Treatment of retinitis pigmentosa using engineered meganucleases

Abstract

To provide recombinant meganucleases engineered to recognize and cleave recognition sequences present in a human rhodopsin gene allele.SOLUTION: Disclosed are recombinant meganucleases engineered to recognize and cleave recognition sequences present in a mutant RHO P23H allele. The invention further relates to the use of such recombinant meganucleases in methods for treating retinitis pigmentosa, wherein the mutant RHO P23H allele is preferentially targeted, cleaved, and inactivated.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed on September 8, 2015, under 35 U.S.C. § 119(e). No. 62 / 215,460, filed on Dec. 16, 2007, the contents of which are incorporated by reference in their entirety. (incorporated herein) claiming the benefit of priority rights.

[0002] FIELD OF THE INVENTION The present invention relates to the fields of molecular biology and recombinant nucleic acid technology. In particular, the present invention relates to the human Recombinant proteins designed to recognize and cleave recognition sequences found in pusin gene alleles The present invention further relates to a meganuclease in a method for treating retinitis pigmentosa. The present invention relates to the use of such recombinant meganucleases. [Background technology]

[0003] Background of the Invention Retinitis pigmentosa (RP) is a severe visual impairment caused by progressive degeneration of photoreceptor cells in the retina. RP is a genetic degenerative eye disease that causes a loss of rod photoreceptors, resulting in peripheral vision loss. It is characterized by impaired peripheral and scotopic vision. After progressive rod degeneration, the retinal pigment epithelium As the disease progresses, patients may experience night blindness, progressive glaucoma, and glaucoma. RP affects approximately 1 in 3,000 people. It can occur alone or in combination with other systemic disorders. Currently, there is no effective treatment for RP. .

[0004] The genetic causes of RP can be autosomal dominant, autosomal recessive, X-linked, or maternal. Autosomal dominant RP accounts for 30-40% of cases (Ma et al. al.(2105),Scientific Reports.18(5:9236): 1-6), and is associated with mutations in genes expressed in rod photoreceptors and the retinal pigment epithelium. The human rhodopsin gene (RHO) has been shown to contribute to the pathogenesis of autosomal dominant RP It is the first gene and the most common gene associated with this form of the disease (McWil liam et al.(1989)Genomics.5:619-622;Dryj a et al.(1990)Nature.343:364-366;Farrar et al. (1990) EMBO Journal.21:857-864). RHO mutations are associated with 30-40% of autosomal dominant RP cases worldwide, with the highest incidence in the US It has been observed in approximately 26.5% of cases (Illing et al. (2002) Journal al of Bio.Chem.277(37):34150-34160).

[0005] Rhodopsin is involved in the first step of phototransduction, converting light stimuli into electrical signals. Rhodopsin is an essential photopigment expressed in retinal rod photoreceptor cells. Light-sensitive G protein-coupled receptors consisting of opsin protein moieties bound to a nal chromophore It is expressed as a receptor and is a major component of the disc membrane of the outer segments of rod photoreceptors.

[0006] The first RHO mutations shown to contribute to autosomal dominant RP were A C to A mutation at position 68 of the encoded sequence (which is at position 23 of the encoded protein) This mutation resulted in a proline to histidine substitution (P23H) in the nucleotide sequence. The mutation is referred to herein as the "RHO P23H mutation," and RHO containing the mutation is The allele is referred to herein as the "mutant RHO P23H allele." The P23H mutation is the most frequently reported RH mutation in autosomal dominant RP cases in North America. O mutation (Mao et al. (2011) Human Gene Ther apy.22:567-575), carrying a single mutant RHO P23H allele Patients can develop RP despite the presence of a functional wild-type RHO allele.

[0007] Rhodopsin proteins containing improper P23H substitution folds are deposited in the endoplasmic reticulum of rod photoreceptors. It accumulates in the retinal chromosome and is not reconstituted by the 11-cis-retinal chromophore. In many cases, misfolded P23H rhodopsin leads to rod photoreceptor degeneration and death. Accumulated P23H rhodopsin undergoes proteasomal and lysosomal degradation, resulting in the formation of E ER-associated unfolded proteins can induce R stress and cell apoptosis It has been shown to stimulate a cellular response (Lin et al. (2007), Science e.318:944-949;Gorbatyuk et al.(2010)PNAS USA107(13):5961-5966). Loading also prevents cell death by interfering with the transport or function of wild-type rhodopsin. (Illing et al., 2002; Lin et al., 200 7) Furthermore, P23H rhodopsin has been shown to exhibit delayed dephosphorylation, and Death may be due to abnormal cytosolic Ca2+ levels (Saito et al. (2008) )Clin.Opthamol.2:821-828).

[0008] Multiple strategies, including nutritional therapy, pharmaceuticals, and gene therapy, treat autosomal dominant RP. Gene therapy approaches are being pursued for the treatment of autosomal dominant RP. These approaches employ either indirect or direct strategies. Indirect approaches involve identifying pathogenic mutations. The aim is to promote the survival of rod photoreceptors without directly affecting protein expression. For example, gene therapy has been used to inhibit apoptosis in rod photoreceptors. In response, neurotrophic factors such as GDNF and anti-apoptotic proteins such as XIAP are secreted into retinal cells. It has been introduced.

