit comprising any one of the compositions described above.In some embodiments, the kit comprises any one of the compositions described above for use in any of the methods described herein.In some embodiments, the kit further comprises instructions for use.

[0053] In some aspects, the invention provides viral particles comprising a viral vector, the viral vector comprising a nucleic acid encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking a deep intron mutation in an individual's gene; and b) a nucleotide sequence encoding a Cas protein, wherein the Cas protein cleaves the target DNA molecule at sites flanking the deep intron mutation, thereby excising a portion of the target DNA comprising the deep intron mutation. In some embodiments, the deep intronic mutation in the individual's gene is a mutation in afibrinogenemia, Alport syndrome, amyotrophic lateral sclerosis, ataxia-telangiectasia, autosomal recessive polycystic kidney disease, Barth syndrome, beta thalassemia, congenital afibrinogenemia, congenital cataract, dysfacial and neuropathy syndrome, congenital glycosylation disorder type Ia, congenital glycosylation disorder type II, cystic fibrosis, dihydropteridine reductase deficiency, Fabry disease, familial platelet disorder with acute myeloid leukemia, Fanconi anemia, Blood, Gitelman syndrome, growth hormone insensitivity, Friedreich's ataxia, hemophilia A, hereditary megaloblastic anemia 1, Hermansky-Pudlak syndrome, homocystinuria, maple syrup urine disease, Marfan syndrome, methionine synthase deficiency, methylmalonic acidemia, mitochondrial trifunctional protein deficiency, mucopolysaccharidosis type II, multiminicore disease, muscular dystrophy, neurofibromatosis type I, Niemann-Pick disease type C, ocular albinism type I, ornithine delta aminotransferase deficiency, predisposition to systemic lupus erythematosus, prostate cancer In some embodiments, the deep intronic mutation in the individual's gene is associated with pionic acidemia, rhabdoid tumor, Schwartz-Jampel syndrome, Stickler syndrome, systemic lupus erythematosus, tuberous sclerosis, Werner syndrome, X-linked hyperimmunoglobulin M syndrome, or X-linked hypophosphatemia. In some embodiments, the deep intronic mutation in the individual's gene is a deep intronic mutation set forth in Table 1.

[0054] In some embodiments, the viral particle is a viral particle that is associated with a disease such as afibrinogenemia, Alport syndrome, amyotrophic lateral sclerosis, ataxia-telangiectasia, autosomal recessive polycystic kidney disease, Barth syndrome, beta-thalassemia, congenital afibrinogenemia, congenital cataract, dysfacial, and neuropathy syndrome, congenital glycosylation disorder type Ia, congenital glycosylation disorder type II, cystic fibrosis, dihydropteridine reductase deficiency, Fabry disease, familial platelet disorder with acute myeloid leukemia, Fanconi anemia, Gitelman syndrome, growth hormone insensitivity syndrome, Friedreich's ataxia, hemophilia A, hereditary megaloblastic anemia 1, Hermansky-Pudlak syndrome, or a combination thereof. It is used to treat individuals with: syndrome, homocystinuria, maple syrup urine disease, Marfan syndrome, methionine synthase deficiency, methylmalonic acidemia, mitochondrial triprotein deficiency, mucopolysaccharidosis type II, multiminicore disease, muscular dystrophy, neurofibromatosis type I, Niemann-Pick disease type C, ocular albinism type I, ornithine delta aminotransferase deficiency, a predisposition to systemic lupus erythematosus, propionic acidemia, rhabdoid tumor, Schwartz-Jampel syndrome, Stickler syndrome, systemic lupus erythematosus, tuberous sclerosis complex, Werner syndrome, X-linked hyperimmunoglobulin M syndrome, or X-linked hypophosphatemia.

[0055] In some aspects, the present invention provides a viral particle comprising a viral vector, the viral vector comprising a nucleic acid encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system, the nucleic acid comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking a deep intron mutation in a gene of an individual associated with an ocular disease or disorder; and b) a nucleotide sequence encoding a Cas protein, wherein the Cas protein cleaves the target DNA molecule at sites flanking the deep intron mutation, thereby excising the portion of the target DNA containing the deep intron mutation. In some embodiments, the ocular disease or disorder is Leber congenital amaurosis, optic atrophy, retinitis pigmentosa, retinoblastoma, Stargardt disease, Usher syndrome, or X-linked retinitis pigmentosa. In some embodiments, the deep intron mutation is a deep intron mutation listed in Table 2. In some embodiments, the viral particles are used to treat Leber's congenital amaurosis, optic atrophy, retinitis pigmentosa, retinoblastoma, Stargardt's disease, Usher syndrome, or X-linked retinitis pigmentosa.

[0056] In some embodiments, the eye disease is Leber's congenital amaurosis. In some embodiments, the first guide RNA and second guide RNA guide sequences hybridize to opposite strands of a target DNA sequence located on either side of a deep intronic mutation in the Centrosomal Protein 290 kDa (CEP290) gene. In some embodiments, the deep intronic mutation is the c.2991+1655A>G mutation. In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:41 (for SpCas9), SEQ ID NO:45 (for SaCas9), SEQ ID NO:46 (for SaCas9), or SEQ ID NO:47 (for SaCas9). In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:19 (for SpCas9), SEQ ID NO:50 (for SaCas9), SEQ ID NO:51 (for SaCas9), or SEQ ID NO:52 (for SaCas9). In some embodiments, the second guide RNA is SEQ ID NO: 42 (for SpCas9), SEQ ID NO: 43 (for SpCas9), SEQ ID NO: 44 (for SpCas9), SEQ ID NO: 48 (for SpCas9), In some embodiments, the second guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:20 (for SpCas9), SEQ ID NO:21 (for SpCas9), SEQ ID NO:22 (for SpCas9), SEQ ID NO:53 (for SaCas9), or SEQ ID NO:54 (for SaCas9). In some embodiments, CEP290 is human CEP290. In some embodiments, CEP290 comprises a deep intron mutation of the sequence set forth in SEQ ID NO:23.

[0057] In some embodiments of the above-described viral particles, the deep intron mutation is located about 1 to 10,000 nucleotides, about 1 to 1,000 nucleotides, or about 100 to 1,000 nucleotides downstream of the 5' splice donor site of the gene. In some embodiments, the deep intron mutation is located about 1 to 10,000 nucleotides, about 1 to 1,000 nucleotides, or about 100 to 1,000 nucleotides upstream of the 3' splice acceptor site of the gene. In some embodiments, the deep intron mutation introduces a splice donor site or a splice acceptor site into the gene.

[0058] In some embodiments of the above viral particles, the Cas protein is a Cas9 protein. In some embodiments, the Cas9 protein is a Streptococcus pyogenes Cas9 protein, a Staphylococcus aureus Cas9 protein, a Streptococcus thermophilus Cas9 protein, a Neisseria meningitidis Cas9 protein, or a Treponema denticola Cas9 protein. In some embodiments, the Cas9 is codon-optimized for expression in a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell.

[0059] In some embodiments of the above viral particles, the CRISPR-Cas system further comprises one or more nuclear localization signals (NLS). In some embodiments, the Cas protein comprises one or more NLS. In some embodiments, the NLS is a C-terminal sequence in the SV40 large T antigen. In some embodiments, the NLS comprises the sequence PKKKRKV (SEQ ID NO: 26) or PKKKRKVEDPKKKRKVD (SEQ ID NO: 27).

[0060] In some embodiments of the above viral particles, the first guide RNA and / or the second guide RNA is fused to a transactivating cr (tracr) sequence. In some embodiments, the tracr sequence comprises a nucleotide sequence encoded by SEQ ID NO:25.

[0061] In some embodiments of the above viral particles, the nucleic acid encoding the first guide RNA, the second guide RNA, and the Cas protein is expressed in a eukaryotic cell. In some embodiments, the first guide RNA, the second guide RNA, and / or the nucleic acid encoding the Cas protein are operably linked to one or more regulatory control elements. In some embodiments, the first guide RNA and / or the second guide RNA is operably linked to an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6, 7SK, or H1 promoter. In some embodiments, the nucleic acid encoding the Cas protein is operably linked to an RNA polymerase II promoter. In some embodiments, the RNA polymerase II promoter is a cytomegalovirus (CMV) immediate early promoter, a minimal promoter fragment derived from the CMV promoter (minCMV promoter), or a CMV promoter fragment. TERT), RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, simian virus 40 (SV40) promoter, CK6 promoter, transthyretin promoter (TTR), TK promoter, tetracycline-responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, chimeric liver-specific promoter (LSP), E2F promoter, EF1α promoter, telomerase (hTERT) promoter, cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG) promoter, rod opsin promoter, cone opsin promoter, β-phosphodiesterase (PDE) promoter, retinitis pigmentosa (RP1) promoter, or interphotoreceptor retinoid-binding protein gene (IRBP) promoter.

[0062] In some embodiments of the above viral particles, the nucleic acids encoding one or more of the first guide RNA, the second guide RNA, or the Cas protein are located on the same or different vectors of the system. In some embodiments, the vector is a plasmid. In some embodiments, the vector is complexed with a delivery system. In some embodiments, the vector is complexed with a lipid, a cationic lipid, a liposome, a polycation, or a substance that enhances cellular uptake of the nucleic acid.

[0063] In some embodiments of the above-mentioned viral particle, the vector is a recombinant adeno-associated virus (rAAV) vector, a recombinant adenovirus vector, a recombinant lentivirus vector, or a recombinant herpes simplex virus (HSV) vector. In some embodiments, the vector is a recombinant adenovirus vector. In some embodiments, the recombinant adenoviral vector is derived from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3. In some embodiments, the recombinant adenoviral vector is derived from a variant of adenovirus serotype 2 or adenovirus serotype 5. In some embodiments, the viral particle is a recombinant adenovirus particle that encapsidates a recombinant adenovirus vector. In some embodiments, the recombinant adenovirus particle comprises a capsid derived from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3. In some embodiments, the recombinant adenovirus particle comprises a mutant of an adenovirus serotype 2 capsid or an adenovirus serotype 5 capsid.

[0064] In some embodiments, the vector is a recombinant lentiviral vector. In some embodiments, the recombinant lentiviral vector is derived from a lentivirus pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD114, or a mutant thereof. In some embodiments, the viral particle is a recombinant lentiviral particle that encapsidates a recombinant lentiviral vector. In some embodiments, the recombinant lentiviral particle is derived from a lentivirus pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD114, or a mutant thereof. The capsid contains a pseudotyped capsid of Mokala virus, rabies virus, RD114 or a mutant thereof.

[0065] In some embodiments, the vector is an rHSV vector. In some embodiments, the rHSV vector is derived from rHSV-1 or rHSV-2. In some embodiments, the viral particle is a recombinant HSV particle that encapsidates a recombinant HSV vector. In some embodiments, the recombinant HSV particle is an rHSV-1 particle or an rHSV-2 viral particle.

[0066] In some embodiments, the vector is a recombinant AAV (rAAV) vector. In some embodiments, the nucleic acid encoding one or more of the first guide RNA, second guide RNA, or Cas protein is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the nucleic acid encoding one or more of the first guide RNA, second guide RNA, or Cas protein is flanked by two AAV ITRs. In some embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV capsid serotype ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the vector is a self-complementary vector. In some embodiments, the viral particle is a recombinant AAV viral particle comprising a recombinant AAV vector. In some embodiments, the recombinant AAV viral particle comprises an AAV serotype capsid from clades A-F. In some embodiments, the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or murine AAV capsid rAAV2 / HBoV1 serotype capsid. In some embodiments, the ITRs and capsid of the rAAV viral particle are derived from the same AAV serotype. In some embodiments, the ITRs and capsid of the rAAV viral particle are derived from different AAV serotypes. In some embodiments, the recombinant AAV viral particle comprises an AAV1, AAV2, AAV8, AAVrh8R, AAV9, and / or AAVrh10 capsid.In some embodiments, the AAV1, AAV2, AAV8, AAVrh8R, AAV9 and / or AAVrh10 capsid comprises a tyrosine mutation or a heparan-binding mutation. In some embodiments, the rAAV vector comprises an AAV2 ITR.

[0067] In some embodiments of the above viral particles, the viral particles are in a pharmaceutical composition.

[0068] In some embodiments, the present invention provides methods for generating in vitro models of eye diseases associated with deep intronic mutations in genes, comprising: a) introducing into a eukaryotic cell a nucleic acid encoding a CRISPR-Cas system, the nucleic acid comprising: i) a single guide RNA that hybridizes to opposite strands of a target DNA sequence of an intron in the gene; ii) a nucleotide sequence encoding a Cas protein; and iii) a single-stranded oligonucleotide comprising a homology-directed repair (HDR) template comprising the desired intron mutation and homology arms flanking a protospacer adjacent motif (PAM); and b) isolating cells containing the mutation integrated into the gene.

[0069] In some embodiments, the deep intron mutation is located about 1-10,000 nucleotides, about 1-1000 nucleotides, or about 100-1000 nucleotides downstream of the 5' splice donor site of the gene. In some embodiments, the deep intron mutation is located about 1-10,000 nucleotides, about 1-1000 nucleotides, or about 100-1000 nucleotides upstream of the 3' splice acceptor site of the gene. In some embodiments, the deep intron mutation introduces a splice donor or splice acceptor site into the gene.

[0070] In some embodiments, the PAM comprises a mutation to avoid cleavage of the single-stranded oligonucleotide by a Cas protein expressed in the cell.

[0071] In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the Cas9 protein is a Cas9 protein that is specific for Streptococcus pyogenes. In some embodiments, the Cas9 is a Bacillus pyogenes Cas9 protein, a Staphylococcus aureus Cas9 protein, a Streptococcus thermophilus Cas9 protein, a Neisseria meningitidis Cas9 protein, or a Treponema denticola Cas9 protein. In some embodiments, the Cas9 is codon-optimized for expression in a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the eukaryotic cell is an ocular cell. In some embodiments, the ocular cell is a retinal cell.

[0072] In some embodiments, the CRISPR-Cas system further comprises one or more nuclear localization signals (NLS). In some embodiments, the Cas protein comprises one or more NLS. In some embodiments, the NLS is a C-terminal sequence in the SV40 large T antigen. In some embodiments, the NLS comprises the sequence PKKKRKV (SEQ ID NO: 26) or PKKKRKVEDPKKKRKVD (SEQ ID NO: 27).

[0073] In some embodiments, the first guide RNA and / or the second guide RNA is fused to a transactivating cr (tracr) sequence. In some embodiments, the tracr sequence comprises the nucleotide sequence encoded by SEQ ID NO:25.

[0074] In some embodiments, the nucleic acid encoding the first guide RNA, the second guide RNA, and the Cas protein is expressed in a eukaryotic cell. In some embodiments, the nucleic acid encoding the first guide RNA, the second guide RNA, and / or the Cas protein is operably linked to one or more regulatory control elements. In some embodiments, the first guide RNA and / or the second guide RNA is operably linked to an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6, 7SK, or H1 promoter. In some embodiments, the nucleic acid encoding the Cas protein is operably linked to an RNA polymerase II promoter. In some embodiments, the RNA polymerase II promoter is selected from the group consisting of a cytomegalovirus (CMV) immediate early promoter, a minimal promoter fragment derived from a CMV promoter (minCMV promoter), an RSV LTR, an MoMLV LTR, a phosphoglycerate kinase-1 (PGK) promoter, a simian virus 40 (SV40) promoter, a CK6 promoter, a transthyretin promoter (TTR), a TK promoter, a tetracycline-responsive promoter (TRE), an HBV promoter, a hAAT promoter, an LSP promoter, a chimeric liver-specific promoter (LSP), an E2F promoter, an EF1α promoter, a telomerase II promoter, an ER ... These include the human TERT promoter, the cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG) promoter, the rod opsin promoter, the cone opsin promoter, the β-phosphodiesterase (PDE) promoter, the retinitis pigmentosa (RP1) promoter, or the interphotoreceptor retinoid-binding protein gene (IRBP) promoter.

[0075] In some embodiments, the nucleic acids encoding one or more of the single guide RNA, Cas protein, or single-stranded oligonucleotide are located on the same or different vectors of the system.

[0076] In some embodiments, the ocular disease is Leber congenital amaurosis, optic atrophy, retinitis pigmentosa, retinoblastoma, Stargardt disease, Usher syndrome, or X-linked retinitis pigmentosa. In some embodiments, the deep intronic mutation is a deep intronic mutation set forth in Table 2. In some embodiments, the viral particle is used to treat Leber congenital amaurosis, optic atrophy, retinitis pigmentosa, retinoblastoma, Stargardt disease, Usher syndrome, or X-linked retinitis pigmentosa.

[0077] In some embodiments, the eye disease is Leber's congenital amaurosis. In some embodiments, the first guide RNA and second guide RNA guide sequences hybridize to opposite strands of a target DNA sequence located on either side of a deep intronic mutation in the Centrosomal Protein 290 kDa (CEP290) gene. In some embodiments, the deep intronic mutation is the c.2991+1655A>G mutation. In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:41 (for SpCas9), SEQ ID NO:45 (for SaCas9), SEQ ID NO:46 (for SaCas9), or SEQ ID NO:47 (for SaCas9). In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:19 (for SpCas9), SEQ ID NO:50 (for SaCas9), SEQ ID NO:51 (for SaCas9), or SEQ ID NO:52 (for SaCas9). In some embodiments, the second guide RNA is encoded by DNA comprising the sequence of SEQ ID NO: 42 (for SpCas9), SEQ ID NO: 43 (for SpCas9), SEQ ID NO: 44 (for SpCas9), SEQ ID NO: 48 (for SaCas9), or SEQ ID NO: 49 (for SaCas9). In some embodiments, the second guide RNA is encoded by DNA comprising the sequence of SEQ ID NO: 20 (for SpCas9), SEQ ID NO: 21 (for SpCas9), SEQ ID NO: 22 (for SpCas9), SEQ ID NO: 53 (for SaCas9), or SEQ ID NO: 54 (for SaCas9). In some embodiments, CEP290 is human CEP290. In some embodiments, CEP290 comprises a deep intron mutation of the sequence set forth in SEQ ID NO: 23.

[0078] In some aspects, the invention provides a method for cleaving a target nucleic acid in a cell, the method comprising delivering to the cell an effective amount of a composition comprising: a) a nucleic acid encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system, the CRISPR system comprising a first guide RNA and a second guide RNA that hybridize to opposite strands of the target DNA sequence flanking a mutation; and b) a Cas expression cassette comprising i) a nucleotide sequence encoding a Cas protein and ii) a first guide RNA target site to which the first guide RNA or the second guide RNA hybridizes, wherein the Cas protein is expressed from the Cas expression cassette, and the Cas protein cleaves the target DNA sequence flanking the mutation, thereby excising a portion of the target DNA comprising the mutation, and the Cas protein cleaves the Cas expression cassette at the first guide RNA target site, thereby reducing expression of the Cas protein compared to expression of the Cas protein before cleavage of the Cas expression cassette. In some embodiments, the present invention provides a method for treating a disease or disorder associated with a mutation in a nucleic acid in an individual, the method comprising: a) using an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic RNA (CRISRNA) gene comprising a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking the mutation; and b) administering to an individual a therapeutically effective amount of a composition comprising: i) a nucleotide sequence encoding a Cas protein; and ii) a Cas expression cassette comprising a first guide RNA target site to which a first guide RNA or a second guide RNA hybridizes; wherein the Cas protein is expressed from the Cas expression cassette; the Cas protein cleaves a target DNA sequence flanking the mutation, thereby excising a portion of the target DNA containing the mutation; and the Cas protein cleaves the Cas expression cassette at the first guide RNA target site, thereby reducing expression of the Cas protein compared to expression of the Cas protein before cleavage of the Cas expression cassette.In some embodiments, the present invention provides a method for treating an ocular disease or disorder associated with a mutation in a nucleic acid in an individual, the method comprising: a) providing an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic RNA (CRISRNA) gene comprising a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking the mutation; and b) administering to an individual a therapeutically effective amount of a composition comprising: i) a nucleotide sequence encoding a Cas protein; and ii) a Cas expression cassette comprising a first guide RNA target site to which a first guide RNA or a second guide RNA hybridizes; wherein the Cas protein is expressed from the Cas expression cassette; the Cas protein cleaves a target DNA sequence flanking the mutation, thereby excising a portion of the target DNA containing the mutation; and the Cas protein cleaves the Cas expression cassette at the first guide RNA target site, thereby reducing expression of the Cas protein compared to expression of the Cas protein before cleavage of the Cas expression cassette. In some embodiments, the Cas expression cassette further comprises iii) a second guide RNA target site to which the first guide RNA or the second guide RNA hybridizes, and the Cas protein cleaves the Cas expression cassette at the first and second guide RNA target sites, thereby reducing expression of the Cas protein compared to expression of the Cas protein before cleavage of the Cas expression cassette.

[0079] In some embodiments of the above methods, the first guide RNA hybridizes with the first guide RNA target site and the second guide RNA target site. In some embodiments, the second guide RNA hybridizes with the first guide RNA target site and the second guide RNA target site. In some embodiments, the first guide RNA hybridizes with the first guide RNA target site, and the second guide RNA hybridizes with the second guide RNA target site. In some embodiments, the Cas expression cassette further comprises a polyadenylation (polyA) sequence operably linked to the nucleotide sequence encoding the Cas protein. In some embodiments, the polyA sequence is an SV40 polyA sequence. In some embodiments, cleavage of the first or second guide RNA target site by the Cas protein disrupts the operably linkage between the nucleotide sequence encoding the Cas protein and the polyA sequence. In some embodiments, the first or second guide RNA target site is between the nucleotide sequence encoding the Cas protein and the polyA sequence. In some embodiments, the nucleotide sequence encoding the Cas protein is operably linked to a nucleotide sequence encoding one or more nuclear localization signals (NLSs), such that the Cas protein expressed from the Cas expression cassette is fused in-frame with the one or more NLSs. The nucleotide sequence encoding one or more NLSs is between the nucleotide sequence encoding the Cas protein and the polyadenylation (polyA) sequence. In some embodiments, the first or second guide RNA target site is between the nucleotide sequence encoding the one or more NLSs and the polyA sequence. In some embodiments, the one or more NLSs comprise a C-terminal sequence in the SV40 large T antigen. In some embodiments, the one or more NLSs comprise the sequence PKKKRKV (SEQ ID NO: 26) or PKKKRKVEDPKKKRKVD (SEQ ID NO: 27). In some embodiments, the nucleic acid encoding the CRISPR-Cas system and / or the Cas expression cassette is operably linked to one or more regulatory control elements. In some embodiments, the nucleotide sequence encoding the Cas protein is operably linked to a promoter. In some embodiments, cleavage of the first or second guide RNA target site by the Cas protein disrupts the operable linkage between the regulatory control element and the nucleotide sequence encoding the Cas protein. In some embodiments, the first or second guide RNA target site is between the promoter and the nucleotide sequence encoding the Cas protein.

[0080] In some embodiments of the above methods, the Cas expression cassette further comprises iii) a second guide RNA target site to which the first guide RNA or the second guide RNA hybridizes, the second guide RNA target site being adjacent to a protospacer adjacent motif (PAM) specific to the Cas protein; wherein cleavage of the first guide RNA target site by the Cas protein disrupts an operable linkage between the regulatory control element and the nucleotide sequence encoding the Cas protein; cleavage of the second guide RNA target site by the Cas protein disrupts an operable linkage between the nucleotide sequence encoding the Cas protein and the polyA sequence; and upon expression of the Cas protein and cleavage of the target DNA sequence, the Cas protein cleaves the Cas expression cassette at the first and second guide RNA target sites, thereby reducing expression of the Cas protein compared to expression of the Cas protein before cleavage of the Cas expression cassette. In some embodiments, the Cas expression cassette further comprises iii) a second guide RNA target site to which the first guide RNA or the second guide RNA hybridizes, wherein the first guide RNA target site is between the nucleotide sequence encoding the Cas protein and the promoter operably linked to the nucleotide sequence encoding the Cas protein, and the second guide RNA target site is between the nucleotide sequence encoding the Cas protein and the polyA sequence operably linked to the nucleotide sequence encoding the Cas protein, and the Cas protein cleaves the Cas expression cassette at the first and second guide RNA target sites, thereby reducing expression of the Cas protein compared to expression of the Cas protein before cleavage of the Cas expression cassette.

[0081] In some embodiments of the above methods, the disease or disorder associated with a deep intronic mutation is afibrinogenemia, Alport syndrome, amyotrophic lateral sclerosis, ataxia-telangiectasia, autosomal recessive polycystic kidney disease, Barth syndrome, beta-thalassemia, congenital afibrinogenemia, congenital cataract, dysfacial, and neuropathy syndrome, congenital glycosylation disorder type Ia, congenital glycosylation disorder type II, cystic fibrosis, dihydropteridine reductase deficiency, Fabry disease, familial platelet disorder with acute myeloid leukemia, Fanconi anemia, Gitelman syndrome, growth hormone deficiency, or growth hormone deficiency. Mon refractory syndrome, Friedreich's ataxia, hemophilia A, hereditary megaloblastic anemia 1, Hermansky-Pudlak syndrome, homocystinuria, maple syrup urine disease, Marfan syndrome, methionine synthase deficiency, methylmalonic acidemia, mitochondrial trifunctional protein deficiency, mucopolysaccharidosis type II, multiminicore disease, muscular dystrophy, neurofibromatosis type I, Niemann-Pick disease type C, ocular albinism type I, ornithine delta-aminotransferase deficiency, predisposition to systemic lupus erythematosus, propionic acidemia, rhabdoid tumor, Schwartz-Jampel syndrome, Stickler syndrome In some embodiments, the deep intronic mutation is a deep intronic mutation shown in Table 1.

[0082] In some aspects, the invention provides compositions for treating an ocular disease or disorder associated with a deep intronic mutation in a gene in an individual, the composition comprising a nucleic acid encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking the deep intronic mutation; and b) a nucleotide sequence encoding a Cas protein, wherein the Cas protein cleaves the target DNA molecule at sites flanking the deep intronic mutation, thereby excising a portion of the target DNA comprising the deep intronic mutation. In some embodiments, the present invention provides a composition for treating an ocular disease or disorder associated with a deep intronic mutation in an individual's gene, the composition comprising an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking the deep intronic mutation; and b) a Cas protein, wherein the Cas protein cleaves the target DNA molecule at sites flanking the deep intronic mutation, thereby excising the portion of the target DNA containing the deep intronic mutation. In some embodiments, the ocular disease is Leber's congenital amaurosis, optic atrophy, retinitis pigmentosa, retinoblastoma, Stargardt disease, Usher syndrome, or X-linked retinitis pigmentosa. In some embodiments, the deep intronic mutation is a deep intronic mutation listed in Table 2.

[0083] In some of the above embodiments, the ocular disease is Leber's congenital amaurosis. In some embodiments, the first guide RNA and second guide RNA guide sequences hybridize to opposite strands of a target DNA sequence located on either side of a deep intronic mutation in the Centrosomal Protein 290 kDa (CEP290) gene. In some embodiments, the deep intronic mutation is the c.2991+1655A>G mutation. In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:41 (for SpCas9), SEQ ID NO:45 (for SaCas9), SEQ ID NO:46 (for SaCas9), or SEQ ID NO:47 (for SaCas9). In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:19 (for SpCas9), SEQ ID NO:50 (for SaCas9), SEQ ID NO:51 (for SaCas9), or SEQ ID NO:52 (for SaCas9). In some embodiments, the second guide RNA is encoded by DNA comprising the sequence of SEQ ID NO: 42 (for SpCas9), SEQ ID NO: 43 (for SpCas9), SEQ ID NO: 44 (for SpCas9), SEQ ID NO: 48 (for SaCas9), or SEQ ID NO: 49 (for SaCas9). In some embodiments, the second guide RNA is encoded by DNA comprising the sequence of SEQ ID NO: 20 (for SpCas9), SEQ ID NO: 21 (for SpCas9), SEQ ID NO: 22 (for SpCas9), SEQ ID NO: 53 (for SaCas9), or SEQ ID NO: 54 (for SaCas9). In some embodiments, CEP290 is human CEP290. In some embodiments, CEP290 comprises a deep intron mutation of the sequence set forth in SEQ ID NO: 23.

[0084] In some of the above embodiments, the deep intronic mutation is located about 1-10,000 nucleotides, about 1-1000 nucleotides, or about 100-1000 nucleotides downstream of the 5' splice donor site of the gene. The deep intronic mutation is located about 1 to 10,000 nucleotides, about 1 to 1000 nucleotides, or about 100 to 1000 nucleotides upstream of the 3' splice acceptor site of the gene. In some embodiments, the deep intronic mutation introduces a splice donor or splice acceptor site into the gene.

[0085] In some of the above embodiments, the Cas protein is a Cas9 protein. In some embodiments, the Cas9 protein is a Streptococcus pyogenes Cas9 protein (SEQ ID NO: 40), a Staphylococcus aureus Cas9 protein (SEQ ID NO: 55), a Streptococcus thermophilus Cas9 protein, a Neisseria meningitidis Cas9 protein, or a Treponema denticola Cas9 protein. In some embodiments, the Cas9 is codon-optimized for expression in a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell.

[0086] In some of the above embodiments, the first guide RNA and / or the second guide RNA is fused to a transactivating cr (tracr) sequence. In some embodiments, the tracr sequence comprises a nucleotide sequence encoded by SEQ ID NO:25.

[0087] In some of the above embodiments, the CRISPR-Cas system (e.g., the first guide RNA, the second guide RNA, and the Cas protein) is complexed with a lipid, a cationic lipid, a liposome, a polycation, or a substance that enhances cellular uptake of nucleic acids and / or proteins.

[0088] In some of the above embodiments, the nucleic acid encoding the first guide RNA, the second guide RNA, and the Cas protein is expressed in a eukaryotic cell. In some embodiments, the nucleic acid encoding the first guide RNA, the second guide RNA, and / or the Cas protein is operably linked to one or more regulatory control elements. In some embodiments, the first guide RNA and / or the second guide RNA is operably linked to an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6, 7SK, or H1 promoter. In some embodiments, the nucleic acid encoding the Cas protein is operably linked to an RNA polymerase II promoter. In some embodiments, the RNA polymerase II promoter is a cytomegalovirus (CMV) immediate early promoter, a minimal promoter fragment derived from the CMV promoter (minCMV promoter), an RSV LTR, an MoMLV LTR, a phosphoglycerate kinase-1 (PGK) promoter, a simian virus 40 (SV40) promoter, a CK6 promoter, a transthyretin promoter (TTR), a TK promoter, a tetracycline-responsive promoter (TRE), an HBV promoter, a hAAT promoter, an LSP promoter, a chimeric liver-specific promoter (LSP), an E2F promoter, an EF1α promoter, a telomerase (hTERT) promoter, a cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG) promoter, a rod opsin promoter, a cone opsin promoter, a β-phosphodiesterase (PDE) promoter, a retinitis pigmentosa (RP1) promoter, or an interphotoreceptor retinoid-binding protein gene (IRBP) promoter.

[0089] In some of the above embodiments, the first guide RNA, the second guide RNA Alternatively, the nucleic acids encoding one or more of the Cas proteins are located on the same or different vectors of the system. In some embodiments, the vector is a plasmid. In some embodiments, the vector is complexed with a delivery system. In some embodiments, the vector is complexed with a lipid, cationic lipid, liposome, polycation, or substance that enhances cellular uptake of the nucleic acid.

[0090] In some of the above embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector, a recombinant adenovirus vector, a recombinant lentivirus vector, or a recombinant herpes simplex virus (HSV) vector. In some embodiments, the vector is a recombinant adenovirus vector. In some embodiments, the recombinant adenoviral vector is derived from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3. In some embodiments, the recombinant adenoviral vector is derived from a variant of adenovirus serotype 2 or adenovirus serotype 5.

[0091] In some embodiments, the vector is a recombinant lentiviral vector, hi some embodiments, the recombinant lentiviral vector is derived from a lentivirus pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD114 or a mutant thereof.

[0092] In some embodiments, the vector is an rHSV vector. In some embodiments, the rHSV vector is derived from rHSV-1 or rHSV-2.

[0093] In some embodiments, the vector is a recombinant AAV (rAAV) vector. In some embodiments, the nucleic acid encoding one or more of the first guide RNA, second guide RNA, or Cas protein is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the nucleic acid encoding one or more of the first guide RNA, second guide RNA, or Cas protein is flanked by two AAV ITRs. In some embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV capsid serotype ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the vector is a self-complementary vector.

[0094] In some embodiments, the vector is encapsidated in a viral particle, hi some embodiments, the viral particle is a recombinant adenoviral particle that encapsidates a recombinant adenoviral vector. In some embodiments, the recombinant adenoviral particle comprises a capsid derived from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3. In some embodiments, the recombinant adenovirus particle comprises a mutant of an adenovirus serotype 2 capsid or an adenovirus serotype 5 capsid.

[0095] In some embodiments, the viral particle is a recombinant lentiviral particle that encapsidates a recombinant lentiviral vector. In some embodiments, the recombinant lentiviral particle comprises a capsid pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD114, or a mutant thereof.

[0096] In some embodiments, the viral particle is a recombinant HSV particle that encapsidates a recombinant HSV vector. In some embodiments, the recombinant HSV particle is a rHSV-1 or rHSV-2 viral particle.

[0097] In some embodiments, the viral particle is a recombinant AAV viral particle comprising a recombinant AAV vector. In some embodiments, the recombinant AAV viral particle comprises an AAV serotype capsid from clades A-F. In some embodiments, the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or murine AAV capsid rAAV2 / HBoV1 serotype capsid. In some embodiments, the ITRs and capsid of the rAAV viral particle are derived from the same AAV serotype. In some embodiments, the ITRs and capsid of the rAAV viral particle are derived from different AAV serotypes. In some embodiments, the recombinant AAV viral particle comprises an AAV1, AAV2, AAV8, AAVrh8R, AAV9, and / or AAVrh10 capsid. In some embodiments, the AAV1, AAV2, AAV8, AAVrh8R, AAV9, and / or AAVrh10 capsid comprises a tyrosine mutation or a heparan binding mutation. In some embodiments, the rAAV vector comprises an AAV2 ITR.

[0098] In some aspects, the invention provides a composition for cleaving a target nucleic acid in a cell, the composition comprising: a) a nucleic acid encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system, the CRISPR system comprising a first guide RNA and a second guide RNA that hybridize to opposite strands of the target DNA sequence flanking a mutation; and b) a Cas expression cassette comprising i) a nucleotide sequence encoding a Cas protein and ii) a first guide RNA target site to which the first guide RNA or the second guide RNA hybridizes, wherein the Cas protein is expressed from the Cas expression cassette, the Cas protein cleaves the target DNA sequences flanking the mutation, thereby excising a portion of the target DNA containing the mutation, and the Cas protein cleaves the Cas expression cassette at the first guide RNA target site, thereby reducing expression of the Cas protein compared to expression of the Cas protein before cleavage of the Cas expression cassette. In some embodiments, the present invention provides a composition for treating a disease or disorder associated with a mutation in a nucleic acid in an individual, the composition comprising: a) an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR hybridization targeting a target DNA sequence, the CRISPR ...

[0003] In some embodiments, the present invention provides a composition for treating an ocular disease or disorder associated with a mutation in a nucleic acid in an individual, the composition comprising: a nucleic acid encoding a CRISPR-associated (Cas) (CRISPR-Cas) system; and b) a Cas expression cassette comprising i) a nucleotide sequence encoding a Cas protein and ii) a first guide RNA target site to which a first guide RNA or a second guide RNA hybridizes, wherein the Cas protein is expressed from the Cas expression cassette, the Cas protein cleaves a target DNA sequence flanking the mutation, thereby excising a portion of the target DNA containing the mutation, and the Cas protein cleaves the Cas expression cassette at the first guide RNA target site, thereby reducing expression of the Cas protein compared to expression of the Cas protein prior to cleavage of the Cas expression cassette. In some embodiments, the present invention provides a composition for treating an ocular disease or disorder associated with a mutation in a nucleic acid in an individual, the composition comprising: a) a genetically engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic RNA (CRISPR-Cas) gene encoding a CRISPR-Cas system; and ii) a Cas expression cassette comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of the target DNA sequence flanking the mutation. and b) a Cas expression cassette comprising: i) a nucleotide sequence encoding a Cas protein; and ii) a first guide RNA target site to which a first guide RNA or a second guide RNA hybridizes; wherein the Cas protein is expressed from the Cas expression cassette; the Cas protein cleaves a target DNA sequence flanking a mutation, thereby excising a portion of the target DNA containing the mutation; and the Cas protein cleaves the Cas expression cassette at the first guide RNA target site, thereby reducing expression of the Cas protein compared to expression of the Cas protein before cleavage of the Cas expression cassette.In some embodiments, the Cas expression cassette further comprises iii) a second guide RNA target site to which the first guide RNA or the second guide RNA hybridizes, and the Cas protein cleaves the Cas expression cassette at the first and second guide RNA target sites, thereby reducing expression of the Cas protein compared to expression of the Cas protein before cleavage of the Cas expression cassette.

[0099] In some embodiments of the above compositions, the first guide RNA hybridizes with the first guide RNA target site and the second guide RNA target site. In some embodiments, the second guide RNA hybridizes with the first guide RNA target site and the second guide RNA target site. In some embodiments, the first guide RNA hybridizes with the first guide RNA target site, and the second guide RNA hybridizes with the second guide RNA target site. In some embodiments, the Cas expression cassette further comprises a polyadenylation (polyA) sequence operably linked to the nucleotide sequence encoding the Cas protein. In some embodiments, the polyA sequence is an SV40 polyA sequence. In some embodiments, cleavage of the first or second guide RNA target site by the Cas protein disrupts the operably linkage between the nucleotide sequence encoding the Cas protein and the polyA sequence. In some embodiments, the first or second guide RNA target site is between the nucleotide sequence encoding the Cas protein and the polyA sequence. In some embodiments, the nucleotide sequence encoding the Cas protein is operably linked to a nucleotide sequence encoding one or more nuclear localization signals (NLSs) such that the Cas protein expressed from the Cas expression cassette is fused in-frame with the one or more NLSs. In some embodiments, the nucleotide sequence encoding the one or more NLSs is between the nucleotide sequence encoding the Cas protein and a polyadenylation (polyA) sequence. In some embodiments, a first or second guide RNA target site is between the nucleotide sequence encoding the one or more NLSs and the polyA sequence. In some embodiments, the one or more NLSs comprise a C-terminal sequence in the SV40 large T antigen. In some embodiments, the one or more NLSs comprise the sequence PKKKRKV (SEQ ID NO: 26) or PKKKRKVEDPKKKRKVD (SEQ ID NO: 27). In some embodiments In some embodiments, the nucleic acid encoding the CRISPR-Cas system and / or the Cas expression cassette is operably linked to one or more regulatory control elements. In some embodiments, the nucleotide sequence encoding the Cas protein is operably linked to a promoter. In some embodiments, cleavage of the first or second guide RNA target site by the Cas protein disrupts the operable linkage between the regulatory control element and the nucleotide sequence encoding the Cas protein. In some embodiments, the first or second guide RNA target site is between the promoter and the nucleotide sequence encoding the Cas protein.

[0100] In some embodiments of the above compositions, the Cas expression cassette further comprises iii) a second guide RNA target site to which the first guide RNA or the second guide RNA hybridizes, the second guide RNA target site being adjacent to a protospacer adjacent motif (PAM) specific to the Cas protein; cleavage of the first guide RNA target site by the Cas protein disrupts an operable linkage between the regulatory control element and the nucleotide sequence encoding the Cas protein; cleavage of the second guide RNA target site by the Cas protein disrupts an operable linkage between the nucleotide sequence encoding the Cas protein and the polyA sequence; and upon expression of the Cas protein and cleavage of the target DNA sequence, the Cas protein cleaves the Cas expression cassette at the first and second guide RNA target sites, thereby reducing expression of the Cas protein compared to expression of the Cas protein before cleavage of the Cas expression cassette. In some embodiments, the Cas expression cassette further comprises iii) a second guide RNA target site to which the first guide RNA or the second guide RNA hybridizes, wherein the first guide RNA target site is between the nucleotide sequence encoding the Cas protein and the promoter operably linked to the nucleotide sequence encoding the Cas protein, and the second guide RNA target site is between the nucleotide sequence encoding the Cas protein and the polyA sequence operably linked to the nucleotide sequence encoding the Cas protein, and the Cas protein cleaves the Cas expression cassette at the first and second guide RNA target sites, thereby reducing expression of the Cas protein compared to expression of the Cas protein before cleavage of the Cas expression cassette.

[0101] In some embodiments of the above compositions, the disease or disorder associated with a deep intronic mutation is afibrinogenemia, Alport syndrome, amyotrophic lateral sclerosis, ataxia-telangiectasia, autosomal recessive polycystic kidney disease, Barth syndrome, beta-thalassemia, congenital afibrinogenemia, congenital cataract, dysfacial, and neuropathy syndrome, congenital glycosylation disorder type Ia, congenital glycosylation disorder type II, cystic fibrosis, dihydropteridine reductase deficiency, Fabry disease, familial platelet disorder with acute myeloid leukemia, Fanconi anemia, Gitelman syndrome, growth hormone insensitivity syndrome, Friedreich's ataxia, hemophilia A, hereditary macroangiopathy, The deep intronic mutation is selected from the group consisting of blastic anemia 1, Hermansky-Pudlak syndrome, homocystinuria, maple syrup urine disease, Marfan syndrome, methionine synthase deficiency, methylmalonic acidemia, mitochondrial triprotein deficiency, mucopolysaccharidosis type II, multiminicore disease, muscular dystrophy, neurofibromatosis type I, Niemann-Pick disease type C, ocular albinism type I, ornithine delta aminotransferase deficiency, predisposition to systemic lupus erythematosus, propionic acidemia, rhabdoid tumor, Schwartz-Jampel syndrome, Stickler syndrome, systemic lupus erythematosus, tuberous sclerosis complex, Werner syndrome, X-linked hyperimmunoglobulin M syndrome, and X-linked hypophosphatemia. In some embodiments, the deep intronic mutation is a deep intronic mutation set forth in Table 1.

[0102] In some embodiments, the present invention provides a composition for treating an ocular disease or disorder associated with a deep intronic mutation in a gene in an individual, the composition comprising: a) a first guide strand that hybridizes to opposite strands of a target DNA sequence flanking the deep intronic mutation; The present invention provides a composition comprising a nucleic acid encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system, the system comprising a) a guide RNA and a second guide RNA; and b) a nucleotide sequence encoding a Cas protein, wherein the Cas protein cleaves a target DNA molecule at sites flanking a deep intron mutation, thereby excising a portion of the target DNA containing the deep intron mutation. In some embodiments, the present invention provides a composition for treating an ocular disease or disorder associated with a deep intronic mutation in an individual's gene, the composition comprising an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking the deep intronic mutation; and b) a Cas protein, wherein the Cas protein cleaves the target DNA molecule at sites flanking the deep intronic mutation, thereby excising the portion of the target DNA containing the deep intronic mutation. In some embodiments, the ocular disease is Leber's congenital amaurosis, optic atrophy, retinitis pigmentosa, retinoblastoma, Stargardt disease, Usher syndrome, or X-linked retinitis pigmentosa. In some embodiments, the deep intronic mutation is a deep intronic mutation listed in Table 2.

[0103] In some embodiments of the above composition, the eye disease is Leber's congenital amaurosis. In some embodiments, the first guide RNA and second guide RNA guide sequences hybridize to opposite strands of a target DNA sequence located on either side of a deep intronic mutation in the Centrosomal Protein 290 kDa (CEP290) gene. In some embodiments, the deep intronic mutation is the c.2991+1655A>G mutation. In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:41 (SpCas9), SEQ ID NO:45 (SaCas9), SEQ ID NO:46 (SaCas9), or SEQ ID NO:47 (SaCas9). In some embodiments, the first guide RNA is encoded by DNA comprising the sequence of SEQ ID NO:19 (SpCas9), SEQ ID NO:50 (SaCas9), SEQ ID NO:51 (SaCas9), or SEQ ID NO:52 (SaCas9). In some embodiments, the second guide RNA is encoded by DNA comprising the sequence of SEQ ID NO: 42 (for SpCas9), SEQ ID NO: 43 (for SpCas9), SEQ ID NO: 44 (for SpCas9), SEQ ID NO: 48 (for SaCas9), or SEQ ID NO: 49 (for SaCas9). In some embodiments, the second guide RNA is encoded by DNA comprising the sequence of SEQ ID NO: 20 (for SpCas9), SEQ ID NO: 21 (for SpCas9), SEQ ID NO: 22 (for SpCas9), SEQ ID NO: 53 (for SaCas9), or SEQ ID NO: 54 (for SaCas9). In some embodiments, CEP290 is human CEP290. In some embodiments, CEP290 comprises a deep intron mutation of the sequence set forth in SEQ ID NO: 23.

[0104] In some embodiments of the above compositions, the deep intron mutation is located about 1 to 10,000 nucleotides, about 1 to 1,000 nucleotides, or about 100 to 1,000 nucleotides downstream of the 5' splice donor site of the gene. In some embodiments, the deep intron mutation is located about 1 to 10,000 nucleotides, about 1 to 1,000 nucleotides, or about 100 to 1,000 nucleotides upstream of the 3' splice acceptor site of the gene. In some embodiments, the deep intron mutation introduces a splice donor or splice acceptor site into the gene.

[0105] In some embodiments of the above compositions, the Cas protein is a Cas9 protein. In some embodiments, the Cas9 protein is a Cas9 protein specific for Streptococcus pyogenes. In some embodiments, the Cas9 is a Staphylococcus pyogenes Cas9 protein (SEQ ID NO: 40), a Staphylococcus aureus Cas9 protein (SEQ ID NO: 55), a Streptococcus thermophilus Cas9 protein, a Neisseria meningitidis Cas9 protein, or a Treponema denticola Cas9 protein. In some embodiments, the Cas9 is codon-optimized for expression in a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell.

[0106] In some embodiments of the above compositions, the first guide RNA and / or the second guide RNA is fused to a transactivating cr (tracr) sequence. In some embodiments, the tracr sequence comprises a nucleotide sequence encoded by SEQ ID NO: 25.

[0107] In some embodiments of the above compositions, the CRISPR-Cas system (e.g., the first guide RNA, the second guide RNA, and the Cas protein) is complexed with a lipid, a cationic lipid, a liposome, a polycation, or a substance that enhances cellular uptake of nucleic acids and / or proteins.

[0108] In some embodiments of the above compositions, the nucleic acid encoding the first guide RNA, the second guide RNA, and the Cas protein is expressed in a eukaryotic cell. In some embodiments, the nucleic acid encoding the first guide RNA, the second guide RNA, and / or the Cas protein is operably linked to one or more regulatory control elements. In some embodiments, the first guide RNA and / or the second guide RNA is operably linked to an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6, 7SK, or H1 promoter. In some embodiments, the nucleic acid encoding the Cas protein is operably linked to an RNA polymerase II promoter. In some embodiments, the RNA polymerase II promoter is a cytomegalovirus (CMV) immediate early promoter, a minimal promoter fragment derived from the CMV promoter (minCMV promoter), an RSV LTR, an MoMLV LTR, a phosphoglycerate kinase-1 (PGK) promoter, a simian virus 40 (SV40) promoter, a CK6 promoter, a transthyretin promoter (TTR), a TK promoter, a tetracycline-responsive promoter (TRE), an HBV promoter, a hAAT promoter, an LSP promoter, a chimeric liver-specific promoter (LSP), an E2F promoter, an EF1α promoter, a telomerase (hTERT) promoter, a cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG) promoter, a rod opsin promoter, a cone opsin promoter, a β-phosphodiesterase (PDE) promoter, a retinitis pigmentosa (RP1) promoter, or an interphotoreceptor retinoid-binding protein gene (IRBP) promoter.

[0109] In some embodiments of the above compositions, the nucleic acids encoding one or more of the first guide RNA, the second guide RNA, or the Cas protein are located on the same or different vectors of the system. In some embodiments, the vector is a plasmid. In some embodiments, the vector is complexed with a delivery system. In some embodiments, the vector is complexed with a lipid, a cationic lipid, a liposome, a polycation, or a substance that enhances cellular uptake of the nucleic acid.

[0110] In some embodiments of the above compositions, the vector is a recombinant adeno-associated virus (r In some embodiments, the vector is a recombinant adenovirus (AAV) vector, a recombinant adenovirus vector, a recombinant lentivirus vector, or a recombinant herpes simplex virus (HSV) vector. In some embodiments, the vector is a recombinant adenovirus vector. In some embodiments, the recombinant adenoviral vector is derived from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3. In some embodiments, the recombinant adenoviral vector is derived from a variant of adenovirus serotype 2 or adenovirus serotype 5.

[0111] In some embodiments, the vector is a recombinant lentiviral vector, hi some embodiments, the recombinant lentiviral vector is derived from a lentivirus pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokala virus, rabies virus, RD114 or a mutant thereof.

[0112] In some embodiments, the vector is an rHSV vector. In some embodiments, the rHSV vector is derived from rHSV-1 or rHSV-2.

[0113] In some embodiments, the vector is a recombinant AAV (rAAV) vector. In some embodiments, the nucleic acid encoding one or more of the first guide RNA, second guide RNA, or Cas protein is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the nucleic acid encoding one or more of the first guide RNA, second guide RNA, or Cas protein is flanked by two AAV ITRs. In some embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV capsid serotype ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the vector is a self-complementary vector.

[0114] In some embodiments, the vector is encapsidated in a viral particle, hi some embodiments, the viral particle is a recombinant adenoviral particle that encapsidates a recombinant adenoviral vector. In some embodiments, the recombinant adenoviral particle comprises a capsid derived from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3. In some embodiments, the recombinant adenovirus particle comprises a mutant of an adenovirus serotype 2 capsid or an adenovirus serotype 5 capsid.

[0115] In some embodiments, the viral particle is a recombinant lentiviral particle that encapsidates a recombinant lentiviral vector. In some embodiments, the recombinant lentiviral particle is a recombinant lentiviral particle that encapsidates a recombinant lentiviral vector, such as vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mocha virus, or the like. Mokala virus, rabies virus, containing capsids pseudotyped with RD114 or its mutants.

[0116] In some embodiments, the viral particle is a recombinant HSV particle that encapsidates a recombinant HSV vector. In some embodiments, the recombinant HSV particle is a rHSV-1 or rHSV-2 viral particle.