[0009] In contrast, direct approaches in gene therapy directly affect the pathogenesis of autosomal dominant RP. The aim is to regulate the levels of proteins that contribute to RHO-associated autosomal dominant RP. In the past, there have been strategies to enhance the proteasomal degradation of P23H rhodopsin, but these have not been successfully demonstrated in animal models. Another strategy is to use targeted RNA therapy to target functional wild-type genes. It silences the mutant RHO allele while maintaining expression of the normal allele. Such an approach uses ribozymes and RNA interference (RNAi) to induce the expression of ribozymes in rats. Specific mRNA transcripts produced by the mutant RHO P23H transgene in It's targeting things.

[0010] A further strategy involves the non-specific identification of both wild-type and mutant RHO alleles. This method selectively silences the wild-type RHO gene and simultaneously delivers an alternative copy of wild-type RHO to inhibit the wild-type transcription factor. They are pursuing a "suppression and substitution" approach by expressing proteins, e.g. O'Reilly et al. used adeno-associated virus (AAV) vectors to achieve heterologous transfection. In Pro23His+ / - mice, both wild-type and mutant RHO alleles The short hairpin RNA is designed to target and suppress the The RHO replacement gene was also delivered and expressed (O'Reilly et al. (2012) 007)Amer.J.of Human Genetics.81:127-135) Similarly, Palfi et al. used AAV vectors to deliver RHO replacement genes into Rho- / demonstrated delivery of α-glucan to knockout mice (Palfi et al. (2010) )Human Gene Therapy.21:311-323). However, Such approaches can lead to toxicity and off-target effects if RHO replacement levels are excessively high. Furthermore, off-target effects of RNAi approaches are known complications. siRNAs longer than 21 base pairs can induce retinal degeneration in animal models. It has been shown that (Kleinman et al. (2012) Mol. Ther .20(1):101-108).

[0011] Previously, U.S. Patent Application Publication No. 2012 / 0204282 to Zhang ("Zhang") The patent application discloses engineered nucleases for cleaving DNA targets in the human RHO gene. The Zhang application discloses the use of a mutated RHO allele to target expression. Several approaches to adjusting the temperature and humidity have been disclosed. Zinc finger proteins (ZFPs) and ATPases act as repressors of RHO gene expression. and engineered DNA-binding domains such as TAL effector (TALE) proteins. In addition, the Zhang application also discusses the creation of regulatory domains or functional domains. Fusion proteins containing operably linked ZFP or TALE binding domains have been described. The functional domain may be a transcriptional repressor domain that downregulates RHO gene expression. Alternatively, the functional domain can be a transcription activation domain. , may contain a nuclease domain. When linked to a nuclease domain, the resulting fusion Proteins include zinc finger nucleases (ZFNs) and TALE nucleases (TALEs). ALEN).

[0012] In addition to ZFNs and TALENs, the Zhang application also provides wild-type and / or mutant RH The use of meganucleases to target and inhibit expression of O alleles has been discussed. The Zhang application also describes a method for catalyzing RHO via non-homologous end joining (NHEJ) at a recognition sequence. To disrupt gene expression, and to introduce an alternative wild-type RHO gene sequence to generate wild-type rhodopsin. The use of such meganucleases to express synproteins has been described. However, the recognition sequence in the RHO gene identified by the Zhang application is The ZFN target sites are limited to three sets of sites found in the wild-type RHO gene (Table 2 in the Zhang application). (See

[0013] Also, U.S. Patent Application Publication No. 2013 / 01832 to Lemaire and Arnould No. 82 (the "Lemaire Application") discloses a method for cleaving a DNA target in the RHO gene. The Lemaire application discloses the use of engineered meganucleases. to target various regions of the RHO gene for use in one of several therapeutic strategies. Designed meganucleases have been disclosed. The first strategy is gene modification, where engineered The meganuclease is specific for a recognition sequence near a particular mutation, and It induces a double-strand break and relies on homologous recombination of the corresponding non-mutated allele sequence into the genome. The second strategy disclosed in the Lemaire application is exon knock-in, which involves the removal of a pathogenic variant. Using meganucleases to synthesize synthetic wild-type coding sequences into genomes while preventing the expression of natural mutations By introducing the protein into the system, functional proteins are reconstituted. A third strategy that has been proposed is gene inactivation by mutagenesis, which targets recognition sequences in the genome. The meganuclease induces double-strand breaks in the nuclease and induces mutations at the break site. It relies on NHEJ.

[0014] Therefore, the art has identified a number of genes that favor the RHO P23H allele for the treatment of RP. There remains a need for methods that can proactively target and inactivate proteins. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] US Patent Application Publication No. 2012 / 0204282 [Patent Document 2] US Patent Application Publication No. 2013 / 0183282 [Non-patent literature]

[0016] [Non-Patent Document 1] Ma et al. (2105), Scientific Reports.18(5:9236):1-6 [Non-patent document 2] McWilliam et al.(1989)Genomics.5:619-622;Dryja et al.(1990)Nature.343:364-366;Farrar et al.(1990)EMBO Journal.21:857-864 [Non-patent document 3] Illing et al. (2002) Journal of Bio.Chem.277(37):34150-34160 [Non-patent document 4] Mao et al.(2011)Human Gene Therapy.22:567-575 [Non-patent document 5] Lin et al.(2007),Science.318:944-949;Gorbatyuk et al.(2010)PNAS USA107(13):5961-5966 [Non-patent document 6] Saito et al.(2008)Clin.Opthamol.2:821-828 [Non-Patent Document 7] O'Reilly et al.(2007)Amer.J.of Human Genetics.81:127-135 [Non-patent document 8] Palfi et al.(2010)Human Gene Therapy.21:311-323 [Non-Patent Document 9] Kleinman et al.(2012)Mol.Ther.20(1):101-108 Summary of the Invention [Problem to be solved by the invention]

[0017] The present invention provides a recombinant meganuclear vector engineered to recognize and cleave the P23H recognition sequence. The present invention further provides a recombinant meganuclease (or the encoded recombinant meganuclease). nuclease) recognizes the P23H recognition sequence present on the mutant RHO P23H allele. Such recombinant meganucleases or such The gene encoding the recombinant meganuclease can be delivered to the cells of patients with RP. and NHEJ occurs at the cleavage site, and mutant RHO P23 This results in mutagenesis and disruption of the H allele, but not the functional wild-type RHO allele. However, the rod photoreceptors in the retina are still intact enough to express wild-type rhodopsin. Preferential inactivation of the mutant RHO P23H allele and P23H rhodopsin expression Destruction of this protein is expected to slow, prevent, or reverse the progression of RP in patients.

[0018] Thus, in some embodiments, the present invention provides a method for treating a mutant RHO P23H allele. It is designed to recognize and cleave the P23H recognition sequence present in but absent from the wild-type RHO allele. The present invention further provides a recombinant meganuclease engineered to A method for treating chromosomal dominant RP, comprising administering a mutant RHO P23H allele The use of such recombinant meganucleases in methods for preferential targeting and cleavage is Thus, expression of P23H rhodopsin is enhanced by the addition of the cleavage site of the meganuclease. Although the RHO allele is suppressed by NHEJ, the functional wild-type RHO allele still contributes to the retinal It is intact enough to express wild-type rhodopsin in rod photoreceptors.

[0019] Thus, the present invention provides a method for the production of a gene encoding a target gene, which is engineered to recognize a specific DNA sequence in a locus of interest. Site-specific rare-cutting homing endonucleases (also known as "meganucleases") Homing endonucleases are unique to plant and fungal genomes. A group of naturally occurring nucleases that recognize commonly occurring cleavage sites of 15 to 40 base pairs. They are a group of genes that contain group 1 self-splicing introns and inteins. They are often associated with living DNA elements. They form double-strand breaks in chromosomes. This allows the DNA to be repaired at specific locations in the host genome by recruiting cellular DNA repair mechanisms. naturally promotes homologous recombination or gene insertion in the target gene (Stoddard (2006) , Q.Rev.Biophys.38:49-95). Homing endonucleases are Generally, there are four families: the LAGLIDADG (SEQ ID NO: 96) family, the GI They are divided into the Y-YIG family, the His-Cys box family, and the HNH family. These families share structural motifs that influence catalytic activity and recognition sequences. For example, members of the LAGLIDADG (SEQ ID NO: 96) family are characterized by: Contains one or two copies of the conserved LAGLIDADG (SEQ ID NO: 96) motif (Chevalier et al. (2001), Nucleic Acids Res.29(18):3757-3774). 1 copy of LAGLIDA LAGLIDADG (SEQ ID NO: 96) homing with DG (SEQ ID NO: 96) motif The endonuclease forms a homodimer, whereas two copies of LAGLIDADG Members with the (SEQ ID NO: 96) motif are seen as monomers.

[0020] Methods for producing engineered site-specific recombinant meganucleases are known in the art. I-CreI (SEQ ID NO: 95) is a gene encoding the alga Chlamydomonas re Homo sapiens recognizes and cuts a 22-base pair recognition sequence in the chloroplast chromosome of B. inhardtii. A member of the LAGLIDADG (SEQ ID NO: 96) family of binding endonucleases Genetic selection techniques were used to alter wild-type I-CreI cleavage site preference. (Sussman et al. (2004), J. Mol. Biol. 342 :31-41;Chames et al.(2005), Nucleic Acids Res.33:e178;Seligman et al.(2002), Nucle ic Acids Res.30:3870-9,Arnould et al.(20 06), J. Mol. Biol. 355:443-58). More recently, monoLAGL A method for rationally designing IDADG (SEQ ID NO: 96) homing endonuclease This allows for the identification of diverse DNA sites (sites in mammalian, yeast, plant, bacterial and viral genomes) I-CreI and other homing endonucleases to target A method has been described in which the No. 9).

[0021] As first described in WO 2009 / 059195, I-CreI and and its engineered derivatives, which are usually dimers, have the C-terminus of the first subunit linked to the C-terminus of the second subunit. a single polypeptide using a short peptide linker connecting the N-terminus of the subunits (See, e.g., Li, et al. (2009) Nucleic Acids Res.37:1650-62;Grizot,et al.(2009)Nucle (See also ic Acids Res. 37:5405-19). "Single-stranded" meganucleases can be expressed from a single transcript. The ganucleases exhibit extremely low off-target cutting. by delivering a gene encoding a leukemia enzyme to retinal cells, preferably rod photoreceptors. , specifically and preferentially targets, cleaves, and inactivates the mutant RHO P23H allele It is possible to suppress the expression of P23H rhodopsin by activating the rhodopsin gene. [Means for solving the problem]