[0117] In some embodiments, the viral particle is a recombinant AAV viral particle comprising a recombinant AAV vector. In some embodiments, the recombinant AAV viral particle comprises an AAV serotype capsid from clades A-F. In some embodiments, the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, or murine AAV capsid rAAV2 / HBoV1 serotype capsid. In some embodiments, the ITRs and capsid of the rAAV viral particle are derived from the same AAV serotype. In some embodiments, the ITRs and capsid of the rAAV viral particle are derived from different AAV serotypes. In some embodiments, the recombinant AAV viral particle comprises an AAV1, AAV2, AAV8, AAVrh8R, AAV9, and / or AAVrh10 capsid. In some embodiments, the AAV1, AAV2, AAV8, AAVrh8R, AAV9, and / or AAVrh10 capsid comprises a tyrosine mutation or a heparan binding mutation. In some embodiments, the rAAV vector comprises an AAV2 ITR.

[0118] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. [Brief explanation of the drawings]

[0119]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6-1

Figure 6-2

Figure 7

Figure 8

[0120] The present invention provides compositions, methods and viral particles for editing deep intron mutation.In some embodiments, the composition for treating the disease or disorder associated with deep intron mutation in individual's gene comprises engineered non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system, which comprises:a) a first guide RNA and a second guide RNA, which hybridize with opposite strands of the target DNA sequence located on both sides of the deep intron mutation;b) a Cas protein, and the Cas protein cuts the target DNA molecule at the site located on both sides of the deep intron mutation, thereby excising the part of the target DNA that comprises the deep intron mutation. In other embodiments, a composition for treating a disease or disorder associated with a deep intronic mutation in a gene in an individual comprises a nucleic acid encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system, the system comprising: a) a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence flanking the deep intronic mutation; and b) a nucleotide sequence encoding a Cas protein, wherein the Cas protein flanks the deep intronic mutation. The target DNA molecule is cleaved at a site located at , thereby excising the portion of the target DNA containing the deep intron mutation.

[0121] In some embodiments, the present invention provides compositions, methods, and viral particles for treating eye diseases. As described above, the most frequent genetic cause of LCA is the deep intronic mutation c.2991+1655A>G in intron 26 of the CEP290 gene, which generates a cryptic splice donor site, resulting in the inclusion of an aberrant exon containing a premature stop codon (p.C998X) in CEP290 mRNA (Figure 1). The present inventors have designed a simple and efficient method for treating LCA patients with the intronic c.2991+1655A>G mutation in the CEP290 gene by targeted genomic DNA deletion in human cells using a pair of single guide RNA (sgRNA) and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) systems (Figure 2). This approach efficiently and permanently deletes the intronic c.2991+1655A>G mutation and prevents splicing of the inserted cryptic exon in the CEP290 mRNA, while leaving the endogenous genomic regulatory elements intact.

[0122] I. General technology The techniques and procedures described or referenced herein are generally well understood and are well documented in, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (F.M.A.usubel et al., eds., 2003); the series Methods in Enzymology (Academic Press, Inc.); PCR2: A Practical Approach (M.J. MacPherson, B.D. Hames, and G.R. Taylor, eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (R.I. Freshney, 6th ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Manual Notebook (ed. J.E.Cellis, Academic Press, 1998); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (eds. A. Doyle, J.B. Griffiths and D.G. Newell, J. Wiley & Sons, 1993-8); Handbook of Experimental Immunology (eds. D.M. Weir and C.C. Blackwell, 1996); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P.Calos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JE Coligan et al., 1991); Short Protocols in Molecular Biology (Ausubel et al., 2002); Immunobiology (CA Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibod. Antibodies are commonly used by those skilled in the art using conventional methodologies such as those widely used methodologies described in *Monoclonal Antibodies: A Practical Approach* (D. Catty, ed., IRL Press, 1988-1989); *Monoclonal Antibodies: A Practical Approach* (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); *Using Antibodies: A Laboratory Manual* (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); *The Antibodies* (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and *Cancer: Principles and Practice of Oncology* (VT DeVita et al., eds., J.B. Lippincott Company, 2011).

[0123] II. Definition As used herein, "CRISPR-Cas" refers to a two-component ribonucleoprotein complex containing a guide RNA and a Cas endonuclease. CRISPR refers to the Clustered Regularly Interspaced Short Palindromic Repeats type II system. CRISPR was discovered as an adaptive defense system that allows bacteria and archaea to detect and silence foreign nucleic acids (e.g., from viruses or plasmids), but has been adapted for use in various cell types to enable sequence-specific polynucleotide editing (see, for example, Jinek, M. et al. (2012) Science 337:816-821 and Ran, FA et al. (2013) Nat. Protoc. 8:2281-2308). In the type II system, the guide RNA interacts with Cas and directs the nuclease activity of the Cas enzyme to a target DNA sequence identical to the guide RNA guide sequence. The guide RNA base pairs with the opposite strand of the target sequence. Cas nuclease activity then generates a double-stranded break in the target DNA. In some embodiments, the Cas protein is a Cas9 protein.

[0124] As used herein, "CRISPR-Cas single guide RNA" (the terms "single guide RNA" and "sgRNA" can be used interchangeably herein) refers to a single RNA species capable of directing Cas-mediated cleavage of target DNA. In some embodiments, the single guide RNA may contain sequences required for Cas (e.g., Cas9) nuclease activity and a guide sequence identical to the target DNA of interest.

[0125] The terms "chimeric RNA," "chimeric guide RNA," "guide RNA," "single guide RNA," and "synthetic guide RNA" may be used interchangeably herein to refer to a polynucleotide sequence comprising a guide sequence, a tracr sequence, and a tracr mate sequence. The term "guide sequence" refers to an approximately 20 bp sequence within a guide RNA that specifies a target site, and may be used interchangeably with the terms "guide," "spacer," or "protospacer." The term "tracr mate sequence" may also be used interchangeably with the term "direct repeat."

[0126] As used herein, "sgRNA guide sequence" can refer to the nucleotide sequence of an sgRNA that binds to the opposite strand of a target DNA sequence and directs Cas (e.g., Cas9) nuclease activity to that locus. In some embodiments, the sgRNA guide sequence is identical to the target sequence. Complete identity is not necessary, as long as there is sufficient similarity to cause hybridization and promote the formation of a CRISPR complex. The guide sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide.

[0127] As used herein, a "Cas" polypeptide is a polypeptide that functions as a nuclease when complexed with a guide RNA, e.g., an sgRNA. In some embodiments, the Cas polypeptide is a Cas9 polypeptide (CRISPR-associated 9, also known as Csn1). When bound to a crRNA:tracrRNA guide or a single guide RNA, a Cas polypeptide (e.g., Cas9) can cleave target DNA at a sequence adjacent to a PAM motif identical to the sgRNA guide sequence. The Cas9 polypeptide, unlike other Cas polypeptides, is characteristic of Type II CRISPR-Cas systems (see Makarova, KS et al. (2011) Nat. Rev. Microbiol. 9(6):467-77 for a description of Cas proteins in various CRISPR-Cas systems). As used herein, "Cas" can refer to a ribonucleoprotein complex with an sgRNA or a polypeptide component of the complex, unless otherwise specified.

[0128] The term "CRISPR RNA (crRNA)" as used herein refers to an RNA containing a guide sequence used by the CRISPR-Cas system to direct cleavage at a target DNA sequence. The term "trans-activating crRNA (tracrRNA)" as used herein refers to an RNA containing a sequence that forms the structure required for a CRISPR-Cas effector complex that mediates DNA cleavage. In endogenous bacterial and archaeal type II CRISPR-Cas systems, the effector CRISPR-Cas complex contains two polyribonucleotide molecules: crRNA and a Cas protein (e.g., Cas9 protein) complexed with the tracrRNA. The crRNA contains an approximately 20-nucleotide guide sequence that mediates target recognition and a sequence that forms a duplex with the tracrRNA. The crRNA:tracrRNA duplex binds to the Cas protein and is required for CRISPR-Cas effector complex function. In some embodiments, the crRNA and tracrRNA functions are carried out by a single RNA (single guide RNA or sgRNA) that contains both sequences that mediate target recognition and sequences that generate the structure required for the CRISPR-Cas effector complex.

[0129] The term " deep intron mutation " as used herein refers to the mutation in the intron sequence of the region outside the wild-type splice acceptor sequence and splice donor sequence.In some cases, deep intron mutation can lead to the change of splicing of related gene, for example, the intron sequence is included in mature mRNA.In a non-limiting example, deep intron mutation is about 100bp more downstream (i.e., 3') of exon, about 100bp more upstream (i.e., 5') of exon, or about 100bp more downstream of the first exon and about 100bp more upstream of the second exon.

[0130] The term "Leber congenital amaurosis (LCA)" as used herein refers to a group of early-onset disorders characterized by vision loss, retinal dysfunction, and nystagmus. Although various mutations have been implicated in LCA, LCA is usually inherited as an autosomal recessive disorder. For further description and exemplary LCA disease genes and loci, see, e.g., OMIM Entry 204000.

[0131] The term "CEP290" as used herein refers to a gene encoding a centrosomal protein involved in ciliogenesis, also known as MKS4, CT87, POC3, rd16, BBS14, LCA10, JBTS5, NPHP6, SLSN6, and 3H11Ag. Mutations in CEP290 are associated with LCA. One example of such a mutation is the c.2991+1655A>G mutation, which introduces a cryptic splice donor site and results in the inclusion of an aberrant exon with a premature stop codon. See, for example, NCBI gene number 80184 and UniProt number O15078 for exemplary human gene and protein sequences, respectively. Other examples of the CEP290 gene include: Examples of suitable CEP290 genes include, but are not limited to, mouse CEP290 (e.g., NCBI gene no. 216274), rat CEP290 (e.g., NCBI gene no. 314787), rhesus monkey CEP290 (e.g., NCBI gene no. 708286), zebrafish CEP290 (e.g., NCBI gene no. 560588), dog CEP290 (e.g., NCBI gene no. 482591), chimpanzee CEP290 (e.g., NCBI gene no. 452113), cat CEP290 (e.g., NCBI gene no. 100113471), chicken CEP290 (e.g., NCBI gene no. 417887), and bovine CEP290 (e.g., NCBI gene no. 282707). In some embodiments, the CEP290 gene comprises a deep intron mutation of the sequence set forth in SEQ ID NO: 23.

[0132] A "vector," as used herein, refers to a recombinant plasmid or virus containing a nucleic acid to be delivered into a host cell either in vitro or in vivo.

[0133] The term "polynucleotide" or "nucleic acid" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically, or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugars and phosphate groups (as typically found in RNA or DNA) or modified or substituted sugars or phosphate groups. Alternatively, the backbone of a polynucleotide may comprise a polymer of synthetic subunits, such as phosphoramidates, and thus may be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer. Additionally, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing the complementary strand and annealing the strands under appropriate conditions or by synthesizing the complementary strand de novo using DNA polymerase with appropriate primers.

[0134] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or unnatural amino acid residues and may include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, "polypeptide" refers to proteins containing modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental due to host mutations resulting from PCR amplification or error-inducing protein amplification.

[0135] "Recombinant viral vector" refers to a recombinant polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of viral origin). In the case of a recombinant AAV vector, the recombinant nucleic acid is flanked by at least one inverted terminal repeat (ITR). In some embodiments, the recombinant nucleic acid is flanked by two ITRs.

[0136] A "recombinant AAV vector (rAAV vector)" is a vector containing one or more heterologous sequences (i.e., The term rAAV refers to a polynucleotide vector containing a nucleic acid sequence (a nucleic acid sequence not of AAV origin). When present in a host cell infected with a suitable helper virus (or expressing suitable helper functions) and expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins), such rAAV vectors are replicated and packaged into infectious viral particles. When the rAAV vector is incorporated into a larger polynucleotide (e.g., into a chromosome or into another vector, such as a plasmid used for cloning or transfection), the rAAV vector is called a "provector" that is "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. The rAAV vector can be in any of several forms, including, but not limited to, a plasmid, a linear artificial chromosome, complexed with lipids, encapsulated in a liposome, or encapsidated into a viral particle, e.g., an AAV particle. The rAAV vector can be packaged into an AAV viral capsid to produce a "recombinant adeno-associated viral particle (rAAV particle)."

[0137] "rAAV virus" or "rAAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated rAAV vector genome.

[0138] A "recombinant adenoviral vector" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not of adenoviral origin) flanked by at least one adenoviral inverted terminal repeat (ITR). In some embodiments, the recombinant nucleic acid is flanked by two inverted terminal repeats (ITRs). Such recombinant viral vectors are replicated and packaged into infectious viral particles when present in a host cell expressing essential adenoviral genes (e.g., E1, E2, E4, etc.) that have been deleted from the recombinant viral genome. When a recombinant viral vector is integrated into a larger polynucleotide (e.g., into a chromosome or into another vector such as a plasmid used for cloning or transfection), the recombinant viral vector is referred to as a "provector," which is "rescued" by replication and encapsidation in the presence of adenoviral packaging functions. Recombinant viral vectors can be in any of several forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsidated in viral particles, such as adenovirus particles. Recombinant viral vectors can be packaged in the viral capsid of adenovirus to produce "recombinant adenovirus particles."

[0139] A "recombinant lentiviral vector" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences not of lentiviral origin) flanked by at least one lentiviral long terminal repeat (LTR). In some embodiments, the recombinant nucleic acid is flanked by two lentiviral long terminal repeat (LTR). Such recombinant viral vectors are replicated and packaged into infectious viral particles when present in infected host cells with appropriate helper functions. Recombinant lentiviral vectors can be packaged into lentiviral capsids to produce "recombinant lentiviral particles."

[0140] "Recombinant herpes simplex vector (recombinant HSV vector)" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not of HSV origin) flanked by HSV terminal repeat sequences. Such recombinant viral vectors are capable of replicating and infecting viruses when present in infected host cells with appropriate helper functions. The recombinant viral vector is packaged into infectious viral particles. When the recombinant viral vector is integrated into a larger polynucleotide (for example, into a chromosome or into another vector, such as a plasmid used for cloning or transfection), the recombinant viral vector is called a "provector," which is "rescued" by replication and encapsidation in the presence of HSV packaging function. The recombinant viral vector can be in any of several forms, including, but not limited to, a plasmid, a linear artificial chromosome, complexed with lipid, encapsulated in a liposome, or encapsidated into a viral particle, such as an HSV particle. The recombinant viral vector can be packaged into an HSV capsid to produce a "recombinant herpes simplex viral particle."

[0141] "Heterologous" means derived from a genotypically distinct entity from that of the remainder of the entity to which it is compared or into which it is introduced or incorporated. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide (which, when expressed, may encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence relative to the vector.

[0142] The term "transgene" refers to a polynucleotide that can be introduced into a cell and, under appropriate conditions, transcribed into RNA, optionally translated, and / or expressed. In some embodiments, the transgene confers a desired characteristic on the cell into which it is introduced, or otherwise leads to a desired therapeutic or diagnostic outcome. In other embodiments, the transgene is transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA, or shRNA.

[0143] The terms "genome particles (gp)," "genome equivalents," or "genome copies," as used in connection with viral titer, refer to the number of virions containing the recombinant AAV DNA genome, regardless of infectiousness or functionality. The number of genome particles in a particular vector preparation can be measured by procedures such as those described in the Examples herein or, for example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0144] The term "vector genome (vg)" as used herein may refer to one or more polynucleotides comprising a set of polynucleotide sequences of a vector, e.g., a viral vector. A vector genome is encapsidated in a viral particle. Depending on the particular viral vector, the vector genome may comprise single-stranded DNA, double-stranded DNA, or single-stranded RNA or double-stranded RNA. A vector genome may comprise endogenous sequences associated with a particular viral vector and / or any heterologous sequences inserted into the particular viral vector by recombinant techniques. For example, a recombinant AAV vector genome may comprise at least one ITR sequence flanking a promoter, a stuffer, a sequence of interest (e.g., RNAi), and a polyadenylation sequence. A complete vector genome may comprise the complete set of polynucleotide sequences of a vector. In some embodiments, the nucleic acid titer of a viral vector can be measured in units of vg / mL. Suitable methods for measuring this titer are known in the art (e.g., quantitative PCR).

[0145] The terms "infectious unit (iu)," "infectious particle," or "replication unit," as used in reference to viral titer, refer to the number of infectious and replicative recombinant AAV vector particles as measured by the infectious center assay, also known as the replication center assay, e.g., as described in McLaughlin et al. (1988) J. Virol. 62:1963-1973.

[0146] The term "transducing unit (tu)" as used in reference to viral titer refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product, as measured in the Examples herein or in a functional assay such as that described, for example, in Xiao et al. (1997) Exp. Neurobiol. 144:113-124; or Fisher et al. (1996) J. Virol. 70:520-532 (LFU assay).

[0147] "Inverted terminal repeat" or "ITR" sequences are a term well understood in the art and refer to relatively short sequences found at the ends of viral genomes in opposite orientations.

[0148] The term "AAV inverted terminal repeat (ITR)" is well understood in the art and refers to a sequence of approximately 145 nucleotides present at both ends of a natural single-stranded AAV genome. The outermost 125 nucleotides of the ITR can exist in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter regions of self-complementarity (represented as A, A', B, B', C, C', and D regions), allowing intrastrand base pairing to occur within this portion of the ITR.

[0149] "Terminal release sequences" or "trs" are sequences in the D region of the AAV ITRs that are cleaved by the AAV rep protein during viral DNA replication. Mutant terminal release sequences are resistant to cleavage by the AAV rep protein.

[0150] The term "helper virus" for AAV refers to a virus that enables AAV (a defective parvovirus) to be replicated and packaged by host cells. Several such helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses such as vaccinia. Adenovirus type 5 (Ad5) of subgroup C is the most well-known, but adenoviruses encompass several different subgroups. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as the ATCC. Similarly, herpes viruses available from depositories such as the ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV).

[0151] "Percent sequence identity (%)" relative to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to those in the reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid or nucleic acid sequence identity can be achieved in a variety of ways within the skill of those in the art, for example, using publicly available computer software programs, including those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Appendix 30, Section 7.7.18, Table 7.7.1, and BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. An example of an alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. For purposes herein, a given amino acid sequence B is a sequence of The % amino acid sequence identity of a given amino acid sequence A (which can be rephrased as a given amino acid sequence A having or containing a particular % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: multiply the fraction X / Y (where X is the number of amino acid residues scored as identical matches by a sequence alignment program in that program's alignment of A and B, and Y is the total number of amino acid residues in B) by 100. It will be recognized that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. For purposes herein, the percent nucleic acid sequence identity of a given nucleic acid sequence C to, with, or to a given nucleic acid sequence D (which can be translated as a given nucleic acid sequence C having or comprising a particular percent nucleic acid sequence identity to, with, or to a given nucleic acid sequence D) is calculated as follows: the fraction W / Z (where W is the number of nucleotides scored as identical matches by a sequence alignment program in that program's alignment of C and D, and Z is the total number of nucleotides in D) multiplied by 100. It will be recognized that if the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, then the percent nucleic acid sequence identity of C to D will not equal the percent nucleic acid sequence identity of D to C.

[0152] An "isolated" molecule (eg, nucleic acid or protein) or cell means that it has been identified and separated and / or recovered from a component of its natural environment.

[0153] An "effective amount" is an amount sufficient to achieve beneficial or desired results, including clinical results (e.g., amelioration of symptoms, achievement of a clinical endpoint, etc.). An effective amount is administered in one or more administrations. In terms of a disease state, an effective amount is an amount sufficient to ameliorate, stabilize, or delay the onset of the disease.

[0154] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0155] As used herein, "treatment" refers to an approach to obtain beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilized (e.g., not worsening) state of disease, prevention of disease spread (e.g., metastasis), delay or slowing of disease progression, amelioration or alleviation and remission (whether partial or total) of disease state. "Treatment" can also mean prolonging survival compared to expected survival if not receiving treatment.

[0156] As used herein, the term "prophylactic treatment" refers to treatment when an individual is known to have, suspected to have, or at risk of having a disorder but does not exhibit symptoms of the disorder or exhibits minimal symptoms of the disorder. An individual receiving prophylactic treatment is treated prior to the onset of symptoms.

[0157] As used herein, a "therapeutic" agent (e.g., a therapeutic polypeptide, nucleic acid, or transgene) is one that provides a beneficial or desired clinical result, such as the exemplary clinical results described above. Thus, a therapeutic agent is used in such treatments.

[0158] The term "central retina" as used herein refers to the outer macula and / or inner macula and / or fovea. The term "central retinal cell type" as used herein refers to cell types of the central retina, such as, for example, RPE and photoreceptor cells.

[0159] The term "macula" refers to a region of the central retina in primates that contains a higher relative concentration of photoreceptor cells, specifically rods and cones, compared to the peripheral retina. The term "external macula" is also referred to herein as "peripheral macula." The term "internal macula" is also referred to herein as "central macula."

[0160] The term "fovea" refers to a small region in the central retina of primates, approximately 0.5 mm in diameter or less, that contains a high relative concentration of photoreceptor cells, specifically cones, compared to the peripheral retina and macula.

[0161] The term "subretinal space" as used herein refers to the location in the retina between photoreceptor cells and retinal pigment epithelial cells. The subretinal space may be a potential space prior to any subretinal injection of fluid, etc. The subretinal space may also contain a fluid that is injected into the potential space. In this case, the fluid is "in contact with the subretinal space." Cells "in contact with the subretinal space" include cells adjacent to the subretinal space, such as RPE and photoreceptor cells.

[0162] The term "bleb" as used herein refers to a fluid space within the subretinal space of the eye. The blebs of the present invention are created by a single injection of fluid into a single space, by multiple injections of one or more fluids into the same space, or by multiple injections into multiple spaces, which, when rearranged, create a total fluid space useful for achieving a therapeutic effect across a desired portion of the subretinal space.

[0163] Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter itself. For example, a description that refers to "about X" includes a description of "X."

[0164] As used herein, the singular articles "a," "an," and "the" include plural references unless specifically stated otherwise.

[0165] Aspects and embodiments of the invention described herein are understood to include "comprising," "consisting of," and / or "consisting essentially of" aspects and embodiments.

[0166] III. CRISPR-Cas Certain aspects of the present disclosure relate to engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) systems. These systems are used, inter alia, to treat diseases or disorders associated with deep intronic mutations in an individual's genes, such as eye diseases or disorders associated with deep intronic mutations. In some embodiments, the CRISPR-Cas system includes a first guide RNA and a second guide RNA that hybridize to opposite strands of a target DNA sequence located on either side of the deep intronic mutation, and a nucleotide sequence encoding a Cas protein, where the Cas protein cleaves the target DNA molecule at sites located on either side of the deep intronic mutation, thereby excising the portion of the target DNA containing the deep intronic mutation.

[0167] As mentioned above, the CRISPR-Cas system was originally discovered as an adaptive defense against foreign nucleic acids in bacteria and archaea. Indeed, CRISPR loci have been found in a variety of organisms, including, but not limited to, Aeropyrum, Pyrobaculum, and other species. culum), Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thernioplasnia, Corynebacterium, Mycobacterium, Streptomyces, Aquifrx, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, The genus Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myrococcus, Campylobacter, mpylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,It has been identified in over 40 species of prokaryotes, including the genera Treponema and Thermotoga (see, e.g., Jansen, R. et al. (2002) Mol. Microbiol. 43:1565-1575 and Mojica, FJ et al. (2005) J. Mol. Evol. 60:174-182).

[0168] In bacteria, Cas (e.g., Cas9) proteins bind to two distinct guide RNAs: CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). The crRNA and tracrRNA ribonucleotides base-pair to form the structure required for Cas-mediated cleavage of target DNA. However, it has recently been demonstrated that a single guide RNA (sgRNA) can be engineered to form a crRNA:tracrRNA structure and direct Cas-mediated cleavage of target DNA (Jinek, M. et al. (2012) Science 337(6096):816-21). Because the specificity of Cas nuclease activity is determined by the guide RNA, the CRISPR-Cas system has been explored as a tool to direct double-stranded DNA breaks in heterologous cells, enabling customizable genome editing (Mali, P. et al. (2013) Science 339(6121):823-6). Further description of exemplary CRISPR-Cas systems and related methods of use can be found, inter alia, in U.S. Patent No. 8,697,359. In some embodiments, a guide RNA (e.g., a first or second guide RNA) as described herein comprises a single guide RNA (sgRNA) comprising a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). In some embodiments, a guide RNA (e.g., a first or second guide RNA) as described herein is fused with a transactivating cr (tracr) sequence. In some embodiments, the transactivating cr (tracr) sequence comprises the sequence of SEQ ID NO: 25.

[0169] Therefore, the CRISPR-Cas system described herein may be contrasted with naturally occurring CRISPR-Cas systems for many reasons.For example, among others, the CRISPR-Cas system of the present disclosure may include one or more guide RNAs that hybridize with non-natural sequences (e.g., eukaryotic introns).Naturally occurring CRISPR-Cas systems recognize sequences that bacteria and archaea are usually exposed to, such as plasmid or phage sequences.In addition, while many of the CRISPR-Cas systems of the present disclosure include a single guide RNA, naturally occurring CRISPR-Cas systems usually include separate CRISPR RNAs (crRNAs) and transactivating crRNAs (tracrRNAs).

[0170] In some embodiments, the CRISPR-Cas systems described herein can be self-limiting. For example, as described below, a CRISPR-Cas system can include one or more guide RNAs that hybridize to target sequence(s) within the system itself, e.g., sequence(s) whose cleavage affects the expression levels of system components, such as Cas proteins. Without wishing to be bound by theory, it is believed that because CRISPR-Cas systems do not need to be persistently expressed in host cells, engineering the system to be "self-limiting" (e.g., characterized by persistence and / or reduced expression) can be advantageous, e.g., to reduce off-target effects, reduce the potential for unwanted immune responses and / or safety issues, etc.

[0171] In the self-limiting CRISPR-Cas system, the CRISPR-Cas complex targets one or more sites in the vector used to express one or more components of the complex itself. Thus, upon expression of guide RNA(s) and Cas protein, the CRISPR-Cas system targets the desired locus (for example, the site of mutation as described herein) and one or more targets in the Cas vector, ultimately leading to the cleavage of the Cas vector and the reduction or elimination of Cas protein expression (after cleavage at the desired locus). Example 3 below demonstrates that such a self-limiting CRISPR-Cas system is characterized by a reduced Cas duration, while still allowing effective cleavage at the desired target sequence(s) (for example, excision of deep intron mutations).

[0172] Certain aspects of the present disclosure relate to methods for treating a disease or disorder associated with a mutation in an individual's gene using a self-limiting CRISPR-Cas system. For example, the mutation can be an unwanted sequence (e.g., a deep intron mutation) that is excised from the individual's gene by the CRISPR-Cas system. In other embodiments, the mutation can be a missense, point, or other mutation that is corrected by the CRISPR-Cas system (e.g., repairing the excised DNA sequence, particularly if a homologous template is included). In some embodiments, the CRISPR-Cas system is in a composition. In some embodiments, the composition is administered to the individual in a therapeutically effective amount.

[0173] In some embodiments, the composition comprises: a) a nucleic acid encoding a CRISPR-Cas system comprising a first guide RNA and a second guide RNA that hybridize with opposite strands of a target DNA sequence flanking a mutation of interest (including, but not limited to, a deep intron mutation as described herein); and b) a Cas expression cassette. In some embodiments, the Cas expression cassette comprises a nucleotide sequence encoding a Cas protein and a guide RNA target site. The first or second guide RNA hybridizes with the guide RNA target site, thus enabling the CRISPR-Cas system to catalyze cleavage at the guide RNA target site. The guide RNA target site also The Cas protein may contain a protospacer adjacent motif (PAM) adjacent to the guide RNA-hybridizing sequence, specific for the Cas protein. Upon expression, the Cas protein cleaves the target DNA sequences flanking the mutation, thereby excising the target DNA containing the mutation. Upon expression, the Cas protein also cleaves the Cas expression cassette at the guide RNA target site, thereby reducing Cas protein expression. Thus, Cas expression initially increases after introduction of the Cas expression cassette into a cell, but as Cas protein accumulates in the cell, the Cas protein cleaves the Cas expression cassette. As more of the Cas expression cassette is blocked by the Cas protein, expression of additional Cas proteins is reduced (i.e., Cas limits the expression of its own expression cassette, considered a self-limiting Cas expression cassette). In some embodiments, Cas protein expression is characterized by an initial increase, followed by a decrease in expression after cleavage at the guide RNA target site. As described herein, reduced Cas protein expression can refer to a reduction in the amount and / or duration of the Cas protein. In some embodiments, Cas protein expression is reduced compared to before cleavage of the Cas expression cassette (e.g., compared to initial Cas expression). In some embodiments, Cas protein expression is reduced compared to use of a Cas expression cassette lacking a guide RNA target site. In some embodiments, a composition comprising a self-limiting Cas expression cassette is used to cleave a target nucleic acid. In some embodiments, the composition is used to cleave a target nucleic acid in vitro or in vivo. In some embodiments, the composition is used to cleave a target nucleic acid containing a mutation (e.g., a deep intron mutation). For example, a self-limiting Cas expression cassette is used to treat a disease or disorder associated with a mutation in a nucleic acid.

[0174] In some embodiments, the Cas expression cassette further comprises a second guide RNA target site, wherein the first guide RNA or the second guide RNA hybridizes to the second guide RNA target site, and the second guide RNA target site is flanked by a protospacer adjacent motif (PAM) specific to the Cas protein. When the Cas protein is expressed, the Cas protein cleaves the target DNA sequence flanked on both sides of the mutation, thereby excising the portion of the target DNA containing the mutation. When the Cas protein is further expressed, the Cas protein cleaves the Cas expression cassette at both guide RNA target sites, thereby reducing expression of the Cas protein. As exemplified below, one guide RNA may hybridize to two guide RNA target sites; a first guide RNA may hybridize to the first guide RNA target site and a second guide RNA may hybridize to the second guide RNA target site; or a second guide RNA may hybridize to the first guide RNA target site and the first guide RNA may hybridize to the second guide RNA site.

[0175] In some embodiments, the Cas expression cassette may include one or more promoters, enhancers, introns, polyadenylation (polyA) sequences, terminators, regulatory elements, etc. present in the 5' or 3' untranslated region useful for directing / promoting expression of the Cas protein. In some embodiments, the nucleotide sequence encoding the Cas protein is operably linked to the promoter. In some embodiments, a guide RNA target site may be between the promoter and the nucleotide sequence encoding the Cas protein, such that, upon cleavage, it results in reduced expression of the Cas protein. In some embodiments, the expression of the Cas protein is reduced by cleavage of the operably linked linkage between the promoter and the nucleotide sequence encoding the Cas protein. In some embodiments, the nucleotide sequence encoding the Cas protein is operably linked to a polyA sequence. In some embodiments, a guide RNA target site may be between the polyA sequence and the nucleotide sequence encoding the Cas protein, such that, upon cleavage, it results in reduced expression of the Cas protein. In some embodiments, the Cas protein is cleaved by cleavage of the operable linkage between the nucleotide sequence encoding the Cas protein and the polyadenylation sequence. In some embodiments, the first guide RNA target site can be between the promoter and the nucleotide sequence encoding the Cas protein, and the second guide RNA target site can be between the polyA sequence and the nucleotide sequence encoding the Cas protein, resulting in reduced expression of the Cas protein upon cleavage. In some embodiments, the Cas protein is fused in-frame with one or more NLSs, and the guide RNA target site can be between the sequence encoding one or more NLSs and the polyA sequence (particularly when the NLS is fused to the C-terminus of the Cas protein), resulting in reduced expression of the Cas protein to which the NLS is fused upon cleavage.

[0176] As described herein, a CRISPR-Cas system, such as a self-limiting CRISPR-Cas system, is encoded on one or more vectors, such as any of the vectors or viral vectors / particles described herein.In some embodiments, the nucleic acid encoding the first and second guide RNAs can be on the same vector as the Cas expression cassette.In other embodiments, the nucleic acid encoding the first and second guide RNAs can be on a vector different from the Cas expression cassette.For example, the first and second guide RNAs are encoded by a first rAAV vector, and the Cas expression cassette is encoded by a second rAAV vector.In some embodiments, target cells are transfected with both vectors, which leads to the expression of the self-limiting CRISPR-Cas system.

[0177] In some embodiments, the Cas protein cleaves the target DNA molecule at sites flanking the mutation (e.g., a deep intron mutation), thereby excising the portion of the target DNA containing the mutation. For example, a DNA repair process such as non-homologous end joining (NHEJ) can repair the cleaved DNA sequence by joining the cleaved ends, thereby excising the portion of the target DNA containing the deep intron mutation. In other examples, homologous DNA repair can repair the cleaved DNA sequence, particularly when a homologous template is involved. As described and exemplified herein, the use of two guide RNAs flanking a target DNA sequence (e.g., a sequence with a deep intron mutation) allows for the excision of a portion of the target DNA sequence, such as a sequence with a deep intron mutation (see also Brandl, C. et al. (2014) FEBS Open Bio. 5:26-35; Zheng, Q. et al. (2014) Biotechniques 57:115-124 for a description of exemplary gene deletion strategies using the CRISPR-Cas system). In some embodiments, the excised portion of the target DNA sequence comprises intron DNA. In some embodiments, the excised portion of the target DNA sequence consists solely of intron DNA.

[0178] In some embodiments, the first and / or second guide RNAs hybridize to opposite strands of the target DNA sequence located on either side of the mutation (e.g., a deep intron mutation). Without wishing to be bound by theory, it is believed that the first and / or second guide RNAs may hybridize to opposite strands of the target DNA sequence located within an intron any distance from the deep intron mutation. In some embodiments, the first and / or second guide RNAs hybridize to opposite strands of the target DNA sequence between 1 base pair and about 10,000 base pairs from the deep intron mutation. In some embodiments, the first and / or second guide RNA hybridizes to the opposite strand of the target DNA sequence located less than approximately any of the following distances (in nucleotides) from the deep intron mutation: 10,000; 9,500; 9,000; 8,500; 8,000; 7,500; 7,000; 6,500; 6,000; 5,500; 5,000; 4 ,500;4,000;3,500;3,000;2,500;2,000;1,500;1,000;950;900;850;800;750;700;650;600;550;500;450;400;350;300;250;200;150;100;95;90;85;80;75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4;3;2; or any value in between. In some embodiments, the first and / or second guide RNA hybridizes to the opposite strand of the target DNA sequence located more than approximately any of the following distances (in nucleotides) from the deep intron mutation: 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 15; 20; 25; 30; 35; 40; 45; 50; 55; 60; 65; 70; 75; 80; 85; 90; 95; 100; 150; 2 00;250;300;350;400;450;500;550;600;650;700;750;800;850;900;950;1,000;1,500;2,000;2,500;3,000;3,500;4,000;4,500;5,000;5,500;6,000;6,500;7,000;7,500;8,000;8,500;9,000;9,500; or any value in between.That is, the first and / or second guide RNAs may be: 10,000; 9,500; 9,000; 8,500; 8,000; 7,500; 7,000; 6,500; 6,000; 5,500; 5,000; 4,500; 4,000; 3,500; 3,000; 2,500; 2,000; 1,500; 1,000; 950; 900; 850; 800; 750; 700; 650; 600;550;500;450;400;350;300;250;200;150;100;95;90;85;80;75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4;3;2; or any value therebetween and an upper limit of 1;2;3;4;5;6;7;8;9;10;15;20;25;30;35; 40;45;50;55;60;65;70;75;80;85;90;95;100;150;200;250;300;350;400;450;500;550;600;650;700;750;800;850;900;950;1,000;1,500;2,000;2,500;3,000;3,500;4,000;4,500;5,000;5,500;6 The target DNA sequence may hybridize to the opposite strand of the target DNA sequence located at a distance from the deep intron mutation that can be any of a range of distances (in nucleotides) having an independently selected lower limit of 1,000; 6,500; 7,000; 7,500; 8,000; 8,500; 9,000; 9,500; or any value therebetween, wherein the lower limit is less than the upper limit.

[0179] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modifications thereof. These enzymes are generally known in the art.

[0180] In some embodiments, the Cas protein (e.g., a CRISPR enzyme) is a Cas9 protein. In some embodiments, an unmodified CRISPR enzyme, such as Cas9, has DNA cleavage activity. Exemplary Cas9 proteins include, but are not limited to, S. pyogenes Cas9 (see, e.g., SwissProt database accession number Q99ZW2), S. aureus Cas9 (see, e.g., GenBank accession number CCK74173), S. thermophilus Cas9 (see, e.g., SwissProt database accession number CCK74174), S. pyogenes Cas9 (see, e.g., SwissProt database accession number Q99ZW2), S. aureus Cas9 (see, e.g., GenBank accession number CCK74174), S. thermophilus Cas9 (see, e.g., SwissProt database ... Examples of Cas9 include N. meningitidis Cas9 (see, e.g., UniProt accession number G3ECR1), N. meningitidis Cas9 (see, e.g., UniProt accession number C9X1G5), and T. denticola Cas9 (see, e.g., GenBank accession number EMB41078). In some embodiments, the Cas9 is derived from S. pyogenes or S. pneumoniae. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of the target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0181] In some embodiments, the enzyme coding sequence encoding the Cas protein of the present disclosure is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be of or derived from a particular organism, such as a mammal, including, but not limited to, a human, mouse, rat, rabbit, dog, or non-human primate. Generally, codon optimization refers to the process of modifying a nucleic acid sequence for enhanced expression in a host cell of interest by replacing at least one codon of the native sequence with a codon more frequently or most frequently used in the host cell's genes while maintaining the native amino acid sequence. Various species exhibit particular biases for particular codons of particular amino acids. Codon usage tables are readily available, for example, in "codon usage databases," and these tables can be adapted in several ways (e.g., Nakamura, Y. et al. (2000) Nucleic Acids Res. 28:292). Computer algorithms for codon-optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.).

[0182] In some embodiments, the Cas protein is a fusion protein containing one or more heterologous protein domains (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains in addition to a CRISPR enzyme, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains). Cas fusion proteins can include any additional protein sequences and, optionally, a linker sequence between any two domains. Examples of protein domains fused to Cas proteins include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Examples of epitope tags include, but are not limited to, histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, and fluorescent proteins (e.g., GFP, CFP, YFP, BFP, etc.). Cas proteins are fused to genetic sequences encoding proteins or protein fragments that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. In some embodiments, Cas proteins are modified for enhanced function.

[0183] In some embodiments, the Cas protein is a mutant Cas protein. In some embodiments, the Cas protein (e.g., a Cas9 protein) is a nickase mutant (Ran et al., 2013 Cell 156(6):1380-9). In some embodiments, a Cas9 nickase mutant is used in conjunction with a pair of guide RNAs to introduce a targeted double-strand break and reduce off-target DNA cleavage.

[0184] In some embodiments, the Cas protein is a highly accurate Cas protein mutant with amino acid alterations that exhibit robust on-target activity but negligible off-target cleavage (Slaymaker, IM et al. (2016) Science 351(6268):84-88; Kleinstiver, BP et al. (2016) Nature 529:490-495).

[0185] In some embodiments, the CRISPR-Cas system further comprises one or more nuclear localization signals (NLSs). For example, a Cas protein (e.g., a Cas9 protein) can comprise one or more NLSs. An exemplary plasmid comprising Cas9 with an NLS can be found in Ran, FA et al. (2013) Nat. Protoc. 8:2281-2308. Various NLSs suitable for various host cells are known in the art. For example, but not limited to, the NLS can be an SV40 NLS (e.g., as described in Mali, P. et al. (2013) Science 339(6121):823-6), an SV40 large T antigen monopartite NLS, a nucleoplasmin NLS, and an hnRNP A1 NLS. Exemplary NLS sequences of use include, but are not limited to, PKKKRKV (SEQ ID NO: 26) or PKKKRKVEDPKKKRKVD (SEQ ID NO: 27) (see, e.g., Jinek, M. et al. (2013) eLife 2:e00471).

[0186] Formation of a CRISPR complex (comprising a guide sequence hybridized to opposite strands of a target sequence and complexed with one or more Cas proteins) typically results in cleavage of one or both strands at or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs of) the target sequence in the endogenous CRISPR system. It is contemplated that a tracr sequence, which may include all or a portion of a wild-type tracr sequence, can also form part of a CRISPR complex, such as by hybridization of all or a portion of a tracr mate sequence along at least a portion of the tracr sequence, e.g., a tracr mate sequence operably linked to a guide sequence.

[0187] Generally, the tracr mate sequence can comprise any sequence that has sufficient complementarity with the tracr sequence to promote the excision of the guide sequence flanked by the tracr mate sequences in cells containing the corresponding tracr sequence; or the formation of a CRISPR complex containing the tracr mate sequence hybridized with the tracr sequence at the target sequence; or both. In some embodiments, the tracr sequence has sufficient complementarity to hybridize with the tracr mate sequence and participate in the formation of a CRISPR complex. As described below with respect to the target sequence, it is believed that at least sufficient complementarity is required to be functional (i.e., perfect complementarity between the tracr and tracr mate sequences is not required).

[0188] Generally, the degree of complementarity refers to the optimal alignment of the tracr mate sequence and the tracr sequence along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm (e.g., as described herein) and may take into account secondary structure, such as self-complementarity, within either the tracr sequence or the tracr mate sequence. In some embodiments, the tracr sequence is about or greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more nucleotides in length. In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned (e.g., as determined by any of the exemplary alignment methods described herein).

[0189] Without wishing to be bound by theory, it is believed that any desired target DNA sequence of interest can be targeted by an sgRNA guide sequence, with the only requirement for the target DNA sequence being the presence of a protospacer adjacent motif (PAM) adjacent to the sgRNA target sequence (Mali, P. et al. (2013) Science 339(6121):823-6). Different Cas complexes are known to have different PAM motifs. For example, Cas9 from Streptococcus pyogenes has a GG dinucleotide PAM motif. By way of further example, the PAM motif for S. aureus Cas9 is GRRT (where R is a purine (A or G)), the PAM motif for N. meningitidis Cas9 is GATT, the PAM motif for S. thermophilus Cas9 is AGAA, and the PAM motif for T. denticola Cas9 is AAAAC.

[0190] Generally, a guide sequence can be any polynucleotide sequence that has sufficient similarity to a target polynucleotide sequence to hybridize with opposite strands of the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of identity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or greater than 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more. Optimal alignment is determined using any suitable algorithm for aligning sequences; non-limiting examples include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), Maq (available at maq.sourceforge.net), and the like.

[0191] In some embodiments, the guide sequence is about any one of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length, or greater than any one of about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length. In some embodiments, the guide sequence is less than any one of about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or fewer nucleotides in length. Assays for determining the ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence are known in the art. For example, sufficient components of a CRISPR system to form a CRISPR complex containing the guide sequence to be tested can be found in, for example, U.S. Patent No. 8,239,949. CRISPR sequences are provided to host cells harboring the corresponding target sequence, such as by transfection of a vector encoding the components of the CRISPR sequence followed by evaluation of desired cleavage within the target sequence, as described in US Pat. No. 6,697,359.

[0192] In some embodiments, a guide RNA (e.g., a first guide RNA) of the present disclosure comprises the sequence of SEQ ID NO: 41 (for SpCas9), SEQ ID NO: 45 (for SaCas9), SEQ ID NO: 46 (for SaCas9), or SEQ ID NO: 47 (for SaCas9). In some embodiments, a guide RNA (e.g., a first guide RNA) of the present disclosure comprises the sequence of SEQ ID NO: 19 (for SpCas9), SEQ ID NO: 50 (for SaCas9), SEQ ID NO: 51 (for SaCas9), or SEQ ID NO: 52 (for SaCas9). In some embodiments, the guide RNA comprises one, two, three, four, or five substitutions, deletions, or insertions of the sequence of SEQ ID NO:41 (for SpCas9), SEQ ID NO:45 (for SaCas9), SEQ ID NO:46 (for SaCas9), SEQ ID NO:47 (for SaCas9), SEQ ID NO:19 (for SpCas9), SEQ ID NO:50 (for SaCas9), SEQ ID NO:51 (for SaCas9), or SEQ ID NO:52 (for SaCas9), while maintaining its function as a guide RNA for Cas cleavage of the CEP290 gene. In some embodiments, the guide RNA is a variant of the guide RNA of SEQ ID NO: 41 (for SpCas9), SEQ ID NO: 45 (for SaCas9), SEQ ID NO: 46 (for SaCas9), SEQ ID NO: 47 (for SaCas9), SEQ ID NO: 19 (for SpCas9), SEQ ID NO: 50 (for SaCas9), SEQ ID NO: 51 (for SaCas9), or SEQ ID NO: 52 (for SaCas9) that has enhanced function as a guide RNA for Cas cleavage of the CEP290 gene.

[0193] In some embodiments, a guide RNA (e.g., a second guide RNA) of the present disclosure comprises the sequence of SEQ ID NO: 42 (for SpCas9), SEQ ID NO: 43 (for SpCas9), SEQ ID NO: 44 (for SpCas9), SEQ ID NO: 48 (for SaCas9), or SEQ ID NO: 49 (for SaCas9). In some embodiments, a guide RNA (e.g., a second guide RNA) of the present disclosure comprises the sequence of SEQ ID NO: 20 (for SpCas9), SEQ ID NO: 21 (for SpCas9), SEQ ID NO: 22 (for SpCas9), SEQ ID NO: 53 (for SaCas9), or SEQ ID NO: 54 (for SaCas9). In some embodiments, a guide RNA comprises one, two, three, four, or five substitutions, deletions, or insertions of the sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, while maintaining its function as a guide RNA for Cas cleavage of the CEP290 gene. In some embodiments, the guide RNA is a variant of the guide RNA of SEQ ID NO: 42 (for SpCas9), SEQ ID NO: 43 (for SpCas9), SEQ ID NO: 44 (for SpCas9), SEQ ID NO: 48 (for SaCas9), SEQ ID NO: 49 (for SaCas9), SEQ ID NO: 20 (for SpCas9), SEQ ID NO: 21 (for SpCas9), SEQ ID NO: 22 (for SpCas9), SEQ ID NO: 53 (for SaCas9), or SEQ ID NO: 54 (for SaCas9) that has enhanced function as a guide RNA for Cas cleavage of the CEP290 gene.

[0194] In some embodiments, the tracr sequence and the tracr mate sequence are contained within a single transcript such that hybridization between the two results in a transcript with a secondary structure (e.g., a hairpin). In some embodiments, the loop-forming sequence for use in the hairpin structure is four nucleotides in length. In some embodiments, the loop-forming sequence has the sequence GAAA. However, longer or shorter loop sequences are used, as are alternative sequences. Examples of other loop-forming sequences include, but are not limited to, CAAA and AAAG. In some embodiments, the transcript or transcribed polynucleotide sequence has at least two or more hairpins, e.g., two, three, four, or five hairpins. In some embodiments, the single transcript further comprises a transcription termination sequence, e.g., a poly-T sequence, such as six T nucleotides.

[0195] IV. Deep intronic mutations Certain aspects of the present disclosure relate to deep intron mutations. As described above, deep intron mutations can result in the abnormal inclusion of intron sequences in mature (e.g., spliced) mRNA. For example, deep intron mutations can introduce splice donor sites, splice acceptor sites, or splicing enhancer sites into genes. As a result, intron sequences are included as cryptic exons. This typically results in mutated polypeptides, especially when the cryptic exons contain frameshift mutations or premature stop codons.

[0196] As mentioned above, deep intronic mutations refer to mutations outside the wild-type splice acceptor and splice donor sequences, as opposed to mutations at the endogenous splice acceptor or splice donor. Typically, deep intronic mutations occur at some distance from the endogenous splice acceptor / splice donor site.

[0197] In some embodiments, the deep intronic mutation is located at least about 100 nucleotides from the 5' splice donor site of the gene. In some embodiments, the deep intronic mutation is located at least about 100 nucleotides from the 3' splice acceptor site of the gene. In some embodiments, the deep intronic mutation is located at least about 100 nucleotides from the 5' splice donor site and at least about 100 nucleotides from the 3' splice acceptor site of the gene.In some embodiments, deep intronic mutations are located more than about 100 nucleotides, more than about 150 nucleotides, more than about 200 nucleotides, more than about 250 nucleotides, more than about 300 nucleotides, more than about 350 nucleotides, more than about 400 nucleotides, more than about 450 nucleotides, more than about 500 nucleotides, more than about 550 nucleotides, more than about 600 nucleotides, more than about 650 nucleotides, more than about 700 nucleotides, more than about 750 nucleotides, more than about 800 nucleotides, more than about 850 nucleotides, more than about 900 nucleotides, more than about 950 nucleotides, more than about 1,000 nucleotides, more than about 1,500 nucleotides, more than about 2,000 nucleotides, more than about 2,500 nucleotides, more than about 3,000 nucleotides, more than about 3,500 nucleotides, more than about 4,000 nucleotides from the endogenous splice acceptor and / or splice donor site (e.g., the 5' splice donor site and / or the 3' splice acceptor site). more than about 4,500 nucleotides, more than about 5,000 nucleotides, more than about 5,500 nucleotides, more than about 6,000 nucleotides, more than about 6,500 nucleotides, more than about 7,000 nucleotides, more than about 7,500 nucleotides, more than about 8,000 nucleotides, more than about 8,500 nucleotides, more than about 9,000 nucleotides, more than about 9,500 nucleotides, more than about 10,000 nucleotides, more than about 15,000 nucleotides, more than about 20,000 nucleotides, It occurs over about 25,000 nucleotides, over about 30,000 nucleotides, over about 35,000 nucleotides, over about 40,000 nucleotides, over about 45,000 nucleotides, over about 50,000 nucleotides, over about 55,000 nucleotides, over about 60,000 nucleotides, over about 65,000 nucleotides, over about 70,000 nucleotides, over about 75,000 nucleotides, over about 80,000 nucleotides, or over about 85,000 nucleotides.

[0198] In some embodiments, deep intronic mutations occur at a distance (in nucleotides) from the endogenous splice acceptor and / or splice donor site (e.g., the 5' splice donor or 3' splice acceptor) that is less than approximately any of the following distances: about 85,000; 80,000; 75,000; 70,000; 65,000; 60,000; 55,000; 50,000; 00;45,000;40,000;35,000;30,000;25,000;20,000;15,000;10,000;9,500;9,000;8,500;8,000;7,500;7,000;6,500;6,000;5,500;5,000;4,500;4,000;3,500;3,000;2,500;2,000;1,500;1,000;950;900;850;8 00; 750; 700; 650; 600; 550; 500; 450; 400; 350; 300; 250; 200; or 150. In some embodiments, deep intronic mutations occur at a distance (in nucleotides) from the endogenous splice acceptor and / or splice donor site that is greater than approximately any of the following distances: about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900; 950; 1,000; 1,500; 2,000; 2,500. ;3,000;3,500;4,000;4,500;5,000;5,500;6,000;6,500;7,000;7,500;8,000;8,500;9,000;9,500;10,000;15,000;20,000;25,000;30,000;35,000;40,000;45,000;50,000;55,000;60,000;65,000;70,000;75,000;or 80,000.That is, the distance from the deep intronic mutation to the endogenous splice acceptor and / or splice donor site (e.g., 5' splice donor site) is 85,000; 80,000; 75,000; 70,000; 65,000; 60,000; 55,000; 50,000; 45,000; 40,000; 35,000; 30,000; 25,000; 20,000; 15,000; 10, 000;9,500;9,000;8,500;8,000;7,500;7,000;6,500;6,000;5,500;5,000;4,500;4,000;3,500;3,000;2,500;2,000;1,500;1,000;950;900;850;800;750;700;650;600;550;500;450;400;350;300;250;200;or 1 There is an upper limit of 50 and independently selected: 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900; 950; 1,000; 1,500; 2,000; 2,500; 3,000; 3,500; 4,000; 4,500; 5,000; 5,500; 6,000; 6,500; 7,000; 7,500; 8,000; , 500; 9,000; 9,500; 10,000; 15,000; 20,000; 25,000; 30,000; 35,000; 40,000; 45,000; 50,000; 55,000; 60,000; 65,000; 70,000; 75,000; or 80,000 distance ranges (in nucleotides) having a lower limit that is less than the upper limit.