[0022] Thus, in one aspect, the present invention provides a method for coding for the P23H substitution in a P23H mutant allele. The present invention provides a recombinant meganuclease that recognizes and cleaves a recognition sequence containing a mutation that inhibits the cleavage. In some embodiments, the recognition sequence is selected from the group consisting of SEQ ID NOs: 1-4 (i.e., the P23H recognition sequence). The recombinant meganuclease is selected by combining a first subunit and a second subunit. The first subunit recognizes one first recognition half-site of the P23H recognition sequence. (a) residues 198 to 344 of any one of SEQ ID NOs: 6 to 69 or SEQ ID NOs: 70 to 9 an amino acid sequence having at least 80% sequence identity with residues 7 to 153 of any one of and (b) the first hypervariable (HVR) region that determines the specificity for the first recognition half-site. 1) region. The recombinant meganuclease contains the second half of one of the P23H recognition sequences. a second subunit that recognizes the nucleotide sequence of any one of SEQ ID NOs: 6 to 69; Residues 7 to 153 or residues 198 to 344 of any one of SEQ ID NOs: 70 to 93 and at least and (b) an amino acid sequence having at least 80% sequence identity to the second recognition half-site. The second subunit further comprises a second hypervariable (HVR2) region that determines the specificity of the antibody. In some embodiments, the P23H recognition sequence is SEQ ID NO: 1, and the recombinant meganuclear The enzyme comprises a first subunit and a second subunit, and the first subunit is a sequence (a) recognizes one first recognition half site of sequence number 1, and Residues 198 to 344 of one of SEQ ID NOs: 70 to 93 and at least one of SEQ ID NOs: 7 to 153 (b) an amino acid sequence having at least 80% sequence identity; and (b) a first recognition half-site. The recombinant meganuclear vector comprises a first hypervariable (HVR1) region that determines the specificity for the recombinant meganuclear vector. ze is a second subunit that recognizes a second half site of SEQ ID NO: 1, and Residues 7 to 153 of any one of SEQ ID NOs: 6 to 69 or any one of SEQ ID NOs: 70 to 93 an amino acid sequence having at least 80% sequence identity with residues 198 to 344 of one of the sequences; and b) a second hypervariable (HVR2) region that determines specificity for the second recognition half-site; The compound further comprises a second subunit comprising:

[0023] In some embodiments, the P23H recognition sequence is SEQ ID NO: 1, and the first subunit residues 198 to 344 of any one of SEQ ID NOs: 6 to 69 or any one of SEQ ID NOs: 70 to 93 have at least 85%, 90%, or 95% sequence identity with residues 7-153 of any one of The second subunit comprises an amino acid sequence corresponding to any one of residues of SEQ ID NOs: 6 to 69. Residues 7 to 153 or residues 198 to 344 of any one of SEQ ID NOs: 70 to 93 and at least It includes amino acid sequences with 85%, 90%, or 95% sequence identity.

[0024] In another embodiment, the P23H recognition sequence is SEQ ID NO: 1 and the HVR1 region is (a) sequence (b) position 215 of any one of sequence numbers 6 to 69; or (b) any one of sequence numbers 70 to 93 It contains W or Y at a position corresponding to position 24. In another embodiment, P23 The H recognition sequence is SEQ ID NO: 1, and the HVR1 region is (a) any one of SEQ ID NOs: 6 to 69 (b) position 219 of one of SEQ ID NOs: 70 to 93; or (b) a position corresponding to position 28 of any one of SEQ ID NOs: 70 to 93 In another embodiment, the P23H recognition sequence is SEQ ID NO: 1 and the HVR One region is (a) position 259 of any one of SEQ ID NOs: 6 to 69; or (b) SEQ ID NO: 7 and V at a position corresponding to position 68 in any one of 0 to 93. The P23H recognition sequence is SEQ ID NO: 1, and the HVR1 region is (a) any of SEQ ID NOs: 6 to 69. (b) any one of positions 215, 219, and 259, respectively; or (b) SEQ ID NOs: 70-93 at positions corresponding to positions 24, 28, and 68, respectively, of any one of Contains one or more of Y, I, and V.

[0025] In another embodiment, the P23H recognition sequence is SEQ ID NO: 1 and the HVR2 region is (a) sequence (b) position 24 of any one of sequence numbers 6 to 69; or (b) any one of sequence numbers 70 to 93 In another embodiment, the P23 The H recognition sequence is SEQ ID NO: 1, and the HVR2 region is (a) any one of SEQ ID NOs: 6 to 69. (b) position 28 of one of SEQ ID NOs: 70 to 93; or (b) position 219 of any one of SEQ ID NOs: 70 to 93 In another embodiment, the P23H recognition sequence is SEQ ID NO: 1 and the HVR The two regions are (a) position 44 of any one of SEQ ID NOs: 6 to 69; or (b) SEQ ID NO: 70 In another embodiment, the compound of formula (I) contains an H at a position corresponding to position 235 of any one of formulas (I) to (I). The P23H recognition sequence is SEQ ID NO: 1, and the HVR2 region is (a) any of SEQ ID NOs: 6 to 69. (b) position 46 of any one of SEQ ID NOs: 70 to 93; or (b) position 237 of any one of SEQ ID NOs: 70 to 93 In another embodiment, the P23H recognition sequence is SEQ ID NO: 1. The HVR2 region is (a) position 70 of any one of SEQ ID NOs: 6 to 69; or (b) the sequence It contains W at a position corresponding to position 261 of any one of numbers 70 to 93. In this embodiment, the P23H recognition sequence is SEQ ID NO: 1, and the HVR2 region is (a) SEQ ID NO: 6 to or (b) positions 24, 28, 44, 46, and 70, respectively, of any one of the sequences 69; Positions 215, 219, 235, 237, and 238 of any one of numbers 70 to 93, respectively 61, and includes one or more of Y, M, F, H, S, and W.