[0199] Certain aspects of the present disclosure relate to methods, kits, compositions, and viral particles that can be used to treat diseases or disorders associated with deep intron mutations in individual genes, for example.A variety of deep intron mutations are known in the art.The exemplary deep intron mutations associated with diseases are provided in Table 1 below (please note that the specific mutations, genes, and intron sizes provided in Table 1 refer to human DNA sequences as reference).

[0200] [Table 1]

[0201] [Table 2]

[0202] [Table 3]

[0203] In some embodiments, the disease or disorder associated with a deep intronic mutation is afibrinogenemia, Alport syndrome, amyotrophic lateral sclerosis, ataxia-telangiectasia, autosomal recessive polycystic kidney disease, Barth syndrome, beta thalassemia, congenital afibrinogenemia, congenital cataract, dysfacial and neuropathy syndrome, congenital glycosylation disorder type Ia, congenital glycosylation disorder type II, cystic fibrosis, dihydropteridine reductase deficiency, Fabry disease, acute myeloid leukemia, or leukemia. Familial platelet disorders with a predisposition to myeloid leukemia, Fanconi anemia, Gitelman syndrome, growth hormone insensitivity syndrome, Friedreich's ataxia, hemophilia A, hereditary megaloblastic anemia 1, Hermansky-Pudlak syndrome, homocystinuria, maple syrup urine disease, Marfan syndrome, methionine synthase deficiency, methylmalonic acidemia, mitochondrial trifunctional protein deficiency, mucopolysaccharidosis type II, multiminicore disease, muscular dystrophy, neurofibromatosis type I, Niemann-Pick disease type C, ocular albinism I In some embodiments, the deep intronic mutation is a deep intronic mutation set forth in Table 1.

[0204] In some embodiments, the disease or disorder associated with deep intron mutations is an ocular disease.As used herein, the term "ocular disease" is used in the broadest sense and can refer to a disease originating from or resulting from a pathological condition of any structure of the eye, including but not limited to the cornea, iris, lens, retina, optic nerve, aqueous humor, conjunctiva, one or more ocular muscles, sclera, vitreous body, macula, fovea, ciliary body, one or more ligaments or zonules of the lens support ligament, pupil, anterior chamber, and / or posterior chamber.Exemplary deep intron mutations associated with ocular diseases are provided in Table 2 below (please note that the specific mutations, genes, and intron sizes provided in Table 2 refer to human DNA sequences as a reference).

[0205] [Table 4]

[0206] In some embodiments, the ocular disease is Leber's congenital amaurosis, optic atrophy, retinitis pigmentosa, retinoblastoma, Stargardt disease, Usher syndrome, or X-linked retinitis pigmentosa. In some embodiments, the deep intronic mutation is a deep intronic mutation set forth in Table 2.

[0207] In some embodiments, the eye disease is Leber's congenital amaurosis. Leber's congenital amaurosis (LCA) refers to a group of eye disorders characterized by symptoms such as severe vision loss, nystagmus, retinal dysfunction, photophobia, oculo-digital signs (e.g., rubbing, poking, and pressing of the eyes), and keratoconus. LCA typically presents at birth with severe vision loss and nystagmus. Cases of LCA are typically inherited as an autosomal recessive disorder, but mutations at various loci are associated. For example, Table 3 lists some of the loci associated with types of LCA.

[0208] [Table 5]

[0209] The most frequent form of LCA is caused by mutations in CEP290, which encodes a centrosomal protein that plays a key role in the development of centrosomes and cilia (see, e.g., NCBI Gene ID No. 80184 and UniProt ID No. O15078 for exemplary human gene and protein sequences, respectively). CEP290 is essential for the formation of primary cilia on cell membranes, which play an important role in the posterior retinal photoreceptors, as well as in the kidney, brain, and many other organs of the body. CEP290 is also known as MKS4, CT87, POC3, rd16, BBS14, LCA10, JBTS5, NPHP6, SLSN6, and 3H11Ag. In some embodiments, the causative mutation is the c.2991+1655A>G mutation, which introduces a cryptic splice donor site, resulting in the inclusion of an aberrant exon with a premature stop codon. In some embodiments, the guide sequences of the first and second guide RNAs hybridize to opposite strands of the target DNA sequence located on either side of a deep intron mutation in the Centrosomal Protein 290kDa (CEP290) gene. In certain embodiments, the deep intron mutation is a c.2991+1655A>G mutation. In some embodiments, CEP290 is human CEP290 (see, for example, the human CEP290 sequence according to NCBI reference sequence NG_008417). In some embodiments, CEP290 comprises a deep intron mutation of the sequence set forth in SEQ ID NO:23.

[0210] Certain aspects of the present disclosure further relate to methods for generating in vitro models of eye diseases associated with deep intronic mutations in genes. In some embodiments, the method comprises introducing into eukaryotic cells a nucleic acid encoding a CRISPR-Cas system, the nucleic acid comprising: i) a single guide RNA directed to a target DNA sequence of an intron in the gene; ii) a nucleotide sequence encoding a Cas protein; iii) a single-stranded oligonucleotide comprising a homology-directed repair (HDR) template comprising the desired intron mutation and homology arms flanking a protospacer adjacent motif (PAM); and isolating cells containing the mutation integrated into the gene. Exemplary methods associated with this approach are illustrated in the following examples. As described above, CRISPR-Cas-mediated DNA cleavage induces N-terminal cleavage. Repair can be achieved by HEJ, which can result in, for example, the excision of the target DNA sequence.However, CRISPR-Cas-mediated DNA cleavage can also be repaired by homology-directed repair (HDR), which can result in, for example, the introduction of the sequence present in the HDR template (for example, the introduction of deep intron mutations).See, for example, Ran, FA et al. (2013) Nat.Protoc.8:2281-2308.These methods can include any feature, aspect, or element described herein for therapeutic methods, viral particles, compositions, and kits.

[0211] Homology-directed repair (HDR) templates can take various forms, such as double-stranded DNA polynucleotides or single-stranded DNA oligonucleotides (ssODNs). HDR templates can include one or more homology arms located on both sides of the desired intron mutation. These homology arms can be in sense or antisense orientation relative to the target locus. In some embodiments, one or more homology arms can be at least about 40 base pairs, at least about 50 base pairs, at least about 60 base pairs, at least about 70 base pairs, at least about 80 base pairs, at least about 90 base pairs, or at least about 100 base pairs away from the target locus.

[0212] The HDR template may also include a protospacer adjacent motif (PAM). In some embodiments, the PAM includes a mutation to prevent cleavage of the single-stranded oligonucleotide by expressed Cas proteins in cells. For example, the PAM may be mutated so that a specific expressed Cas protein does not cleave the HDR template, or may be mutated so that an identification sequence (e.g., a unique restriction site, as illustrated in the following examples) is introduced during CRISPR-Cas-mediated editing of the genomic locus.

[0213] Any suitable eukaryotic cell may be used for the in vitro model. In some embodiments, the eukaryotic cell is a mammalian or human cell. In some embodiments, the eukaryotic cell is a cell line, such as a human cell line (e.g., HeLa, A549, 293, etc.), a mammalian cell line, a vertebrate cell line, or an insect cell line (e.g., Sf9 or S2). In some embodiments, the eukaryotic cell is a retinal cell, such as a photoreceptor cell (e.g., a WERI cell).

[0214] V. Delivery method Certain aspects of the present disclosure relate to treating a disease or disorder associated with a deep intronic mutation in an individual's gene. In some aspects, the present invention provides a method of treating a disease or disorder associated with a deep intronic mutation in an individual's gene, comprising administering to the individual a therapeutically effective amount of a composition of the present disclosure, e.g., a composition comprising a nucleic acid encoding an engineered, non-naturally occurring CRISPR-Cas system of the present disclosure. In some embodiments, the nucleic acid encoding the engineered, non-naturally occurring CRISPR-Cas system can be DNA or RNA. In some embodiments, the Cas protein is delivered as a protein.

[0215] vector In some embodiments, nucleic acids encoding one or more first guide RNAs, second guide RNAs, or Cas proteins are located on the same or different vectors of the system. In some embodiments, one or more vectors driving the expression of one or more elements of the CRISPR system are introduced into cells so that expression of the elements of the CRISPR system leads to the formation of CRISPR complexes at one or more target sites. For example, the Cas enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more These or more of the above elements can be expressed from the same or different regulatory elements, and / or can be combined in a single vector, optionally with one or more additional vectors that provide any component of the CRISPR system that is not included in the first vector.The elements of the CRISPR system that are combined in vector can be arranged in any suitable direction.For example, the coding sequence of one element can be located on the same or opposite strand of the coding sequence of the second element, and / or can be oriented in the same or opposite direction.

[0216] In some embodiments, a single promoter drives expression of transcripts encoding the Cas protein and one or more guide sequences, a tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more of at least one intron, or all in a single intron). In some embodiments, the Cas protein, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter. In some embodiments, the nucleic acid encoding the first guide RNA, second guide RNA, and / or Cas protein is operably linked to one or more regulatory control elements and / or promoters.

[0217] In some embodiments, the vector is a plasmid.

[0218] In some embodiments, the first guide RNA, the second guide RNA, and the Cas protein are expressed in eukaryotic cells.In some embodiments, the first guide RNA, the second guide RNA, and the Cas protein are operably linked to one or more promoters that allow expression in eukaryotic cells.A variety of promoters that can be expressed in eukaryotic cells are known in the art.Non-limiting exemplary promoters are provided below.

[0219] In some embodiments, the first guide RNA and / or the second guide RNA is operably linked to an RNA polymerase III promoter. The RNA polymerase III promoter may comprise a full-length promoter or a fragment thereof sufficient to drive transcription by RNA polymerase III. For a more detailed description of the types, structural features, and interactions with RNA polymerase III of RNA polymerase III promoters, as well as suitable RNA polymerase III promoters, see Schramm, L. and Hernandez, N. (2002) Genes Dev. 16:2593-620. Any suitable RNA polymerase III promoter known in the art may be used, including, but not limited to, tRNA, 5S RNA, U6 snRNA, H1, 7SK, RNase P, RNA components of the signal recognition particle, and snoRNA (see, for example, Ma, H. et al. (2014) Mol. Ther. Nucleic Acids 3:e161). In some embodiments, the RNA polymerase III promoter is a U6, H1, or 7SK promoter. In some embodiments, the first guide RNA and / or the second guide RNA is operably linked to an RNA polymerase III terminator. Examples of RNA polymerase III terminators include, but are not limited to, a stretch of uridine nucleotides at least 5-6 bases in length (for more detailed information on RNA polymerase III terminators, see Marck, C., et al. (2006) Nucleic Acids Res 34(6):1816-35).

[0220] In some embodiments, the nucleic acid encoding the Cas protein is an RNA polymerase. The RNA polymerase II promoter may comprise a full-length promoter or a fragment thereof sufficient to drive transcription by RNA polymerase II. Any suitable RNA polymerase II promoter known in the art may be used, including the cytomegalovirus (CMV) immediate early promoter, the minimal promoter fragment derived from the CMV promoter (minCMV promoter), the RSV LTR, the MoMLV LTR, the phosphoglycerate kinase-1 (PGK) promoter, the simian virus 40 (SV40) promoter, and the CK6 promoter, the transthyretin promoter (TTR), the TK promoter, the tetracycline-responsive promoter (TRE), the HBV promoter, the hAAT promoter, the LSP promoter, the chimeric liver-specific promoter (LSP), the E2F promoter, the telomerase (hTERT) promoter, the cytomegalovirus enhancer / chicken beta-actin / rabbit beta-globin promoter (CAG promoter; Niwa et al., Gene, 1991, 108(2):193-9), and the elongation factor 1-alpha promoter (EF1-alpha) promoter (Kim et al., Gene, 1991, 108(2):193-9). al., Gene, 1990, 91(2):217-23 and Guo et al., Gene Ther., 1996, 3(9):802-10. In some embodiments, the promoter comprises a cytomegalovirus enhancer linked to the human beta-glucuronidase promoter or the chicken beta-actin (CBA) promoter. The promoter can be a constitutive, inducible, or repressible promoter. In some embodiments, the promoter is capable of expressing a heterologous nucleic acid in ocular cells. In some embodiments, the promoter is capable of expressing a heterologous nucleic acid in photoreceptor cells or RPE. In embodiments, the promoter is a rhodopsin kinase (RK) promoter, such as a human RK promoter. In some embodiments, the promoter is an opsin promoter, such as a human opsin promoter or a mouse opsin promoter.In some embodiments, the promoter is a rod opsin promoter, a cone opsin promoter, a beta phosphodiesterase (PDE) promoter, a retinitis pigmentosa (RP1) promoter, or an interphotoreceptor retinoid binding protein gene (IRBP) promoter.

[0221] Inducible promoters allow for the regulation of gene expression and can be regulated by externally supplied compounds, environmental factors such as temperature, or specific physiological conditions such as the presence of acute phase, a particular differentiation state of cells, or only in replicating cells. Inducible promoters and inducible systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by one skilled in the art. Examples of inducible promoters regulated by an exogenously supplied promoter include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMMV) promoter, the T7 polymerase promoter system (WO 98 / 10088), the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995), as well as the system described by Harvey et al. al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), an RU486-inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997), and Wang et al., Gene Ther., 4:432-441 (1997)), and a rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997). Still other types of inducible promoters that may be useful herein are promoters that are regulated by specific physiological conditions, such as temperature, acute phase, a particular differentiation state of a cell, or only in replicating cells.

[0222] In another embodiment, the native promoter of the transgene or a fragment thereof is used. The native promoter can be used when it is desired that the expression of the transgene mimics native expression. The native promoter can also be used when the expression of the transgene must be regulated temporally or developmentally, tissue-specifically, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, can also be used to mimic native expression.

[0223] In some embodiments, the regulatory sequence confers tissue-specific gene expression. In some cases, the tissue-specific regulatory sequence binds to a tissue-specific transcription factor that induces transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)). In some embodiments, the tissue-specific promoter is a promoter of a gene selected from neuronal nucleus (NeuN), glial fibrillary acidic protein (GFAP), adenomatous polyposis coli (APC), and ionized calcium-binding adaptor molecule 1 (Iba-1). Other suitable tissue-specific promoters will be apparent to those skilled in the art. In some embodiments, the promoter is a chicken beta-actin promoter.

[0224] The present invention contemplates the use of recombinant viral genomes to introduce one or more nucleic acid sequences (e.g., nucleotide sequences encoding guide RNAs and / or Cas proteins) for packaging into viral particles, such as those described below. The recombinant viral genome can include any elements for establishing expression of the nucleotide sequences encoding the guide RNAs and / or Cas proteins, such as promoters, ITRs, ribosome binding elements, terminators, enhancers, selectable markers, introns, polyA signals, and / or origins of replication. Exemplary viral genome elements and various viral particle delivery methods are described in more detail below.

[0225] Non-viral delivery systems Conventional non-viral gene transfer methods can also be used to introduce nucleic acid into cells or target tissues.Non-viral vector delivery systems include DNA plasmids, RNA (e.g., nucleotide sequences encoding guide RNA or Cas proteins), naked nucleic acid (e.g., DNA or RNA), and nucleic acid (e.g., DNA or RNA) complexed with a delivery system.For example, the vector can be complexed with lipids (e.g., cationic or neutral lipids), liposomes, polycations, nanoparticles, or agents that enhance the cellular uptake of nucleic acid.The vector can be complexed with agents suitable for any delivery method described herein.

[0226] Non-viral methods for delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, nanoparticles (e.g., Jin, S. et al. (2009) Methods Mol. Biol. 544:547-557), virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agents to enhance DNA uptake. Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Lipofectamine®, Transfectam™, and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those described by Felgner, WO 91 / 17424; WO 91 / 16024.

[0227] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those of skill in the art (e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52: pp. 4817-4820 (1992); see U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0228] Other compounds that can be complexed with the vectors of the present disclosure include, but are not limited to, cationic peptides (e.g., poly-L-lysine), salts (e.g., calcium phosphate), DEAE dextran, dendrimers (e.g., polyamidoamine or PAMAM), polyethylene glycol, polyethyleneimine (PEI), and conjugates thereof. For a more detailed discussion of such agents, see, e.g., Luo, D. and Saltzman, WM (2000) Nature Biotechnology 18:33-37.

[0229] In some embodiments, the nucleic acid is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation includes a pharmaceutically acceptable carrier. Such carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pages 1035-1038 and 1570-1580). In some embodiments, pharmaceutical compositions comprising the nucleic acids described herein and a pharmaceutically acceptable carrier are suitable for ocular injection. Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, etc. Saline solutions and aqueous dextrose, polyethylene glycol (PEG), and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions can further include additional ingredients, such as preservatives, buffers, isotonicity agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, thickeners, etc. The pharmaceutical compositions described herein can be packaged in single unit doses or multiple unit doses. The compositions are generally formulated as sterile, substantially isotonic solutions.

[0230] virus particles In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. vector, a recombinant adenoviral vector, a recombinant lentiviral vector, or a recombinant herpes simplex virus (HSV) vector.

[0231] rAAV particles In some embodiments, the vector is a recombinant AAV (rAAV) vector. In some embodiments, the nucleic acid encoding one or more of the first guide RNA, second guide RNA, or Cas protein is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the viral particle is a recombinant AAV particle comprising a nucleic acid comprising a transgene flanked by one or two ITRs. In some embodiments, the nucleic acid encoding one or more of the first guide RNA, second guide RNA, or Cas protein is flanked by two AAV ITRs.

[0232] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding one or two guide RNAs and / or a Cas protein of the present disclosure, and a control sequence including transcription start and stop sequences, with the components operably linked in the direction of transcription, thereby forming an expression cassette. The expression cassette is flanked by at least one functional AAV ITR sequence on the 5' and 3' ends. By "functional AAV ITR sequence," we mean that the ITR sequence functions as intended for the rescue, replication, and packaging of AAV virions. See Davidson et al., PNAS, 2000, 97(7)3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16, the entire contents of which are incorporated herein by reference. For the implementation of some embodiments of the present invention, recombinant vectors contain at least all of the AAV sequences essential for encapsidation and the physical structure for infection by rAAV. AAV ITRs for use in the vectors of the present invention need not have wild-type nucleotide sequences (e.g., as described in Kotin, Hum. Gene Ther., 1994, 5:793-801) but may be altered by nucleotide insertions, deletions, or substitutions, or may be derived from any of several AAV serotypes. More than 40 serotypes of AAV are currently known, and new serotypes and variants of existing serotypes are constantly being identified. See Gao et al., PNAS, 2002, 99(18):11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003, 77(12):6799-810.

[0233] The use of any AAV serotype is contemplated within the scope of the present invention. In some embodiments, the rAAV vector is a vector derived from an AAV serotype, including, but not limited to, AAV ITRs, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV In some embodiments, the nucleic acid in the AAV comprises an ITR of an AAV ITR, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or murine AAV. In certain embodiments, the AAV ITRs are AAV2 ITRs.

[0234] In some embodiments, the vector may include a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid may encode a green fluorescent protein. In some embodiments, the stuffer nucleic acid may comprise a promoter and one or more of the following: The nucleotide sequence may be located between one or two of the guide RNAs and / or the nucleotide sequence encoding the Cas protein of the present disclosure.

[0235] In some embodiments, the present invention provides a viral particle comprising a recombinant self-complementary genome.In some embodiments, the vector is a self-complementary vector.AAV viral particles with self-complementary genomes and methods for using self-complementary AAV genomes are described in U.S. Patent Nos. 6,596,535; 7,125,717; 7,765,583; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang Z., et al., (2003) Gene Ther 10:2105-2111, which are incorporated herein by reference in their entirety.rAAV comprising self-complementary genomes rapidly form double-stranded DNA molecules due to their partially complementary sequences (for example, the complementary coding and non-coding strands of transgenes). In some embodiments, the present invention provides AAV viral particles comprising an AAV genome, wherein the rAAV genome comprises a first heterologous polynucleotide sequence (e.g., a nucleotide sequence encoding one or two guide RNAs of the present disclosure and / or a Cas protein of the present disclosure) and a second heterologous polynucleotide sequence (e.g., a nucleotide sequence encoding the non-coding or antisense strand of one or two guide RNAs of the present disclosure and / or a Cas protein of the present disclosure), wherein the first heterologous polynucleotide sequence is capable of intrastrand base pairing with the second polynucleotide sequence along most or all of its length.

[0236] In some embodiments, the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence are linked by a sequence that promotes intrastrand base pairing, such as a hairpin DNA structure. For example, hairpin structures in siRNA molecules are known in the art. In some embodiments, the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence are linked by a mutated ITR (e.g., the right ITR). The mutated ITR comprises a deletion of the D region containing the terminal release sequence. As a result, when the AAV viral genome replicates, the rep protein does not cut the viral genome at the mutated ITR, and therefore, a recombinant viral genome that includes the following in 5' to 3' order is packaged into the viral capsid: AAV ITR, the first heterologous polynucleotide sequence containing a regulatory sequence, the mutated AAV ITR, the second heterologous polynucleotide in the opposite direction to the first heterologous polynucleotide, and the third AAV ITR.

[0237] In some embodiments, the first heterologous nucleic acid sequence and the second heterologous nucleic acid sequence are linked by a mutated ITR (e.g., the right ITR). In some embodiments, the ITR comprises the polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO: 24). The mutated ITR comprises a deletion of the D region, which includes the terminal release sequence. As a result, when the AAV viral genome replicates, the rep protein does not cleave the viral genome at the mutated ITR, and thus a recombinant viral genome comprising, in 5' to 3' order, the following is packaged into the viral capsid: the AAV ITR, the first heterologous polynucleotide sequence including the regulatory sequence, the mutated AAV ITR, the second heterologous polynucleotide in the opposite orientation to the first heterologous polynucleotide, and the third AAV ITR.

[0238] In some embodiments, the vector is encapsidated into a viral particle. In some embodiments, the viral particle is a recombinant AAV viral particle comprising a recombinant AAV vector. Different AAV serotypes can be used to optimize transduction of specific target cells or to target specific cell types within a particular target tissue (e.g., ocular tissue). rAAV particles can be composed of viral proteins and viral vectors of the same serotype or mixed serotypes. The rAAV particles may contain a rAAV serotype nucleic acid. For example, in some embodiments, the rAAV particles may contain an AAV2 capsid protein of the present invention and at least one AAV2 ITR, or may contain an AAV2 capsid protein and at least one AAV1 ITR. Any combination of AAV serotypes for the production of rAAV particles is provided herein as if each combination were expressly set forth herein. In some embodiments, the present invention provides rAAV particles comprising an AAV2 capsid of the present invention. In some embodiments, the present invention provides rAAV particles comprising an AAVrh8R capsid of the present invention.

[0239] In some embodiments, the rAAV particles are AAV1 capsids, AAV2 capsids, AAV3 capsids, AAV4 capsids, AAV5 capsids, AAV6 capsids (e.g., wild-type AAV6 capsids or mutant AAV6 capsids such as ShH10 described in U.S. Pregrant Publication No. 2012 / 0164106), AAV7 capsids, AAV8 capsids, AAVrh8 capsids, AAVrh8R capsids, or AAVrh8R capsids. capsid, AAV9 capsid (e.g., wild-type AAV9 capsid or a modified AAV9 capsid described in U.S. Pregrant Publication No. 2013 / 0323226), AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, tyrosine capsid mutant, hairpin junction capsid mutant, AAV2R471A capsid, AAV2 / 2-7m8 capsid, AAV The mutant capsid protein comprises a DJ capsid (e.g., an AAV-DJ / 8 capsid, an AAV-DJ / 9 capsid, or any other capsid described in U.S. Pregrant Publication No. 2012 / 0066783), an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1 / AAV2 chimeric capsid, a bovine AAV capsid, a murine AAV capsid, a rAAV2 / HBoV1 capsid, or an AAV capsid described in U.S. Patent No. 8,283,151 or International Application WO 2003 / 042397. In some embodiments, the mutant capsid protein maintains the ability to form an AAV capsid. In some embodiments, the rAAV particles comprise an AAV5 tyrosine mutant capsid (Zhong L. et al., (2008) Proc Natl Acad Sci USA 105(22):7827-7832). In further embodiments, the rAAV particles comprise capsid proteins of AAV serotypes of clades A-F (Gao, et al., J. Virol. 2004, 78(12):6381). In some embodiments, the rAAV particle comprises an AAV1 capsid protein or a mutant thereof. In other embodiments, the rAAV viral particle comprises an AAV2 capsid protein or a mutant thereof. In some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some embodiments, the rAAV particle comprises an AAV serotype 1 (AAV1) capsid. In some embodiments, the rAAV particle comprises an AAV serotype 2 (AAV2) capsid. In some embodiments, the recombinant AAV viral particle comprises an AAV1, AAV2, AAV8, AAVrh8R, AAV9, and / or AAVrh10 capsid. In some embodiments, the AAV1, AAV2, AAV8, AAVrh8R, AAV9, and / or AAVrh10 capsid comprises a tyrosine mutation or a heparan-binding mutation, e.g., as described below.