[0026] In some embodiments, the recombinant meganuclease comprises a first subunit and a second subunit. It is a single-chain meganuclease containing a linker that covalently connects the subunits.

[0027] In some embodiments, the P23H recognition sequence is SEQ ID NO: 1, and the first subunit residues 198 to 344 of any one of SEQ ID NOs: 6 to 69 or any one of SEQ ID NOs: 70 to 93 In some embodiments, the P23H recognition sequence comprises any one of residues 7 to 153 of the sequence No. 1, and the second subunit is residues 7 to 15 of any one of SEQ ID NOs: 6 to 69. 3 or residues 198 to 344 of any one of SEQ ID NOs: 70 to 93.

[0028] In some specific embodiments, the P23H recognition sequence is SEQ ID NO: 1, and The enzyme comprises any one of the amino acid sequences of SEQ ID NOs: 6 to 93.

[0029] In some embodiments, the recombinant meganuclease is present in a wild-type RHO allele. The P23H recognition sequence (SEQ ID NOS: 1-4) is 100% identical to the corresponding 22 base pair recognition sequence. It preferentially recognizes and cleaves one of the two (eg, SEQ ID NO: 1 over SEQ ID NO: 5).

[0030] In another aspect, the present invention provides a method for producing a recombinant meganuclease encoding the recombinant meganuclease described herein. An isolated polynucleotide comprising the nucleic acid sequence is provided.

[0031] In another aspect, the present invention provides a method for producing a recombinant meganuclease encoding the recombinant meganuclease described herein. Recombinant DNA constructs are provided that include isolated polynucleotides that include nucleic acid sequences. In some embodiments, the recombinant DNA construct encodes a viral vector. In such embodiments, the viral vector may be a retroviral vector, a lentiviral vector, or vector, adenovirus vector, or adeno-associated virus (AAV) vector In certain embodiments, the recombinant DNA construct is a recombinant AAV vector. Code the .

[0032] In another aspect, the present invention provides a method for producing a recombinant meganuclease encoding the recombinant meganuclease described herein. A viral vector comprising an isolated polynucleotide comprising a nucleic acid sequence is provided. In some embodiments, the viral vector is a retroviral vector, a lentiviral vector, or The vector may be a vector, an adenovirus vector, or an AAV vector. In some embodiments, the viral vector may be a recombinant AAV vector.

[0033] In another aspect, the present invention relates to a method for treating RP, preferably caused by the P23H mutation. A pharmaceutical composition for the treatment of a subject with autosomal dominant RP is provided. The pharmaceutical composition comprises: a pharmaceutically acceptable carrier and: (a) in vivo in target cells (b) a nucleic acid encoding a recombinant meganuclease described herein that is expressed; or This antibody has specificity for one of the P23H recognition sequences (SEQ ID NOs: 1 to 4) in target cells. It may comprise a recombinant meganuclease protein as described herein.

[0034] In some embodiments, the nucleic acid encoding the recombinant meganuclease is mRNA. It is possible.

[0035] In other embodiments, the pharmaceutical composition comprises a recombinant DNA construct comprising the nucleic acid.

[0036] In some embodiments, the pharmaceutical composition comprises a viral vector that includes the nucleic acid. In one such embodiment, the viral vector is a retroviral vector, a lentiviral vector, or The vector may be an adenovirus vector, an adenovirus vector, or an AAV vector. In embodiments, the viral vector may be a recombinant AAV vector.

[0037] In another aspect, the present invention provides a recombinant gene as described herein for use as a pharmaceutical. The present invention further provides a ganucleases comprising RP, preferably P23H mutation. Preferably, for treating autosomal dominant RP caused by the P23H mutation allele. Heterozygous for the gene and functional, normal, or wild-type allele and the use of a recombinant meganuclear compound as described herein in the manufacture of a medicament for a subject to which the compound is administered. Provides for the use of Ze.

[0038] In another aspect, the invention is an isolated polynucleotide for use as a pharmaceutical. Thus, an isolated nucleic acid sequence encoding the recombinant meganuclease described herein is provided. The present invention further provides a polynucleotide comprising a RP, preferably a P23H mutation. Preferably, for treating autosomal dominant RP caused by the P23H mutation Heterogeneous and functional alleles, normal alleles, or wild-type alleles Use of the isolated polynucleotide in the manufacture of a medicament for a telozygous subject. wherein the isolated polynucleotide is a recombinant meganuclear vector as described herein. The present invention provides uses comprising a nucleic acid sequence encoding the enzyme.

[0039] In another aspect, the present invention provides a recombinant AAV vector for use as a medicament, comprising: An isolated polypeptide comprising a nucleic acid sequence encoding the recombinant meganuclease described herein. The present invention further provides a recombinant AAV vector comprising a RP, preferably a ribonucleotide. is a preferred drug for treating autosomal dominant RP caused by the P23H mutation. P23H mutant allele and functional allele, normal allele, or wild type Use of recombinant AAV vectors in the manufacture of medicines for subjects heterozygous for the allele 10. The use of a recombinant AAV vector as defined herein. Uses are provided that include an isolated polynucleotide comprising a nucleic acid sequence encoding a nuclease. do.