[0240] The capsid of AAV (e.g., AAV2, AAVrh8R, etc.) is known to contain three capsid proteins: VP1, VP2, and VP3. These proteins contain a significant amount of overlapping amino acid sequence and unique N-terminal sequences. The AAV2 capsid contains 60 subunits arranged in icosahedral symmetry (Xie, Q., et al. (2002) Proc. Natl. Acad. Sci. 99(16):10 405-10). VP1, VP2, and VP3 have been found to exist in a ratio of 1:1:10.

[0241] The binding between AAV2 capsid proteins and HSPGs is known to occur through electrostatic interactions between basic AAV2 capsid protein residues and negatively charged glycosaminoglycan residues (Opie, SR et al., (2003) J. Virol. 77:6995-7006; Kern, A et al., (2003) J. Virol. 77:11072-11081). Specific capsid residues involved in these interactions include R484, R487, K532, R585, and R588. Mutations at these residues have been shown to reduce AAV2 binding to HeLa cells and to heparan itself (Opie, SR et al., (2003) J. Virol. 77:6995-7006; Kern, A et al., (2003) J. Virol. 77:11072-11081; WO 2004 / 027019 A2, U.S. Patent No. 7,629,322). Furthermore, without wishing to be bound by theory, it is believed that amino acid substitutions at one or more residues corresponding to amino acid positions 484, 487, 532, 585, or 588, numbered based on the AAV2 VP1 numbering, may modulate the transduction properties of AAV capsid forms that do not bind HSPGs or may modulate the transduction properties of AAV capsid forms independently of their HSPG binding ability.

[0242] In some embodiments, the rAAV particles have mutations in the capsid protein at residues that interact with HSPG or at one or more residues corresponding to amino acids 484, 487, 532, 585, or 588 based on the AAV2 VP1 numbering system. Thus, in some embodiments, upon delivery, the heterologous nucleic acid encoded by the rAAV vector is expressed at an increased expression level compared to the expression level of the heterologous nucleic acid of an rAAV particle comprising an rAAV capsid containing a reference rAAV capsid protein (e.g., a wild-type rAAV capsid protein). In some embodiments, nucleic acid expression is increased by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, upon delivery, the rAAV particles cause reduced neuroinflammation compared to rAAV particles comprising a reference rAAV capsid protein (e.g., a wild-type rAAV capsid protein). In some embodiments, neuroinflammation is reduced by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. Suitable reference rAAV capsid proteins can include any capsid protein lacking one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans (thus, the reference capsid can contain one or more "background" substitutions that do not alter binding to HSPGs).

[0243] In some embodiments, the rAAV particle comprises: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence.

[0244] In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particles to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particles to heparan sulfate proteoglycans by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, or at least about 45%. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, or about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% (compared to binding of rAAV particles comprising wild-type capsids). 80% to approximately 100%, approximately 90% to approximately 100%, approximately 10% to approximately 90%, approximately 20% to approximately 90%, approximately 30% to approximately 90%, approximately 40% to approximately 90%, approximately 50% to approximately 90%, approximately 60% to approximately 90%, approximately 70% to approximately 90%, approximately 80% to approximately 90%, approximately 10% to approximately 80%, approximately 20% to approximately 80%, approximately 30% to approximately 80%, approximately 40% to approximately 80%, approximately 50% to approximately 80%, approximately 60% to approximately 80%, approximately 70% to approximately 80%, approximately 10% to approximately 70%, approximately 20% to approximately 70%, approximately 30% to approximately 70%, approximately 40% to approximately 70%, approximately 50% to approximately 70%, approximately 60% to approximately 70%, approximately 10% to approximately 60%, approximately 20% to approximately 60%, approximately 30% to approximately 60%, approximately 40% to approximately 60%, approximately 50% to approximately 60%, approximately 10 The binding of rAAV particles containing wild-type capsids is reduced by any of the following percentages: about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, about 10% to about 40%, about 20% to about 40%, about 30% to about 40%, about 10% to about 30%, about 20% to about 30%, or about 10% to about 20% (compared to binding of rAAV particles containing wild-type capsids). In some embodiments, one or more amino acid substitutions result in no detectable binding of rAAV particles to heparan sulfate proteoglycans compared to binding of wild-type rAAV particles. Means for measuring binding of AAV particles to HSPGs, for example, binding to heparan sulfate chromatography media or binding to cells known to express HSPGs on their surface, are known in the art.See, e.g., Opie, SR et al., (2003) J. Virol. 77:6995-7006 and Kern, A et al., (2003) J. Virol. 77:11072-11081.

[0245] In some embodiments, the rAAV particles comprise one or more amino acid substitutions in a capsid protein that reduce or eliminate binding of the rAAV particle to heparan sulfate proteoglycans, and / or the one or more amino acid substitutions are at amino acid positions 484, 487, 532, 585, or 588 based on AAV2 VP1 numbering. As used herein, "AAV2 VP1 numbering" refers to the recited capsid protein amino acids that correspond to the recited amino acids of AAV2 VP1. For example, if one or more amino acid substitutions are present at positions 347, 350, 390, 395, 448, 451, 484, 487, 527, 532, 585, and / or 588 of the numbering based on VP1 of AAV2, the one or more amino acid substitutions are in amino acids of the recited capsid protein corresponding to amino acids 347, 350, 390, 395, 448, 451, 484, 487, 527, 532, 585, and / or 588 of VP1 of AAV2. In some embodiments, the one or more amino acid substitutions are present at positions 484, 487, 532, 585, or 588 of VP1 of AAV2. In some embodiments, one or more amino acid substitutions are present at positions 484, 487, 532, 585, or 588 of AAV3 VP1 numbering based on AAV2 VP1. In some embodiments, one or more amino acid substitutions are present at positions 485, 488, 528, 533, 586, or 589 of AAVrh8R VP1 numbering. In some embodiments, one or more amino acids at positions corresponding to amino acids 585 and / or 588 (AAV2 VP1 numbering) are substituted with an arginine residue (e.g., S586 and / or T589 for AAV1 or AAV6; S586 and / or A589 for AAV9; A586 and / or T589 for AAVrh8R; A586 and / or T589 for AAV8). Q588 and / or T591; and Q588 and / or A591 for AAVrhlO). In other embodiments, one or more amino acids (e.g., arginine or lysine) at positions corresponding to amino acids 484, 487, 527, and / or 532 (numbering based on VP1 of AAV2) are substituted with a non-positively charged amino acid, such as alanine (e.g., R485, R488, K528, and / or K533 for AAV1 or AAV6; R485, R488, K528, and / or R533 for AAV9 or AAVrhlO; and R487, R490, K530, and / or R535 for AAV8 or AAVrhlO).

[0246] Other virus particles In some embodiments, the vector is a recombinant adenovirus vector. In some embodiments, the viral particle is an adenovirus particle. In some embodiments, the adenovirus particle is a recombinant adenovirus particle, such as a polynucleotide vector comprising one or two guide RNAs and / or a nucleotide sequence encoding a Cas protein of the present disclosure between two ITRs. In some embodiments, the adenovirus particle lacks or contains a defective copy of one or more E1 genes, which renders the adenovirus replication-deficient. Adenoviruses contain a linear, double-stranded DNA genome within a large (approximately 950 Å) non-enveloped icosahedral capsid. Adenoviruses have large genomes that can incorporate heterologous sequences of more than 30 kb (e.g., in place of the E1 and / or E3 regions), making them uniquely suitable for use with larger heterologous genes. They are also known to infect dividing and non-dividing cells and do not naturally integrate into the host genome (although hybrid mutants may retain this ability). In some embodiments, the adenoviral vector may be a first-generation adenoviral vector with a heterologous sequence in place of E1. In some embodiments, the adenoviral vector may be a second-generation adenoviral vector with additional mutations or deletions in E2A, E2B, and / or E4. In some embodiments, the adenoviral vector may be a third-generation or gutted adenoviral vector that lacks all viral coding genes but retains only the ITRs and packaging signal and requires a helper adenovirus in trans for replication and packaging. Adenoviral particles have been investigated for use as vectors for transient transfection of mammalian cells and as gene therapy vectors. For detailed descriptions, see Danthinne, X. and Imperiale, MJ (2000) Gene Ther. 7:1707-14 and Tatsis, N. and Ertl, HC (2004) Mol. Ther. 10:616-29.

[0247] In some embodiments, the viral particle is a recombinant adenoviral particle comprising a nucleic acid comprising a nucleotide sequence encoding one or two guide RNAs of the present disclosure and / or a Cas protein of the present disclosure. The use of any adenoviral serotype is contemplated within the scope of the present invention. In some embodiments, the recombinant adenoviral vector is a vector derived from an adenovirus serotype, including, but not limited to, adenovirus serotypes 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3. In some embodiments, the recombinant adenovirus vector is derived from a variant of adenovirus serotype 2 or adenovirus serotype 5.

[0248] In some embodiments, the vector is encapsidated in a viral particle. In some embodiments, the viral particle is a recombinant adenoviral particle that encapsidates a recombinant adenoviral vector. In some embodiments, the recombinant viral particle comprises an adenoviral particle in combination with one or more foreign viral capsid proteins. Such a combination is referred to as a pseudotyped recombinant adenoviral particle. In some embodiments, the foreign viral capsid protein used in the pseudotyped recombinant adenoviral particle is derived from a foreign virus or another adenovirus serotype. In some embodiments, the foreign viral capsid protein is derived from a virus, including, but not limited to, reovirus type 3. Examples of vector and capsid protein combinations used in pseudotyped adenoviral particles can be found in the following references (Tatsis, N. et al. (2004) Mol. Ther. 10(4):616-629, and Ahi, Y. et al. (2011) Curr. Gene Ther. 11(4):307-320). Different HSV serotypes can be used to optimize transduction of specific target cells or to target specific cell types within a particular target tissue (e.g., diseased tissue). Tissues or cells targeted by specific adenovirus serotypes include, but are not limited to, lung (e.g., HuAd3), spleen and liver (e.g., HuAd37), smooth muscle, synoviocytes, dendritic cells, cardiovascular cells, tumor cell lines (e.g., HuAd11), and dendritic cells (e.g., HuAd5, HuAd30, or HuAd35 pseudotyped with reovirus type 3). For detailed descriptions, see Ahi, Y. et al. (2011) Curr. Gene Ther. 11(4):307-320; Kay, M. et al. (2001) Nat. Med. 7(1):33-40; and Tatsis, N. et al. (2004) Mol. Ther. 10(4):616-629.In some embodiments, the recombinant adenoviral particle may comprise a capsid from an adenovirus serotype, including, but not limited to, adenovirus serotypes 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3. In some embodiments, the recombinant adenovirus particle comprises a mutant of an adenovirus serotype 2 capsid or an adenovirus serotype 5 capsid.

[0249] In some embodiments, the vector is a recombinant lentiviral vector. In some embodiments, the viral particle is a lentiviral particle. In some embodiments, the lentiviral particle is a recombinant lentiviral particle, e.g., a polynucleotide vector comprising a nucleotide sequence encoding one or two guide RNAs and / or a Cas protein of the present disclosure between two LTRs. Lentiviruses are positive-sense ssRNA retroviruses with a genome of approximately 10 kb. Lentiviruses are known to integrate into the genomes of dividing and non-dividing cells. Lentiviral particles can be produced, for example, by transfecting multiple plasmids (typically, the lentiviral genome and genes required for replication and / or packaging are separated to prevent viral replication) into a packaging cell line that packages the modified lentiviral genome into lentiviral particles. In some embodiments, lentiviral particles can refer to first-generation vectors lacking envelope proteins. In some embodiments, lentiviral particles can refer to second-generation vectors lacking all genes except the gag / pol and tat / rev regions. In some embodiments, the lentiviral particles are transducing particles that contain only the endogenous rev, gag, and pol genes and no tat gene. (See Dull, T. et al. (1998) J. Virol. 72:8463-71.) For a detailed description, see Durand, S. and Cimarelli, A. (2011) Viruses 3:132-59.

[0250] In some embodiments, the viral particle is a recombinant lentiviral particle comprising a nucleic acid comprising a nucleotide sequence encoding one or two guide RNAs and / or a Cas protein of the present disclosure. The use of any lentiviral vector is contemplated within the scope of the present invention. In some embodiments, the lentiviral vector is derived from a lentivirus, including, but not limited to, human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), visna virus (VV), and caprine arthritis-encephalitis virus (CAEV).

[0251] In some embodiments, the vector is encapsidated into a viral particle. In some embodiments, the viral particle is a recombinant lentiviral particle that encapsidates a recombinant lentiviral vector. In some embodiments, the recombinant viral particle comprises a lentiviral vector in combination with one or more foreign viral capsid proteins. Such a combination is referred to as a pseudotyped recombinant lentiviral particle. In some embodiments, the foreign viral capsid protein used in the pseudotyped recombinant lentiviral particle is derived from a foreign virus. In some embodiments, the foreign viral capsid protein used in the pseudotyped recombinant lentiviral particle is vesicular stomatitis virus glycoprotein (VSV-GP). VSV-GP interacts with a ubiquitous cellular receptor, providing broad tissue tropism to the pseudotyped recombinant lentiviral particle. In addition, VSV-GP is believed to provide greater stability to the pseudotyped recombinant lentiviral particle. In other embodiments, the foreign viral capsid protein is a virus selected from the group consisting of Chandipura virus, rabies virus, Mokola virus, lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Sindbis virus, Semliki Forest virus (SFV), Venezuelan equine encephalitis virus, Ebola virus Reston strain, Ebola virus Zaire strain, Marburg virus, Lassa virus, avian leukosis virus (ALV), Jaagsiekte sheep retrovirus (JSRV), Moloney murine leukemia virus (MLV), and the like. V), gibbon ape leukemia virus (GALV), feline endogenous retrovirus (RD114), human T-lymphotropic virus (HTLV-1), human foamy virus, Maedi-visna virus (MVV), SARS-CoV, Sendai virus, respiratory syncytial virus (RSV), human parainfluenza virus type 3, hepatitis C virus (HCV), influenza virus, avian plague virus (FPV), or silkworm nuclear polyhedrosis virus (AcMNPV).

[0252] In some embodiments, the recombinant lentiviral vector is derived from a lentivirus pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokara virus, rabies virus, RD114, or a mutant thereof. Examples of vector and capsid protein combinations used in pseudotyped lentiviral particles can be found, for example, in Cronin, J. et al. (2005). Curr. Gene Ther. 5(4):387-398. Different pseudotyped recombinant lentiviral particles can be used to optimize transduction of specific target cells or to target specific cell types within a specific target tissue (e.g., diseased tissue). For example, tissues targeted by specific pseudotyped recombinant lentiviral particles include the liver (e.g., VSV) and the thymus (vulva) of the liver (vulva). These include, but are not limited to, cells pseudotyped with VSV-G, LCMV, RRV, or SeV F proteins), lung (e.g., pseudotyped with Ebola, Marburg, SeV F and HN, or JSRV proteins), pancreatic islet cells (e.g., pseudotyped with LCMV proteins), central nervous system (e.g., pseudotyped with VSV-G, LCMV, rabies, or Mokara proteins), retina (e.g., pseudotyped with VSV-G or Mokara proteins), monocytes or muscle (e.g., pseudotyped with Mokara or Ebola proteins), hematopoietic system (e.g., pseudotyped with RD114 or GALV proteins), or cancer cells (e.g., pseudotyped with GALV or LCMV proteins). For a detailed description, see Cronin, J. et al. (2005). Curr. Gene Ther. 5(4):387-398 and Kay, M. et al. (2001) Nat. Med. 7(1):33-40. In some embodiments, the recombinant lentiviral particle comprises a capsid pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokara virus, rabies virus, RD114, or a mutant thereof.

[0253] In some embodiments, the vector is an rHSV vector. In some embodiments, the viral particle is a herpes simplex virus (HSV) particle. In some embodiments, the HSV particle is an rHSV particle, e.g., a polynucleotide vector comprising a nucleotide sequence encoding one or two guide RNAs and / or a Cas protein of the present disclosure between two TRs. HSV is an enveloped, double-stranded DNA virus with a genome of approximately 152 kb. Advantageously, approximately half of its genes are nonessential and may be deleted to accommodate heterologous sequences. HSV particles infect non-dividing cells. In addition, they naturally reside latent in neurons, migrate via retrograde transport, and are transported across synapses, making them useful for neuronal transfection and / or gene therapy approaches involving the nervous system. In some embodiments, HSV particles can be replication-deficient or replication-competent (e.g., competent for a single replication cycle through inactivation of one or more late genes). For a detailed description, see Manservigi, R. et al. (2010) Open Virol. J. 4:123-56.

[0254] In some embodiments, the viral particle is a rHSV particle comprising a nucleic acid comprising a nucleotide sequence encoding one or two guide RNAs and / or a Cas protein of the present disclosure. The use of any HSV vector is considered within the scope of the present invention. In some embodiments, the HSV vector is derived from an HSV serotype, including, but not limited to, HSV-1 and HSV-2.

[0255] In some embodiments, the vector is encapsidated into a viral particle. In some embodiments, the viral particle is a recombinant HSV particle that encapsidates a recombinant HSV vector. In some embodiments, the recombinant viral particle comprises an HSV vector combined with one or more foreign viral capsid proteins. Such combinations are referred to as pseudotyped rHSV particles. In some embodiments, the foreign viral capsid protein used in the pseudotyped rHSV particle is derived from a foreign virus or another HSV serotype. In some embodiments, the foreign viral capsid protein used in the pseudotyped rHSV serotype is vesicular stomatitis virus glycoprotein (VSV-GP). VSV-GP interacts with a ubiquitous cellular receptor, providing broad tissue tropism to the pseudotyped rHSV particle. In some embodiments, VSV-GP is believed to provide greater stability to the pseudotyped rHSV particle. In other embodiments, the foreign viral capsid protein can be derived from a different HSV serotype. For example, an HSV-1 vector can be derived from one or more foreign viral capsid proteins. The recombinant HSV particles may comprise one or more HSV-2 capsid proteins. Different HSV serotypes can be used to optimize transduction of specific target cells or to target specific cell types within a particular target tissue (e.g., diseased tissue). Tissues or cells targeted by specific adenovirus serotypes include, but are not limited to, the central nervous system and neurons (e.g., HSV-1). For further details, see Manservigi, R. et al. (2010) Open Virol J 4:123-156; Kay, M. et al. (2001) Nat. Med. 7(1):33-40; and Meignier, B. et al. (1987) J. Infect. Dis. 155(5):921-930. In some embodiments, the recombinant HSV particles are rHSV-1 or rHSV-2 viral particles.

[0256] Production of viral particles Numerous methods are known in the art for producing adenoviral vector particles.For example, for gutted adenoviral vectors, adenoviral vector genome and helper adenoviral genome can be transfected into packaging cell lines (e.g., 293 cell lines).In some embodiments, the helper adenoviral genome contains recombination sites located on both ends of its packaging signal, and both genomes can be transfected into packaging cell lines that express recombinase (e.g., Cre / loxP system can be used) so that the desired adenoviral vector can be packaged more efficiently than the helper adenovirus (see, for example, Alba, R. et al. (2005) Gene Ther. 12 Suppl 1:S18-27).Adenoviral vectors can be recovered and purified using standard methods, such as those described herein.

[0257] For the production of lentiviral vector particles, many methods are known in the art.For example, for the third generation lentiviral vector, the vector comprising the target lentiviral genome with gag and pol gene can be co-transfected with the vector comprising rev gene into packaging cell line (for example, 293 cell line).The target lentiviral genome also comprises chimeric LTR that promotes transcription in the absence of Tat (see Dull, T. et al. (1998) J.Virol.72:8463-71).Lentiviral vector can be recovered and used by the method described herein (for example, Segura MM, et al., (2013) Expert Opin Biol Ther.13(7):987-1011).

[0258] For the production of HSV particles, many methods are known in the art. HSV vectors can be recovered and purified using standard methods, such as the methods described herein. For example, for replication-defective HSV vectors, the target HSV genome, which lacks all immediate early (IE) genes, can be transfected into a complementing cell line that provides genes necessary for virus production, such as ICP4, ICP27, and ICP0 (see, for example, Samaniego, LA et al. (1998) J. Virol. 72:3307-20). HSV vectors can be recovered and purified using the methods described (see, for example, Goins, WF et al., (2014) Herpes Simplex Virus Methods in Molecular Biology 1144:63~79).

[0259] For the production of rAAV vectors, transfection, stable cell line production, and the production of infectious hybrids including adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, JE et al., (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids have been used. Numerous methods, including lid virus production systems, are known in the art. rAAV production cultures for producing rAAV viral particles all require 1) suitable host cells, 2) suitable helper virus functions, 3) AAV rep and cap genes and gene products, 4) nucleic acid (e.g., therapeutic nucleic acid) flanked by at least one AAV ITR sequence (e.g., an oversized rAAV vector genome), and 5) suitable media and media components to support rAAV production. In some embodiments, suitable host cells are primate host cells. In some embodiments, suitable host cells are human-derived cell lines such as HeLa, A549, 293, or Perc.6 cells. In some embodiments, suitable helper virus functions are provided by wild-type or mutant adenovirus (such as a temperature-sensitive adenovirus), herpesvirus (HSV), baculovirus, or a plasmid construct providing helper functions. In some embodiments, the AAV rep and cap gene products can be derived from any AAV serotype. Typically, but not necessarily, the AAV rep gene product is of the same serotype as the ITRs of the rAAV vector genome, so long as the rep gene product is capable of replicating and packaging the rAAV genome. Suitable media known in the art may be used for the production of rAAV vectors. These media include modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), Hyclone These include, but are not limited to, media manufactured by JRH Laboratories and JRH, custom formulations such as those described in U.S. Patent No. 6,566,118, and Sf-900 II SFM medium described in U.S. Patent No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom media formulations for use in producing recombinant AAV vectors. In some embodiments, AAV helper functions are provided by adenovirus or HSV. In some embodiments, AAV helper functions are provided by baculovirus, and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cell). Examples of adenovirus helper functions for AAV replication These include E1A, E1B, E2A, VA, and E4orf6 functions. Baculoviruses available from depositories include Bombyx mori nuclear polyhedrosis virus.

[0260] rAAV particles can be produced using methods known in the art.See, for example, U.S. Patent Nos. 6,566,118; 6,989,264; and 6,995,006.In the practice of the present invention, the host cells for producing rAAV particles include mammalian cells, insect cells, plant cells, microorganisms, and yeast.Host cells can also be packaging cells in which AAV rep and cap genes are stably maintained in host cells, or producer cells in which AAV vector genomes are stably maintained.Exemplary packaging and producer cells are derived from 293, A549, or HeLa cells.AAV vectors are purified and formulated using standard techniques known in the art.

[0261] In some embodiments, rAAV particles can be produced by a triple transfection method, such as the exemplary triple transfection method provided below. Briefly, a plasmid containing the rep and capsid genes, along with a helper adenovirus plasmid, is transfected into a cell line (e.g., HEK293 cells) (e.g., using calcium phosphate), and the virus can be harvested and optionally purified.

[0262] In some embodiments, rAAV particles may be produced by producer cell line methods (see, e.g., Martin et al., (2013) Human Gene Therapy Methods 24:253-269). In some embodiments, a cell line (e.g., a HeLa cell line) may be stably transfected with a plasmid containing the rep gene, capsid gene, and promoter-transgene sequence. The cell line may be screened to select a lead clone for rAAV production, which may then be grown in a production bioreactor and infected with adenovirus (e.g., wild-type adenovirus) as a helper to initiate rAAV production. The virus may then be harvested, the adenovirus inactivated (e.g., by heat) and / or removed, and the rAAV particles purified. In some embodiments, the producer cell line is derived from HeLa, 293, A549, or Perc.6 cells. In some embodiments, the producer cell line is adapted for growth in suspension culture. In some embodiments, AAV helper functions are provided by adenovirus, HSV, or baculovirus.

[0263] In some embodiments, the rAAV particles are recovered between about 48 hours and about 96 hours after the provision of helper functions. For example, in some embodiments, the rAAV particles are recovered about 48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours after the provision of helper functions. In some embodiments, the rAAV particles are recovered about 48 hours and about 96 hours, about 48 hours and about 84 hours, about 48 hours and about 72 hours, about 48 hours and about 60 hours, about 60 hours and about 96 hours, about 60 hours and about 84 hours, about 60 hours and about 72 hours, about 72 hours and about 96 hours, about 72 hours and about 84 hours, or about 84 hours and about 96 hours after the provision of helper functions.

[0264] Suitable rAAV production culture media of the present invention may be supplemented with serum or serum-derived recombinant proteins at levels of 0.5% to 20% (v / v or w / v). Alternatively, as is known in the art, rAAV vectors may be produced in serum-free conditions, also referred to as animal-derived product-free media. Those skilled in the art will recognize that to increase the titer of rAAV in production cultures, commercially available or custom media designed to support rAAV vector production may be supplemented with one or more cell culture components known in the art, including, but not limited to, glucose, vitamins, amino acids, and / or growth factors.

[0265] rAAV production cultures can be grown under a variety of conditions (wide temperature ranges, various time periods, etc.) suitable for the particular host cells utilized. As is known in the art, rAAV production cultures include attachment-dependent cultures, which can be cultured in suitable attachment-dependent vessels such as roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV vector production cultures can also include host cells adapted to suspension culture, such as HeLa, 293, and SF-9 cells, which can be cultured in a variety of ways, including spinner flasks, stirred-tank bioreactors, and disposable systems such as the Wave bag system.

[0266] The rAAV vector particles of the invention can be recovered from the rAAV production culture by lysis of the host cells of the production culture or by recovering spent medium from the production culture, provided that the cells are cultured under conditions known in the art to release the rAAV particles into the medium from intact cells, as described in more detail in U.S. Patent No. 6,566,118. Suitable methods for lysing cells are also known in the art and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0267] In a further embodiment, the viral particles are purified. As used herein, the term "purified" refers to a viral particle that is purified, similar to the location where the viral particle is naturally found or originally prepared from. The term "isolated viral particles" includes the preparation of viral particles free from at least some of the other components that may be present in the original mixture. Thus, for example, isolated viral particles can be prepared using purification techniques to enrich them from a source mixture such as a culture lysate or production culture supernatant. Enrichment can be measured in a variety of ways, such as the percentage of DNase-resistant particles (DRPs) or genome copies (gc) present in solution, or the degree of infectivity, or can be measured relative to a second, possibly interfering substance present in the original mixture, such as a production culture contaminant including a helper virus, media components, etc., or a processing contaminant.

[0268] In some embodiments, the viral production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is filtered through filters such as Grade DOHC Millipore Millistak+HC Pod filters, Grade A1HC Millipore Millistak+HC Pod filters, and 0.2 μm filters such as Opticap XL1O Millipore Express Clarification is achieved by filtration through a series of depth filters, including SHC hydrophilic membrane filters. Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate filter with a pore size of 0.2 μm or greater known in the art.

[0269] In some embodiments, the viral production culture harvest is further treated with Benzonase® to digest any high molecular weight DNA present in the production culture. In some embodiments, Benzonase® digestion is carried out under standard conditions known in the art, including, for example, a final concentration of 1-2.5 units / ml of Benzonase® at a temperature ranging from ambient to 37°C for a period of 30 minutes to several hours.

[0270] rAAV particles can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) to concentrate rAAV particles; rAAV capture by apatite chromatography; heat inactivation of helper virus; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps may be used alone, in various combinations, or in different orders. Methods for purifying rAAV particles can be found, for example, in Xiao et al. (1998) Journal of Virology 72:2224-2232; U.S. Patent Nos. 6,989,264 and 8,137,948; and International Publication No. WO 2010 / 148143. Methods for purifying adenoviral particles can be found, for example, in Bo, H et al., (2014) Eur. J. Pharm. Sci. 67C: 119-125. Methods for purifying lentiviral particles can be found, for example, in Segura MM, et al., (2013) Expert Opin. Biol Ther. 13(7):987-1011. Methods for purifying HSV particles can be found, for example, in Goins, WF et al., (2014) Herpes Simplex Virus Methods in Molecular Biology 1144:63-79.

[0271] In some embodiments, the viral particles are in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises a pharmaceutically acceptable carrier. Such carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pages 1035-1038 and 1570-1580). In some embodiments, the viral particles described herein are Pharmaceutical compositions comprising rux particles and a pharmaceutically acceptable carrier are suitable for ocular injection. Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, etc. Saline solutions and aqueous dextrose, polyethylene glycol (PEG), and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions can further contain additional ingredients, such as preservatives, buffers, isotonicity agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, thickeners, etc. The pharmaceutical compositions described herein can be packaged in single unit doses or multiple unit doses. The compositions are generally formulated as sterile, substantially isotonic solutions.

[0272] VI. Treatment method Certain aspects of the present disclosure include administering to an individual a therapeutically effective amount of a composition comprising a nucleic acid encoding an engineered, non-naturally occurring CRISPR-Cas system of the present disclosure, e.g., as described above.

[0273] In some embodiments, aspects of the present disclosure involve administering to an individual a therapeutically effective amount of a composition comprising a Cas protein of the present disclosure and one or more nucleic acids, including a first guide RNA and a second guide RNA, where the first guide RNA and the second guide RNA hybridize to opposite strands of a target DNA sequence flanking a mutation, including, for example, the deep intron mutations described above. That is, with respect to any of the methods, compositions, and kits of the present disclosure, the Cas protein can be provided as a nucleotide sequence encoding the Cas protein or as a polypeptide. As a non-limiting example, the Cas protein can be administered with one or more guide RNAs (e.g., sgRNAs) using cationic lipid-mediated delivery (see, e.g., Zuris, JA et al. Nat Biotechnol. 33:73-80). In some embodiments, the Cas protein can be administered in a complex with one or more guide RNAs, e.g., as a CRISPR-Cas effector complex. It is recognized that any of the delivery and / or administration methods described below may be used for delivery of a Cas protein administered with one or more guide RNAs. In some embodiments, the Cas protein is expressed from a self-limiting expression cassette as described above.

[0274] In some embodiments, a composition comprising a nucleic acid encoding an engineered, non-naturally occurring CRISPR-Cas system of the present disclosure (or a composition comprising a Cas protein of the present disclosure and one or more guide RNAs of the present disclosure) is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intracerebroventricularly, or intranasally.

[0275] In some embodiments, a composition comprising a nucleic acid encoding the engineered, non-naturally occurring CRISPR-Cas system of the present disclosure (or a composition comprising a Cas protein of the present disclosure and one or more guide RNAs of the present disclosure) is administered subretinally or intravitreally. Gene therapy protocols for retinal diseases, such as eye diseases associated with deep intronic mutations, require local delivery of nucleic acids to cells of the retina. The therapeutic target cells in these diseases are either photoreceptor cells of the retina or cells of the RPE underlying the neurosensory retina. Delivery of nucleic acids to these cells requires injection into the subretinal space between the retina and the RPE.

[0276] In some embodiments, the present invention provides compositions comprising any of the nucleic acids described herein, optionally in a pharmaceutically acceptable excipient. As is well known in the art, a pharmaceutically acceptable excipient is a relatively inert excipient that facilitates administration of a pharmacologically active substance. Pharmaceutically acceptable excipients can be supplied as liquid solutions or suspensions, emulsions, or solid dosage forms suitable for dissolution or suspension in liquid before use. For example, excipients can provide form or consistency or act as diluents. Suitable excipients include, but are not limited to, stabilizers, wetting agents and emulsifiers, salts for varying osmotic pressure, encapsulating agents, pH buffering substances, and buffers. Such excipients include any agent suitable for direct delivery to the eye that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various TWEEN compounds, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable excipients can include pharmaceutically acceptable salts, such as inorganic salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and organic salts such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991).

[0277] Generally, these compositions are formulated to be administered by subretinal injection. Thus, these compositions can be combined with a pharmaceutically acceptable vehicle such as saline, Ringer's buffered saline (pH 7.4), etc. Although not required, the compositions can optionally be supplied in unit dosage forms suitable for administering precise amounts.

[0278] Subretinal delivery method Subretinal delivery method is known in the art.For example, see International Publication No. 2009 / 105690, which is incorporated herein by reference.In brief, the general method of delivering composition (for example, the nucleic acid encoding the genetically engineered, non-naturally occurring CRISPR-Cas system of the present disclosure, which can be delivered through the above-mentioned viral or non-viral delivery) to the subretinal area of ​​macula and fovea is illustrated by the following brief summary.This example is merely intended to illustrate certain features of the method, and is not intended to be limiting in any way.

[0279] Generally, the vector can be delivered in the form of a composition that is injected intraocularly (subretinal) under direct observation using a surgical microscope. This technique can involve, after vitrectomy, injection of the vector suspension into the subretinal space through one or more small retinal incisions using a thin cannula.

[0280] Briefly, an infusion cannula can be placed and sutured to maintain normal ocular volume through infusion (e.g., of saline solution) throughout the procedure. Vitrectomy is performed using a cannula of appropriate bore size (e.g., 20-27 gauge), and the volume of removed vitreous gel is replaced by infusing saline or other isotonic solution through the infusion cannula. Vitrectomy is advantageous because (1) removal of the cortex (posterior hyaloid membrane) facilitates penetration of the retina by the cannula; (2) its removal and replacement with fluid (e.g., saline) creates a space suitable for intraocular injection of vector; and (3) its controlled removal reduces the likelihood of retinal tears and unplanned retinal detachment.

[0281] In some embodiments, the vector composition is injected directly into the subretinal space outside the central retina by utilizing a cannula of appropriate bore size (e.g., 27-45 gauge), thus creating a bleb in the subretinal space. In other embodiments, prior to the subretinal injection of the vector composition, a small amount (e.g., about 0.1 to about 0.5 ml) of an appropriate fluid (such as saline or Ringer's solution) is subretinal injected into the subretinal space outside the central retina. This initial injection into the subretinal space establishes an initial fluid bleb within the subretinal space, and localized bleb formation occurs at the location of the initial bleb. This initial fluid bleb facilitates targeted delivery of the vector composition to the subretinal space (by defining the injection plane prior to vector delivery) and can minimize potential vector administration to the choroid and the possibility of injection or reflux into the vitreous cavity. In some embodiments, this initial fluid bleb can be further injected with fluids containing one or more vector compositions and / or one or more additional therapeutic agents by administering these fluids directly into the initial fluid bleb through either the same or additional fine bore cannulas.

[0282] Intraocular administration of the vector composition and / or initial small volume of fluid can be performed using a microbore cannula (e.g., 27-45 gauge) attached to a syringe. In some embodiments, the syringe plunger can be driven by a mechanical device, such as by pressing a foot pedal. The microbore cannula is advanced through the sclerectomy, beyond the vitreous cavity, to a predetermined retinal site in each subject according to the target retinal region (but outside the central retina). Under direct visualization, the vector suspension is mechanically injected beneath the neurosensory epithelium, causing a localized retinal detachment via a self-sealing, non-expanding retinectomy. As described above, the vector composition can be injected directly into the subretinal space, creating a bleb outside the central retina, or the vector can be injected into an initial bleb outside the central retina, causing it to expand (and the area of ​​retinal detachment). In some embodiments, injection of the vector composition is followed by another injection of fluid into the bleb.

[0283] Without wishing to be bound by theory, the speed and location of subretinal injection can result in local shear forces that can cause damage to the macula, fovea, and / or underlying RPE cells. Subretinal injection can be performed at a speed that minimizes or avoids shear forces. In some embodiments, the vector composition is injected over a period of about 15-17 minutes. In some embodiments, the vector is injected over a period of about 17-20 minutes. In some embodiments, the vector composition is injected over a period of about 20-22 minutes. In some embodiments, the vector composition is injected at a rate of about 35 to about 65 μl / min. In some embodiments, the vector composition is injected at a rate of about 35 μl / min. In some embodiments, the vector composition is injected at a rate of about 40 μl / min. In some embodiments, the vector composition is injected at a rate of about 45 μl / min. In some embodiments, the vector composition is injected at a rate of about 50 μl / min. In some embodiments, the vector composition is injected at a rate of about 55 μl / min. In some embodiments, the vector composition is injected at a rate of about 60 μl / min. In some embodiments, the vector composition is injected at a rate of about 65 μl / min. One skilled in the art will recognize that the injection rate and time of the bleb will be dictated by, for example, the volume of vector composition or size of the bleb necessary to create a sufficient retinal detachment to access cells of the central retina, the size of the cannula used to deliver the vector composition, and the ability to safely maintain the position of the cannula of the present invention.

[0284] In some embodiments of the invention, the volume of the composition injected into the subretinal space of the retina is greater than any one of about 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 15 μl, 20 μl, 25 μl, 50 μl, 75 μl, 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, or 1 mL, or any amount in between.

[0285] In some embodiments, the method comprises administering to the eye (e.g., by subretinal and / or intravitreal administration) an effective amount of recombinant viral particles comprising a vector of the present disclosure. In some embodiments, the viral titer of the composition is about 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 10×10 12 , 11×10 12 , 15×10 12 , 20×10 12 , 25×10 12 , 30×10 12 , or 50 × 10 12 Ge In some embodiments, the viral titer of the composition is about 5 x 10 12 ~6×10 12 , 6×10 12~ 7×10 12 , 7×10 12 ~8×10 12 , 8×10 12 ~9×10 12 , 9×10 12 ~10×10 12 , 10×10 12 ~11×10 12 , 11×10 12 ~15×10 12 , 15×10 12 ~20×10 12 , 20×10 12 ~25×10 12 , 25×10 12 ~30×10 12 , 30×10 12 ~50×10 12 , or 50 × 10 12 ~100×10 12 In some embodiments, the viral titer of the composition is about 5 x 10 genome copies / mL. 12 ~10×10 12 , 10×10 12 ~25×10 12 , or 25 × 10 12 ~50×10 12In some embodiments, the viral titer of the composition is about 5 x 10 genome copies / mL. 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 10×10 9 , 11×10 9 , 15×10 9 , 20×10 9 , 25×10 9 , 30×10 9 , or 50 × 10 9 In some embodiments, the viral titer of the composition is about 5 x 10 transducing units / mL or at least one of: 9 ~6×10 9 , 6×10 9 ~7×10 9 , 7×10 9 ~8×10 9 , 8×10 9 ~9×10 9 , 9×10 9 ~10×10 9 , 10×10 9 ~11×10 9 , 11×10 9 ~15×10 9 , 15×10 9 ~20×10 9 , 20×10 9 ~25×10 9 , 25×10 9 ~30×10 9 , 30×10 9 ~50×10 9 , or 50 × 10 9 ~100×10 9 In some embodiments, the viral titer of the composition is about 5 x 10 transducing units / mL. 9 ~10×10 9 , 10×10 9 ~15×10 9 , 15×10 9 ~25×10 9 , or 25 × 10 9 ~50×10 9In some embodiments, the viral titer of the composition is about 5 x 10 transducing units / mL. 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 10×10 10 , 11×10 10 , 15×10 10 , 20×10 10 , 25×10 10 , 30×10 10 , 40×10 10 , or 50 × 10 10 In some embodiments, the viral titer of the composition is about 5 x 10 infectious units / mL or at least one of: 10 ~6×10 10 , 6×10 10 ~7×10 10 , 7×10 10 ~8×10 10 , 8×10 10 ~9×10 10 , 9×10 10 ~10×10 10 , 10×10 10 ~11×10 10 , 11×10 10 ~15×10 10 , 15×10 10 ~20×10 10 , 20×10 10 ~25×10 10 , 25×10 10 ~30×10 10 , 30×10 10 ~40×10 10 , 40×10 10 ~50×10 10 , or 50 × 10 10 ~100×10 10 In some embodiments, the viral titer of the composition is about 5 x 10 infectious units / mL or at least one of: 10 ~10×10 10 , 10×10 10 ~15×10 10 , 15×10 10 ~25×10 10 , or 25 × 1010 ~50×10 10 infectious units / mL.

[0286] In some embodiments, the method includes administering to the eye (e.g., subretinal and / or intravitreal administration) of an individual (e.g., a human) an effective amount of recombinant viral particles comprising a vector of the present disclosure. In some embodiments, the dose of viral particles administered to the individual is about 1 x 10 8 ~Approx. 1×10 13 In some embodiments, the dose of viral particles administered to an individual is about 1 x 10 genome copies / kg body weight or about 1 x 10 8 ~Approx. 1×10 13 genome copies / kg body weight.

[0287] One or more (e.g., two, three, or more) blebs can be created. Generally, the total volume of the bleb or blebs created by the methods and systems of the present invention should not exceed the fluid volume of the eye, e.g., about 4 ml in a typical human subject. The total volume of each individual bleb can be at least about 0.3 ml, or at least about 0.5 ml, to promote retinal detachment of sufficient size to expose central retinal cell types and to create a bleb that is sufficiently dependent for optimal manipulation. Those skilled in the art will recognize that appropriate intraocular pressure must be maintained during bleb creation according to the methods and systems of the present invention to avoid damage to ocular structures. The size of each individual bleb can be, for example, about 0.5 to about 1.2 ml, about 0.8 to about 1.2 ml, or about 0.5 to about 1.2 ml. The volume may be about 0.9 to about 1.2 ml, about 0.9 to about 1.0 ml, about 1.0 to about 2.0 ml, or about 1.0 to about 3.0 ml. Thus, in one example, three blebs of about 1 ml each can be established to inject a total of 3 ml of vector composition suspension. The total volume of all combined blebs may be, for example, about 0.5 to about 3.0 ml, about 0.8 to about 3.0 ml, about 0.9 to about 3.0 ml, about 1.0 to about 3.0 ml, about 0.5 to about 1.5 ml, about 0.5 to about 1.2 ml, about 0.9 to about 3.0 ml, about 0.9 to about 2.0 ml, or about 0.9 to about 1.0 ml.

[0288] To safely and efficiently transduce a region of the target retina (e.g., the central retina) outside the edge of the bleb's original location, the bleb may be manipulated to reposition the bleb to the target region for transduction. Bleb manipulation can be achieved by the dependency of the bleb created by the bleb's volume, repositioning the eye containing the bleb, repositioning the eye containing one or more blebs or the head of a human with multiple eyes, and / or fluid-air exchange. This is particularly relevant to the central retina, as this region typically tolerates detachment from subretinal injections. In some embodiments, fluid-air exchange is utilized to reposition the bleb by temporarily exchanging fluid from the infusion cannula for air, for example, by flowing air over the surface of the retina. Fluid in the vitreous cavity can flow out of the cannula as the air volume displaces the vitreous cavity fluid from the surface of the retina. The temporary lack of pressure from the vitreous cavity fluid causes the bleb to move and be drawn to the dependent portion of the eye. By properly positioning the eye, the bleb of subretinal vector composition is manipulated to engulf adjacent areas (e.g., the macula and / or fovea). In some cases, the mass of the bleb is sufficient to attract it even without the use of fluid-air exchange. Movement of the bleb to the desired location can be further facilitated by changing the subject's head position to attract the bleb to the desired location within the eye. Once the desired shape of the bleb is achieved, the fluid is returned to the vitreous cavity. The fluid is an appropriate fluid, such as fresh saline. Generally, the subretinal vector composition will remain in place without retinal reattachment following retinal resection and without intraocular tamponade, and the retina will spontaneously reattach within approximately 48 hours.

[0289] By safely and effectively transducing ocular cells (e.g., RPE and / or photoreceptor cells of the macula and / or fovea) with the vectors of the present disclosure, the methods of the present invention can be used to treat individuals, e.g., humans, with ocular disorders associated with deep intronic mutations, and the transduced cells produce sufficient quantities of the CRISPR-Cas system to treat the ocular disorder.

[0290] An effective amount of vector (in some embodiments, in the form of viral particles) is administered depending on the goal of treatment. For example, if a low percentage of transduction can achieve the desired therapeutic effect, the goal of treatment is generally to meet or exceed this level of transduction. In some instances, this level of transduction can be achieved by transduction of only about 1-5% of target cells, and in some embodiments, at least about 20%, in some embodiments, at least about 50%, in some embodiments, at least about 80%, in some embodiments, at least about 95%, and in some embodiments, at least about 99% of cells of the desired tissue type. As a guide, the number of viral particles administered per injection is generally 1x10 6 ~approx. 1x10 14 between particles, approximately 1x10 7 ~1x10 13 between particles, approximately 1x10 9 ~1x10 12 particles, or about 1x10 11 The vector composition may be administered by one or more subretinal injections during the same procedure or spaced apart by days, weeks, months, or years. In some embodiments, multiple vectors may be used to treat a person.

[0291] In some embodiments, administration of an effective amount of a vector or nucleic acid of the present disclosure to the retina transduces photoreceptor cells at or near the site of administration. In some embodiments, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or greater than 100% of the photoreceptor cells are transduced. In some embodiments, about 5% to about 100%, about 10% to about 50%, about 10% to about 30%, about 25% to about 75%, about 25% to about 50%, or about 30% to about 50% of the photoreceptor cells are transduced. Methods for identifying transduced photoreceptor cells are known in the art; for example, transduction can be detected using immunohistochemistry or markers such as enhanced green fluorescent protein.

[0292] In some embodiments of the present invention, the method comprises administering an effective amount of a vector or nucleic acid of the present disclosure subretinally (e.g., subretinal space) to a mammal to treat an individual with an ocular disorder, such as a human with an ocular disorder associated with a deep intron mutation. In some embodiments, the composition is injected into one or more subretinal locations to express the nucleic acid in photoreceptor cells. In some embodiments, the composition is injected into any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 subretinal locations.

[0293] In some embodiments, the composition is administered simultaneously or sequentially to more than one location. In some embodiments, the multiple injections are spaced apart by no more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, or 24 hours. In some embodiments, the multiple injections are spaced apart by 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 15 days, 20 days, 25 days, or 30 days. In some embodiments, the multiple injections are spaced apart by 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 11 months. In some embodiments, the multiple injections are spaced apart by 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 8 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 55 years, or 60 years.

[0294] Intravitreal injection method A general intravitreal injection method is illustrated by the following brief outline. This example is meant to illustrate certain features of the method and is not meant to be limiting in any way. Intravitreal injection techniques are known in the art (see, e.g., Peyman, GA, et al., J. Immunol. 1999, 144:131-132). al. (2009) Retina 29(7):875-912 and Fagan, XJ and Al-Qureshi, S. (2013) Clin. Experiment. Ophthalmol. 41(5):500-7).

[0295] Briefly, subjects undergoing intravitreal injection are prepared for the procedure by dilating the pupil, disinfecting the eye, and administering an anesthetic. Any suitable mydriatic agent known in the art can be used to dilate the pupil. Adequate pupil dilation can be confirmed prior to treatment. Disinfection may be achieved by applying an antiseptic eye treatment, e.g., an iodide-containing solution such as povidone-iodine (BETADINE®). Similar solutions are used to cleanse the eyelids, eyelashes, and any other nearby tissues (e.g., skin). Any suitable anesthetic, such as lidocaine or proparacaine, may be used in any suitable concentration. The anesthetic may be administered by any method known in the art, including, but not limited to, topical drops, gels, or jellies, and subconjunctival application of the anesthetic.

[0296] Prior to injection, the eyelashes in the area may be cleaned using a sterile eyelid speculum. The injection site may be marked with a syringe. The injection site is selected based on the patient's lens. For example, the injection site may be 3-3.5 mm from the limbus in pseudophakic or aphakic patients, or 3.5-4 mm from the limbus in phakic patients. The patient should be aware of the injection site and the reflex. You may also look in the opposite direction.

[0297] In some embodiments, the method comprises administering to the eye (e.g., by subretinal and / or intravitreal administration) an effective amount of recombinant viral particles comprising a vector of the present disclosure. In some embodiments, the viral titer of the composition is about 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 10×10 12 , 11×10 12 , 15×10 12 , 20×10 12 , 25×10 12 , 30×10 12 , or 50 × 10 12In some embodiments, the viral titer of the composition is about 5 x 10 genome copies / mL or more. 12 ~6×10 12 , 6×10 12~ 7×10 12 , 7×10 12 ~8×10 12 , 8×10 12 ~9×10 12 , 9×10 12 ~10×10 12 , 10×10 12 ~11×10 12 , 11×10 12 ~15×10 12 , 15×10 12 ~20×10 12 , 20×10 12 ~25×10 12 , 25×10 12 ~30×10 12 , 30×10 12 ~50×10 12 , or 50 × 10 12 ~100×10 12 In some embodiments, the viral titer of the composition is about 5 x 10 genome copies / mL. 12 ~10×10 12 , 10×10 12 ~25×10 12 , or 25 × 10 12 ~50×10 12 In some embodiments, the viral titer of the composition is about 5 x 10 genome copies / mL. 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 10×10 9 , 11×10 9 , 15×10 9 , 20×10 9 , 25×10 9 , 30×10 9 , or 50 × 10 9 In some embodiments, the viral titer of the composition is about 5 x 10 transducing units / mL or at least one of: 9 ~6×109 , 6×10 9 ~7×10 9 , 7×10 9 ~8×10 9 , 8×10 9 ~9×10 9 , 9×10 9 ~10×10 9 , 10×10 9 ~11×10 9 , 11×10 9 ~15×10 9 , 15×10 9 ~20×10 9 , 20×10 9 ~25×10 9 , 25×10 9 ~30×10 9 , 30×10 9 ~50×10 9 , or 50 × 10 9 ~100×10 9 In some embodiments, the viral titer of the composition is about 5 x 10 transducing units / mL. 9 ~10×10 9 , 10×10 9 ~15×10 9 , 15×10 9 ~25×10 9 , or 25 × 10 9 ~50×10 9 In some embodiments, the viral titer of the composition is about 5 x 10 transducing units / mL. 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 10×10 10 , 11×10 10 , 15×10 10 , 20×10 10 , 25×10 10 , 30×10 10 , 40×10 10 , or 50 × 10 10 In some embodiments, the viral titer of the composition is about 5 x 10 infectious units / mL or at least one of: 10 ~6×10 10 , 6×1010 ~7×10 10 , 7×10 10 ~8×10 10 , 8×10 10 ~9×10 10 , 9×10 10 ~10×10 10 , 10×10 10 ~11×10 10 , 11×10 10 ~15×10 10 , 15×10 10 ~20×10 10 , 20×10 10 ~25×10 10 , 25×10 10 ~30×10 10 , 30×10 10 ~40×10 10 , 40×10 10 ~50×10 10 , or 50 × 10 10 ~100×10 10 In some embodiments, the viral titer of the composition is about 5 x 10 infectious units / mL or at least one of: 10 ~10×10 10 , 10×10 10 ~15×10 10 , 15×10 10 ~25×10 10 , or 25 × 10 10 ~50×10 10 infectious units / mL.