[0040] In another aspect, the present invention provides a method for treating a cancer of the brain by the P23H mutation in a subject in need thereof. Preferably, the P23H mutant allele and mechanism for treating RP caused by A subject heterozygous for a defective allele, a normal allele, or a wild-type allele The method comprises combining DNA from a target cell of the subject with DNA from a P23H mutant. The present invention relates to a method for cleaving a recognition sequence containing a mutation (e.g., SEQ ID NOs: 1 to 4) comprising the steps of: The method comprises contacting a target cell of the subject with a recombinant meganuclease that expresses the RHO gene. The gene contains a P23H recognition sequence in the offspring allele, and cleavage of the recognition sequence results in the RHO gene allele. inhibits the expression of

[0041] In some embodiments, the method further comprises detecting autosomal dominant staining caused by the P23H mutation. for treating chromosome dominant RP, preferably the P23H mutant allele and and subjects heterozygous for the functional, normal, or wild-type allele. It is for experimental purposes.

[0042] In some embodiments, the target cells in the subject are retinal target cells. In embodiments, the retinal cells are rod photoreceptor cells.

[0043] In some embodiments of the method, the nucleic acid encoding the recombinant meganuclease is a target In some such embodiments, the nucleic acid may be mRNA. In other such embodiments, the nucleic acid is introduced into the cell using a recombinant DNA construct. In yet other such embodiments, the nucleic acid is obtained from a retroviral vector, a lentiviral vector, or a Viral vectors such as HIV vectors, adenovirus vectors, or AAV vectors are used. In some particular embodiments, the recombinant meganuclei can be introduced into target cells using the The gene encoding the enzyme is delivered to target cells using a recombinant AAV vector.

[0044] In another embodiment of the method, the recombinant meganuclease protein of the invention is will be introduced.

[0045] In various embodiments of the method, a recombinant meganuclease protein or a recombinant meganuclease is Nucleic acids encoding the nucleases can be administered to a subject in pharmaceutical compositions described herein. do.

[0046] In another aspect, the present invention provides a method for producing a genetically modified cell. (a) obtaining cells containing at least one P23H RHO allele; and (b)(i) a nucleic acid encoding a recombinant meganuclease of the invention that is expressed in said cell; or (ii) introducing into said cell a nucleic acid sequence; or (ii) a recombinant meganuclease protein. wherein the recombinant meganuclease is a P23 RHO allele present on the P23H RHO allele. H recognition sequence, and the recombinant meganuclease recognizes the P23H recognition sequence. The P23H RHO allele is recognized and cleaved, and expression of the P23H RHO allele is due to the non-homologous end at the cleavage site. Preferably, the cells are depleted of the P23H mutant allele prior to modification. and heterozygous for the functional, normal, or wild-type allele. be.

[0047] In some embodiments of the method, the cell may be a eukaryotic cell. In such embodiments, the eukaryotic cell may be a pluripotent cell. The germ cells may be induced pluripotent stem (iPS) cells. In some particular embodiments, iPS cells The PS cells may be human iPS cells.

[0048] In other embodiments of the method, the nucleic acid may be mRNA.

[0049] In some embodiments of the method, the nucleic acid is introduced into the cell using a recombinant DNA construct. It can be introduced.

[0050] In some embodiments of the method, the nucleic acid is introduced into the cell using a viral vector. In some such embodiments, the viral vector may be a retrovirus. vector, lentiviral vector, adenoviral vector, or AAV vector. In certain embodiments, the viral vector is a recombinant AAV vector. could be.

[0051] In another aspect, the invention provides a method for the detection of RHO-related HIV-1-associated ... A genetically modified cell that expresses wild-type RHO protein and P23H RHO protein. The genetically modified cells do not express the gene and are produced according to the methods of the invention described herein. In some embodiments, the disrupted P23H allele is a meganuclease and This includes deletion mutations caused by NHEJ cleavage. In some embodiments, the genetically modified cells may be pluripotent cells, iPS cells, or human iPS cells. .

[0052] In another aspect, the present invention provides a method for treating a subject with RP caused by the P23H mutation. Preferably, the P23H mutant allele and the functional allele for treating the body, Pharmaceutical Compositions for Subjects Heterozygous for Normal or Wild-Type Alleles In a different embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a compound of the present invention. Any genetically modified cell of the present invention and / or any genetically modified cell produced according to the methods of the present invention. a wild-type RHO allele that expresses a wild-type RHO protein and a P23H and a genetically modified cell containing a disrupted P23H allele that does not express a RHO protein. In some embodiments, the disrupted P23H allele is obtained by the use of a meganuclease and an NH This includes deletion mutations caused by EJ cleavage.

[0053] In another aspect, the present invention provides a method for treating a cancer of the brain by the P23H mutation in a subject in need thereof. Preferably, the P23H mutant allele and mechanism for treating RP caused by A subject heterozygous for a defective allele, a normal allele, or a wild-type allele The method comprises administering to a subject a pharmaceutically acceptable carrier and a genetically modified organism of the present invention. mutant cells, which express a wild-type RHO protein and a wild-type RHO allele, and and genetically modified cells containing a disrupted P23H allele that does not express the H RHO protein. In some embodiments, the method further comprises administering to the subject a pharmaceutical composition described herein comprising: In this state, the disrupted P23H allele is cleaved by meganucleases and NHEJ. This includes the deletion mutations that occur.

[0054] In some embodiments of the method, the genetically modified cells can be delivered to a target tissue. Such target tissues may include the eye, particularly the retina.