[0298] In some embodiments, the method includes administering to the eye (e.g., subretinal and / or intravitreal administration) of an individual (e.g., a human) an effective amount of recombinant viral particles comprising a vector of the present disclosure. In some embodiments, the dose of viral particles administered to the individual is about 1 x 10 8 ~Approx. 1×10 13 In some embodiments, the dose of viral particles administered to an individual is about 1 x 10 genome copies / kg body weight or about 1 x 10 8 ~Approx. 1×10 13 genome copies / kg body weight.

[0299] During injection, the needle may be inserted perpendicular to the sclera and pointed toward the center of the eye. The needle may be inserted so that the tip terminates in the vitreous rather than the subretinal space. Any suitable amount may be used. After injection, the needle may be treated with a disinfectant, such as an antibiotic, and the eye may be rinsed to remove excess disinfectant.

[0300] Means for determining retinal structure and efficacy of nucleic acid delivery The retina is known to contain multiple layers. Retinal cell layers may include the inner limiting membrane, nerve fibers, ganglion cells, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, outer limiting membrane, photoreceptors, and retinal pigment epithelium. The layer closest to the vitreous body is the inner limiting membrane. This layer may contain Müller cells, a type of glial cell. The nerve fiber layer may contain axons from ganglion cells that form the optic nerve. The ganglion cell layer may contain ganglion cells and amacrine cells. The inner plexiform layer may contain synapses between dendrites of ganglion and amacrine cells and axons of bipolar cells. The inner nuclear layer may contain nuclei of amacrine, bipolar, and horizontal cells. The outer plexiform layer may contain synapses between horizontal cell dendrites and processes of photoreceptor cells. The outer nuclear layer may contain photoreceptor cell bodies. The outer or inner limiting membrane may contain cellular connections, such as adherens junctions and desmosomes in the apical processes of Müller cells, as well as between these processes and the inner segments of photoreceptor cells. The photoreceptor layer, also known as the rod-cone layer and Jacob's membrane, may contain photoreceptor cells, including rods and vertebrates. The most distal retinal layer to the vitreous is the retinal pigment epithelium (RPE), which may contain a layer of hexagonal epithelial cells containing pigment granules.

[0301] The retina is also known to contain many different cell types. Retinal neurons can include photoreceptor cells, bipolar cells, ganglion cells, amacrine cells, and horizontal cells. Photoreceptor cells are sensitive to light. Photoreceptor cells can sense light and respond by transmitting signals to the optic nerve through bipolar cells and ganglion cells. Photoreceptor cells can include rod cells, which generally sense light in low light conditions, and vertebrate cells, which generally sense color and brighter light perception. Bipolar cells receive input from photoreceptor cells and transmit it via synapses to amacrine or ganglion cells. Ganglion cells can receive information from amacrine or horizontal cells, the axons of which form the optic nerve. Horizontal cells can integrate input from multiple photoreceptors and help regulate light levels. Amacrine cells are interneurons that help regulate bipolar cells and provide input to ganglion cells. Retinal glial cells can include Müller cells, astrocytes, and microglia.

[0302] The effectiveness of nucleic acid delivery by subretinal or intravitreal injection can be monitored by several criteria described herein. In some embodiments, effectiveness is assayed by detecting deep intron mutations and / or deletions of sequences flanking the nucleic acid in a sample containing one or more cells into which the nucleic acid has been delivered. Deletions can be detected by any means known in the art, including but not limited to Southern blotting, PCR, qPCR, DNA sequencing (e.g., Sanger sequencing and next-generation sequencing), in situ hybridization, DNA microarray, and Surveyor nuclease assay (see, for example, Ran, FA et al. (2013) Nat. Protoc. 8:2281-2308). Exemplary methods are illustrated in the following examples.

[0303] In some embodiments, efficacy is assayed functionally. For example, after treatment of a subject using the methods of the present invention, the subject may be evaluated for improvement and / or stabilization and / or slowing of progression of one or more signs or symptoms of the disease state, e.g., by one or more clinical parameters. Examples of such tests are known in the art and include objective measures as well as subjective measures (e.g., subject-reported). For example, to measure the efficacy of a treatment on a subject's visual function, one or more of the following may be assessed: improvement in the subject's subjective quality of vision or central visual function (e.g., the ability of the subject to see clearly); In some embodiments, visual performance may be measured by various measures, such as: improved ability to read clearly and recognize faces), a subject's ocular mobility (e.g., reduced time required to navigate a maze), visual acuity (e.g., improved subject's LogMAR score), microperimetry (e.g., improved subject's dB score), scotopic perimetry (e.g., improved subject's dB score), fine matrix mapping (e.g., improved subject's dB score), Goldman perimetry (e.g., reduced size of the scotoma area (i.e., blind spot) and improved ability to resolve smaller targets), flicker susceptibility (e.g., improved Hertz), autofluorescence, and electrophysiology measurements (e.g., improved ERG). In some embodiments, visual performance is measured by the subject's visual mobility. In some embodiments, visual performance is measured by the subject's visual acuity. In some embodiments, visual performance is measured by microperimetry. In some embodiments, visual performance is measured by scotopic perimetry. In some embodiments, visual performance is measured by ERG. In some embodiments, visual performance is measured by the subject's subjective quality of vision.

[0304] In diseases that result in progressive visual degeneration, treating subjects at a young age can not only slow or halt disease progression but also reverse or prevent visual loss due to acquired amblyopia. Amblyopia exists in two forms. In studies of nonhuman primates and kittens kept in complete darkness for several months after birth, exposing the animals to light after birth resulted in irreversible functional blindness, despite the retina still sending functional signals. This blindness occurs because cortical neural connections and "teaching" are developmentally halted postnatally due to cessation of stimulation. It is unclear whether this function can be restored. In retinal degenerative diseases, normal visual cortical circuits are initially "learned" or developmentally appropriate until degeneration causes significant dysfunction. The loss of visual stimuli in terms of signal transduction in the dysfunctional eye results in "acquired" or "learned" dysfunction ("acquired amblyopia"), which results in the brain's inability to interpret signals or "use" that eye. In these "acquired amblyopia" cases, it is unclear whether improved signaling from the retina as a result of gene therapy of the amblyopic eye will result in the acquisition of more normal function in addition to slowing or stabilizing the progression of the disease state. In some embodiments, the treated human is under 30 years of age. In some embodiments, the treated human is under 20 years of age. In some embodiments, the treated human is under 18 years of age. In some embodiments, the treated human is under 15 years of age. In some embodiments, the treated human is under 14 years of age. In some embodiments, the treated human is under 13 years of age. In some embodiments, the treated human is under 12 years of age. In some embodiments, the treated human is under 10 years of age. In some embodiments, the treated human is under 8 years of age. In some embodiments, the treated human is under 6 years of age.

[0305] In some eye disorders, there is a "nurse cell" phenomenon, where improving the function of one cell type improves the function of another. For example, transducing the RPE of the central retina with the nucleic acid of the present invention improves the function of rods, and then the improved rod function improves rod function. Thus, treatment of one cell type can improve the function of another cell type.

[0306] The selection of a particular vector and composition will depend on many different factors, including, but not limited to, the individual's medical history and the characteristics of the condition and individual being treated. The evaluation of such characteristics and the design of an appropriate treatment regimen is ultimately the responsibility of the attending physician.

[0307] In some embodiments, the human being treated has an inherited eye disorder (e.g., associated with a deep intronic mutation) but has not yet developed clinical signs or symptoms. In some embodiments, the human being treated exhibits one or more signs or symptoms of an ocular disorder (e.g., associated with a deep intronic mutation). In some embodiments, a deep intronic mutation has been identified in the human being treated.

[0308] The compositions of the present invention can be used either alone or in combination with one or more additional therapeutic agents to treat eye disorders. The interval between successive administrations can be at least a few minutes, a few hours, or a few days (or even less).

[0309] In some embodiments, one or more additional therapeutic agents may be administered subretinally or vitreously (e.g., via intravitreal administration). Non-limiting examples of additional therapeutic agents include polypeptide neurotrophic factors (e.g., GDNF, CNTF, BDNF, FGF2, PEDF, EPO), polypeptide anti-angiogenic factors (e.g., sFlt, angiostatin, endostatin), anti-angiogenic nucleic acids (e.g., siRNA, miRNA, ribozymes), e.g., anti-angiogenic nucleic acids against VEGF, anti-angiogenic morpholinos, e.g., anti-angiogenic morpholinos against VEGF, anti-angiogenic antibodies, and / or antibody fragments (e.g., Fab fragments), e.g., anti-angiogenic antibodies and / or antibody fragments against VEGF.

[0310] VII. Kit The compositions, nucleic acids, and viral particles described herein can be included in kits designed for use in one of the methods of the invention described herein.

[0311] The compositions, nucleic acids, and viral particles of the invention may be further packaged into kits, which may further include instructions for use. In some embodiments, the instructions include instructions for following one of the methods described herein.

[0312] In some embodiments, the kit further includes a buffer and / or a pharmaceutically acceptable excipient. As is well known in the art, a pharmaceutically acceptable excipient is a relatively inert substance that facilitates administration of a pharmacologically active substance and can be provided as a liquid solution or suspension, an emulsion, or a solid dosage form suitable for dissolution or suspension in a liquid prior to use. For example, an excipient can give shape or consistency or act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting agents and emulsifiers, salts for varying osmotic pressure, encapsulating agents, pH buffering substances, and buffers. Such excipients can include any pharmaceutical agent suitable for direct delivery to the eye that can be administered without causing undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various TWEEN compounds, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable excipients can include pharmaceutically acceptable salts, e.g., salts of inorganic acids such as hydrochlorides, hydrobromides, phosphates, sulfates, etc., as well as salts of organic acids such as acetates, propionates, malonates, benzoates, etc. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991).

[0313] In some embodiments, the pharmaceutically acceptable excipient may include a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, etc. Saline solutions and aqueous dextrose, polyethylene glycol (PEG), and Glycerol solution can also be used as a liquid carrier, particularly for injectable solutions. Additional ingredients, such as preservatives, buffers, isotonicity agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, thickeners, etc., can also be used. The kits described herein can be packaged in single unit doses or multiple unit doses. The contents of the kit are generally formulated as a sterile, substantially isotonic solution. [Example]

[0314] The present invention will be more fully understood with reference to the following examples. However, the following examples should not be construed as limiting the scope of the present invention. The examples and embodiments described herein are for illustrative purposes only, and various changes or modifications will be suggested to those skilled in the art in light thereof, which are understood to be within the spirit and scope of this application and the appended claims. [Example]

[0315] Creation of an in vitro model of Leber congenital amaurosis using Crispr-Cas9 technology method Plasmid The pSpCas9 plasmid expressing pSpCas9 was ordered from Sigma (catalog number: CAS9P-1EA). The BbsI restriction site in the BGH polyA was removed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene) and a pair of mutagenesis primers (SEQ ID NOs: 28-29) according to the manufacturer's protocol. The U6 promoter-BbsI:BbsI-sgRNA scaffold-U6 terminator cassette (SEQ ID NO: 30) was synthesized by GeneArt (Life Technologies) and inserted into the PciI and NruI restriction sites of the pSpCas9-BbsI null plasmid to generate the pSpCas9(BB) plasmid. The sgRNA oligos (SEQ ID NOs: 1-2) were subcloned into the two BbsI restriction sites of the pSpCas9(BB) plasmid according to a previously described protocol ( Ran, FA et al. (2013) Nat. Protoc. 8:2281-2308 ).

[0316] Nucleofection Two and a half micrograms of pSpCas9(BB)-U6-sgRNA plasmid DNA and five microliters of ssODN (10 micromolar) (SEQ ID NO: 3) were transfected into 1x10 cells using the Amaxa SF cell line 4D-Nucleofector X Kit L (Lonza) and program CM-130 of the 4D-Nucleofector system (Lonza) according to the manufacturer's protocol. 6 HEK 293FT cells were co-transfected with the vector.

[0317] screening To identify clones carrying the CEP290 c.2991+1655A>G mutation, cells were dissociated into single cells after 48 hours of co-transfection and serially diluted to a final concentration of 0.5 cells per 100 microliters to reduce the possibility of having multiple cells per well. 100 microliters of diluted cells were seeded into each well of nine 96-well plates. Cells were grown in a 5% CO2, 37°C incubator for 2 weeks.

[0318] 235 single-cell clones were identified and screened for the c.2991+1655A>G mutation in CEP290. Genomic DNA was extracted using QuickExtract DNA extraction solution (Epicentre) and purified using GoTaq Hot Star The PCR product was amplified using t Green Master Mix (Promega) and PCR primers (SEQ ID NOs: 4-5) flanking the intron mutation. Amplification of the PCR product was achieved with the following cycling parameters: 1 cycle at 95°C for 2 minutes; 35 cycles at 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 3 minutes; and 1 cycle at 72°C for 15 minutes. The PCR product was subjected to SnaBI digestion and Sanger sequencing with the sequencing primer (SEQ ID NO: 6).

[0319] RT-qPCR mRNA was extracted from WT, Het, and MT cells using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's protocol. One microgram of total RNA was used to synthesize cDNA using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's protocol. The cDNA was subjected to real-time PCR amplification in an ABI Prism 7500 Real-Time PCR System (Applied Biosystems) in a buffer containing Fast Plus EvaGreen qPCR Master Mix containing low concentrations of ROX (Biotium) and primers specifically detecting wild-type CEP290 mRNA (SEQ ID NOs: 7-8) and mutant CEP290 mRNA (SEQ ID NOs: 9-10). The following conditions were used: 1 cycle of 50°C for 2 minutes; 1 cycle of 95°C for 10 minutes; and 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The specificity of the amplified products was determined from melting curve analysis performed at the end of each run using cycles of 95°C for 15 seconds, 60°C for 60 seconds, 95°C for 15 seconds, and 60°C for 15 seconds. Data were analyzed using SDS 2.3 software (Applied Biosystems). CEP290 expression levels were normalized to that of PPIA mRNA (see SEQ ID NOs: 31-32 for primer sequences).

[0320] Western blot analysis Cells were lysed on ice in RIPA lysis buffer (Cell Signaling Technology) supplemented with 1 mmol / L phenylmethylsulfonyl fluoride (PMSF; Cell Signaling Technology) and 1X protease inhibitor cocktail (Cell Signaling Technology). Cells were then scraped and collected in Eppendorf tubes, and the lysate was cleared by centrifugation at 13,000 rpm for 6 minutes at 4°C. Samples were prepared by adding NuPage 4X LDS sample buffer and NuPage 10X reducing agent (both Life Technologies), heating at 70°C for 10 minutes, and centrifuging at 13,000 rpm for 1 minute. Protein samples were loaded onto a NuPAGE 3-8% Tris-acetate gel along with HiMark prestained protein standards (both Life Technologies). Samples were separated by gel electrophoresis at 180 volts for 1 hour. The running buffer used was Tris-acetate SDS running buffer (Life Technologies). For transfer, polyvinylidene fluoride (PVDF) membranes were briefly treated in methanol to make them hydrophilic and then rinsed with water. A transfer sandwich was prepared by sandwiching the PVDF membrane and gel between filter paper and a sponge in a Life Technologies II blot module. The transfer buffer used was NuPage 20X transfer buffer (Life Technologies) containing 20% ​​methanol. Transfer was performed for 2 hours at 30 volts in an XCell SureLock Mini-Cell (Life Technologies). After transfer, the PVDF membrane was blocked in Pierce TBST buffer (Tris-buffered saline with Tween 20 detergent; Thermo Fisher Scientific) containing 1% nonfat dry milk for 1 hour at room temperature with rocking. The blot was incubated in a primary antibody solution made in blocking solution and rocked overnight at 4°C. The primary antibody used was rabbit polyclonal anti-CEP290 antibody (Ustaglandin E). The primary antibodies were: mouse monoclonal anti-Cas9 antibody (clone 7A9; Millipore), a gift from Professor Hemant Khanna, University of Massachusetts; mouse monoclonal anti-Cas9 antibody (clone 7A9; Millipore); and HRP-conjugated rabbit monoclonal anti-beta-actin antibody (clone 13E5; Cell Signaling Technology). Unbound primary antibodies were washed three times with TBST for 10 min each. Secondary antibodies (Alexa Fluor 647-conjugated anti-rabbit or anti-mouse IgG; Cell Signaling Technology) in blocking solution were added to the membrane and maintained at room temperature for 1 h on a shaker. To reduce nonspecific background, the membrane was washed three times with TBST for 10 min each. The membrane was developed for 4 min using Pierce Enhanced Chemiluminescence (ECL) Western Blot Substrate (Thermo Fisher Scientific). Finally, protein bands in the blot were visualized by exposing the film for various time intervals in a Kodak X-OMAT 2000 processor. To reprobe the blots with anti-beta-actin antibody, the membranes were first stripped by incubation in retrieval Western blot stripping buffer (Thermo Fisher Scientific) for 30 min at 37°C, followed by reprobing with anti-beta-actin antibody. The blotting data shown in this study were representative of at least three independent experiments.

[0321] result First, we used CRISPR-Cas9 genome editing technology to generate a cell model harboring the intronic splice mutation c.2991+1655A>G in CEP290, which was a valuable tool for evaluating therapeutic agents to treat LCA patients with the c.2991+1655A>G mutation in CEP290.

[0322] Genome editing using the bacterial type II CRISPR-Cas9 system begins with the introduction of a double-strand break (DSB) at a targeted genomic locus defined by an sgRNA target sequence and a protospacer adjacent motif (PAM), followed by repair of the DSB through either homology-directed repair (HDR) or non-homologous end joining (NHEJ) (Jinek, M. et al. (2012) Science 337:816-821; Ran, FA et al. (2013) Nat. Protoc. 8:2281-2308). In the presence of an HDR template, the CRISPR-Cas9 system can be used to perform precise and defined genetic manipulations at the target locus through the HDR process.

[0323] To achieve targeted genomic DNA replacement, a plasmid expressing both the sgRNA and Streptococcus pyogenes Cas9 (SpCas9) was introduced into 293FT cells by nucleofection along with a linear HDR template. The HDR template was a single-stranded DNA oligonucleotide (ssODN; SEQ ID NO: 3) containing a mutant PAM (c.2991+1666C>G) and 75-bp homology arms flanking the c.2991+1655A>G mutation. The mutant PAM prevented the donor ssODN from being degraded by Cas9 in the cells while introducing a unique SnaBI restriction site into intron 26 of CEP290.

[0324] To obtain cells carrying the c.2991+1655A>G mutation in CEP290, 235 single-cell clones were isolated and screened from sgRNA / SpCas9 and ssODN co-transfected cells. Genomic DNA was extracted and amplified with PCR primers (SEQ ID NOs: 4-5) flanking the intronic mutation. The PCR product was subjected to SnaBI digestion and Sanger sequencing with sequencing primer (SEQ ID NO: 6).

[0325] Of 235 single-cell clones, one clone was found to be c in both CEP290 alleles. One clone contained the c.2991+1655A>G and c.2991+1666C>G mutations (hereinafter referred to as "mutant cells" or "MT cells"). Another clone contained two mutations on one CEP290 allele and endogenous wild-type CEP290 DNA on the other allele (hereinafter referred to as "heterozygous cells" or "Het cells"). Cells containing two alleles of endogenous wild-type CEP290 are hereinafter referred to as "wild-type cells" or "WT cells."

[0326] The expression levels of wild-type and mutant CEP290 mRNA in wild-type, heterozygous, and mutant cells were measured by reverse transcription quantitative PCR (RT-qPCR) using primers specifically detecting wild-type CEP290 mRNA (SEQ ID NOs: 7-8) and mutant CEP290 mRNA (SEQ ID NOs: 9-10), respectively. The results were normalized to the expression level of PPIA mRNA (SEQ ID NOs: 31-32).

[0327] Compared to wild-type cells, wild-type CEP290 mRNA levels were reduced by 27% and 48%, respectively, in heterozygous and mutant cells (Figure 3A). As expected, wild-type cells did not express mutant CEP290 mRNA, but its level was 24% higher in mutant cells than in heterozygous cells (Figure 3B). Compared to heterozygous cells, mutant cells expressed significantly lower levels of wild-type CEP290 mRNA and significantly highe...

Claims

1. A composition for restricting expression of a Cas9 protein in a cell, comprising: a) a nucleic acid encoding at least one guide RNA, the nucleic acid comprising a first guide RNA that hybridizes to a first target site, the first target site being located at a locus in the genome of the cell; b) a Cas expression cassette located on a vector and comprising: i) a nucleotide sequence encoding a Cas9 protein, and ii) a cleavage site, which is a nucleotide sequence comprising said first target site; Including, wherein the cleavage site can be cleaved by the Cas9 protein, thereby reducing the expression of the Cas9 protein compared to the expression of the Cas9 protein before cleavage of the Cas expression cassette; The above composition.

2. 2. The composition of claim 1, wherein the Cas expression cassette further comprises one or more regulatory control elements.

3. The composition described in claim 2, wherein the one or more regulatory control elements are promoters operably linked to the nucleotide sequence encoding the Cas9 protein, and wherein the cleavage site is between the one or more regulatory control elements and the nucleotide sequence encoding the Cas9 protein.

4. 3. The composition of claim 1 or 2, wherein the Cas expression cassette further comprises a polyadenylation (polyA) sequence operably linked to the nucleotide sequence encoding the Cas9 protein, wherein the cleavage site is between the polyA sequence and the nucleotide sequence encoding the Cas9 protein.

5. The composition of any one of claims 1 to 4, wherein the nucleic acid encoding the at least one guide RNA and the Cas expression cassette are located on the same vector.

6. The composition of any one of claims 1 to 4, wherein the nucleic acid encoding the at least one guide RNA and the Cas expression cassette are located on different vectors.

7. 7. The composition of claim 6, wherein the vector comprising the Cas expression cassette and the vector comprising the nucleic acid encoding the at least one guide RNA are independently selected from a recombinant adeno-associated virus (rAAV) vector, a recombinant adenovirus vector, a recombinant lentivirus vector, or a recombinant herpes simplex virus (HSV) vector.

8. 7. The composition of claim 5 or 6, wherein the vector comprising the Cas expression cassette and the vector comprising the nucleic acid encoding the at least one guide RNA are both rAAV vectors.

9. 9. The composition of any one of claims 1 to 8, wherein the at least one guide RNA further comprises a second guide RNA that hybridizes to a second target site, wherein the second target site is located at a locus within the genome of the cell.

10. A composition for restricting expression of a Cas9 protein in a cell, comprising: a) a nucleic acid encoding at least one guide RNA comprising a first guide RNA that hybridizes to a first target site and a second guide RNA that hybridizes to a second target site, wherein the first and second target sites are located at a locus within the genome of the cell; b) a Cas expression cassette located on a vector and comprising: i) a nucleotide sequence encoding a Cas9 protein, and ii) a first cleavage site, which is a nucleotide sequence comprising said first or second target site; Including, wherein the first cleavage site can be cleaved by the Cas9 protein, thereby reducing expression of the Cas9 protein compared to expression of the Cas9 protein before cleavage of the Cas expression cassette; The above composition.

11. 11. The composition of claim 10, wherein the Cas expression cassette further comprises a second cleavage site that is a nucleotide sequence comprising the first or second target site, wherein the second cleavage site is capable of being cleaved by the Cas9 protein, thereby reducing expression of the Cas9 protein compared to expression of the Cas9 protein prior to cleavage of the Cas expression cassette.

12. 12. The composition of claim 10 or 11, wherein the Cas expression cassette further comprises one or more regulatory control elements.

13. The composition described in claim 12, wherein the one or more regulatory control elements are promoters operably linked to the nucleotide sequence encoding the Cas9 protein, and wherein the first cleavage site is between the one or more regulatory control elements and the nucleotide sequence encoding the Cas9 protein.

14. 13. The composition of Claim 11 or 12, wherein the Cas expression cassette further comprises a polyadenylation (polyA) sequence operably linked to the nucleotide sequence encoding the Cas9 protein, and wherein the second cleavage site is between the polyA sequence and the nucleotide sequence encoding the Cas9 protein.

15. The composition of any one of claims 11 to 14, wherein the first and second cleavage sites comprise the same target site.

16. The composition of any one of claims 11 to 14, wherein the first and second cleavage sites comprise different target sites.

17. 17. The composition of any one of claims 10 to 16, wherein the first and second target sites are located on opposite strands of the target DNA.

18. 18. The composition of claim 17, wherein the target DNA comprises a gene containing a deep intronic mutation.

19. A composition for restricting expression of a Cas9 protein in a cell, comprising: a) a nucleic acid encoding at least one guide RNA comprising a first guide RNA that hybridizes to a first target site and a second guide RNA that hybridizes to a second target site, wherein the first and second target sites are located at a locus in the genome of the cell and are located on opposite strands of a target gene, the target gene comprising a gene containing a deep intron mutation; b) a Cas expression cassette located on a vector and comprising: i) a nucleotide sequence encoding a Cas9 protein, and ii) a first cleavage site, which is a nucleotide sequence comprising said first or second target site; Including, wherein the first cleavage site can be cleaved by the Cas9 protein, thereby reducing expression of the Cas9 protein compared to expression of the Cas9 protein before cleavage of the Cas expression cassette; The above composition.

20. 20. The composition of claim 18 or 19, wherein the gene containing the deep intronic mutation is CEP290.

21. 21. The composition of any one of claims 18 to 20, wherein the deep intronic mutation is the c.2991+1655A>G mutation.

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