[0055] Further, in some embodiments of the method, the genetically modified cells are genetically modified iPS cells. In such an embodiment, the genetically modified iPS cells are delivered to the target tissue. In some particular embodiments, the cells can be differentiated into cells that express wild-type RHO protein. The genetically modified iPS cells express wild-type rhodopsin protein but not P23H rhodopsin. They can differentiate into retinal cells, particularly rod photoreceptors, that do not express the syn protein. [Brief explanation of the drawings]

[0056] [Figure 1] P23H Recognition Sequence. A) Alignment of the 22-base pair P23H recognition sequence of SEQ ID NO: 1 with the corresponding 22-base pair recognition sequence (SEQ ID NO: 5) present in the wild-type human RHO gene allele. These sequences span nucleotides 49-70 of the P23H mutant or wild-type RHO gene coding sequence (SEQ ID NOs: 98 and 97, respectively). The C68A nucleotide substitution within the P23H recognition sequence is highlighted. B) The P23H recognition sequence of SEQ ID NO: 1 contains two recognition half-sites, designated RHO1 and RHO2. As shown, each recognition half-site contains 9 base pairs. The half-sites in the recognition sequence are separated by a 4-base pair central region. C) The recombinant meganuclease of the present invention contains two subunits: the first subunit binds to the first half-site (e.g., RHO1) of the P23H recognition sequence (e.g., SEQ ID NO: 1), and the second subunit binds to the second half-site (e.g., RHO2). In embodiments where the recombinant meganuclease is a single-chain meganuclease, the first subunit can be arranged as either an N-terminal or a C-terminal subunit. Similarly, the second subunit can be arranged as either an N-terminal or a C-terminal subunit. [Figures 2A-2F]Amino acid alignment of RHO1 binding subunits. A-F) Recombinant meganucleases encompassed by the present invention contain one subunit that binds to the 9 base pair RHO1 recognition half-site of SEQ ID NO: 1. Amino acid sequence alignments are shown for the RHO1 binding subunits (SEQ ID NOs: 102-188) of recombinant meganucleases set forth in SEQ ID NOs: 6-93. As shown, the RHO1 binding subunits of SEQ ID NOs: 6-69 contain residues 198-344, whereas the RHO1 binding subunits of SEQ ID NOs: 70-93 contain residues 7-153. As shown, each RHO1 binding subunit contains a 56 amino acid hypervariable region. Variable residues within the hypervariable region are shaded, further highlighting the most frequent amino acid at each position; the most frequently occurring residue is in bold, whereas the second most frequently occurring is in bold italics. With the exception of the Q or E residues at positions 80 or 271, residues outside the hypervariable regions are identical in each subunit (but this is not required) (see U.S. Patent No. 8,021,867). Nearly all of the RHO1-binding subunits shown in Figures 2A-2F share at least 90% sequence identity with the RHO1-binding subunit (residues 198-344) of the RHO2-L3-59 meganuclease (SEQ ID NO: 102). Residue numbers shown are those of SEQ ID NOs: 6-93. [Figures 3A-3F]Amino acid alignment of RHO2 binding subunits. A-F) Recombinant meganucleases encompassed by the present invention contain one subunit that binds to the 9-base pair RHO2 recognition half-site of SEQ ID NO:1. Amino acid sequence alignments are shown for the RHO2 binding subunits (SEQ ID NOs:190-277) of recombinant meganucleases set forth in SEQ ID NOs:6-93. As shown, the RHO2 binding subunits of SEQ ID NOs:6-69 contain residues 7-153, whereas the RHO2 binding subunits of SEQ ID NOs:70-93 contain residues 198-344. As shown, each RHO2 binding subunit contains a 56-amino acid hypervariable region. Variable residues within the hypervariable region are shaded, further highlighting the most frequent amino acid at each position; the most frequently occurring residue is in bold, whereas the second most frequently occurring is in bold italics. With the exception of the Q or E residues at positions 80 or 271, residues outside the hypervariable regions are identical in each subunit (but this is not required) (see U.S. Patent No. 8,021,867). Nearly all of the RHO2-binding subunits shown in Figures 3A-3F share at least 90% sequence identity with the RHO2-binding subunit (residues 7-153) of the RHO2-L3-59 meganuclease (SEQ ID NO: 190). Residue numbers shown are those of SEQ ID NOs: 6-93. [Figure 4]Schematic diagram of a reporter assay in CHO cells to evaluate recombinant meganucleases targeting the P23H recognition sequence. For the recombinant meganucleases described herein, CHO cell lines were produced in which a reporter cassette was stably integrated into the cell genome. The reporter cassette contained, from 5' to 3', an SV40 early promoter, the 5'2 / 3 of the GFP gene, a recognition sequence for an engineered meganuclease of the present invention, e.g., the P23H recognition sequence of any one of SEQ ID NOS: 1-4, the recognition sequence for the CHO-23 / 24 meganuclease (WO 2012 / 167192), and the 3'2 / 3 of the GFP gene. Cells stably transfected with this cassette did not express GFP in the absence of a DNA cleavage-inducing agent. Meganucleases were introduced by transfection of plasmid DNA or mRNA encoding the respective meganuclease. When DNA cleavage is induced in either of the meganuclease recognition sequences, the overlapping regions of the GFP gene recombine with each other to generate a functional GFP gene. The percentage of GFP-expressing cells can then be determined by flow cytometry as an indirect measure of the frequency of genome cleavage by the meganuclease. [Figure 5] Efficiency of recombinant meganucleases to recognize and cleave the P23H recognition sequence in a CHO cell reporter assay. A)-J) Each recombinant meganuclease shown in SEQ ID NOs: 6-93 was engineered to target the P23H recognition sequence and screened for efficacy in a CHO cell reporter assay. The results shown provide the percentage of GFP-expressing cells observed in each assay, which indicates the efficacy of each meganuclease for cleaving the P23H or CHO-23 / 24 recognition sequence. A negative control (RHO1-2bs) was also included in each assay. [Figure 6]Recombinant meganuclease efficacy over time in a CHO cell reporter assay. Recombinant meganucleases encompassed by the present invention were evaluated in a CHO reporter assay to determine the percentage of GFP-expressing cells 1, 4, 6, and 8 days after introduction of meganuclease-encoding mRNA into CHO reporter cells. At each time point, CHO-23 / 24 meganuclease was included as a positive control. [Figure 7] Selectivity of recombinant meganucleases. A)-F) A CHO cell reporter assay was used to determine the selectivity of recombinant meganucleases for the P23H recognition sequence (SEQ ID NO: 1) and the corresponding wild-type RHO recognition sequence (SEQ ID NO: 5). Recombinant meganucleases encompassed by the invention were introduced into cells containing the P23H recognition sequence ("RHO1-2 cells", gray bars) or the corresponding wild-type RHO recognition sequence ("RHO3-4 cells", black bars) to determine whether they could distinguish between mutant and wild-type targets. [Figure 8] Generation and expression of recombinant AAV vectors. A) Schematic representation of the recombinant AAV vector genome, with inverted terminal repeats (ITRs) at the 5' and 3' ends. The vector contains the coding sequence for the recombinant meganuclease RHO-1 / 2-L2-49 (SEQ ID NO: 8) operably linked to a cytomegalovirus early (CMV) promoter. The nuclease expression cassette was incorporated into a "packaging" plasmid, which was used in combination with an Ad helper plasmid to produce recombinant AAV capable of delivering the gene encoding the RHO-1 / 2-L2-49 meganuclease. B) Immunoblot of RHO-1 / 2-L2-49 meganuclease expression in recombinant AAV-transduced CHO cells after 24 hours. [Figure 9]Efficiency and selectivity of RHO-1 / 2-L2-49 meganuclease for the P23H recognition sequence (SEQ ID NO: 1) when expressed by recombinant AAV in a CHO cell reporter assay. CHO cells carrying either the wild-type recognition sequence (SEQ ID NO: 5; "RHO3-4 cells," black bars) or the P23H recognition sequence (SEQ ID NO: 1; "RHO1-2 cells," gray bars) were transduced with three different doses of an AAV2 vector encoding RHO-1 / 2-L2-49 meganuclease operably linked to a CMV promoter. [Figure 10] Stability of RHO-1 / 2-L2-49 meganuclease in CHO reporter cells after treatment with cycloheximide. [Figure 11] A) Vector map of pDS CMV RHO2_L3_59 plasmid (SEQ ID NO: 278). B) Vector map of pDS CMV RHO2_L5_14 plasmid (SEQ ID NO: 279). [Figure 12] CHO GFFP reporter assay demonstrating the specificity of RHO1-2 meganuclease for the RHO1-2 recognition sequence. CHO-K cells (control), WT RHO cells (containing the wild-type RHO sequence of SEQ ID NO: 5), or P23H RHO cells (containing the P23H RHO sequence of SEQ ID NO: 1) were transduced with low-, medium-, or high-titer recombinant AAV vectors encoding GFP protein, RHO2-L3-59 meganuclease (SEQ ID NO: 6), or RHO2-L5-14 meganuclease (SEQ ID NO: 7). The percentage of GFP-positive cells was determined in each cell line after transduction as a measure of recognition sequence cleavage. [Figure 13]Western blot analysis of meganuclease expression in CHO RHO1-2 cells after transduction with recombinant AAV vectors. P23H RHO cells containing the RHO1-2 recognition sequence were transduced with recombinant AAV vectors encoding RHO2-L3-59 meganuclease (SEQ ID NO: 6) or RHO2-L5-14 meganuclease (SEQ ID NO: 7) at 1e8, 1e9, or 2e9 viral genomes per cell. Cell lysates were analyzed for RHO1-2 recognition sequence cleavage (upper panel), GFP as a measure of meganuclease expression (lower panel), and β-actin expression as a loading control. Lanes 1-6 were transduced to express RHO2-L3-59 meganuclease. Lanes 7-12 were transduced to express RHO2-L5-14 meganuclease. [Figure 14] Vector map of pDS GRK1 RHO2_L5_14 plasmid (SEQ ID NO: 281). [Figure 15] Western blot analysis of meganuclease expression in mouse retinal cells after subretinal AAV injection. Wild-type mice were administered a recombinant AAV vector encoding RHO2-L5-14 meganuclease (SEQ ID NO: 7) by subretinal injection. Retinal cells were obtained from the left eye (OS) and right eye (OD) of five mice, and cell lysates were analyzed for the expression of RHO2-L5-14 meganuclease and β-actin by Western blot. DETAILED DESCRIPTION OF THE INVENTION

[0057] A brief description of arrays SEQ ID NO: 1 shows the nucleotide sequence of one P23H recognition sequence (RHO-1 / 2) .

[0058] SEQ ID NO: 2 shows the nucleotide sequence of one P23H recognition sequence (RHO-9 / 10). vinegar.

[0059] SEQ ID NO: 3 shows the nucleotide sequence of one P23H recognition sequence (RHO-11 / 12). show.

[0060] SEQ ID NO: 4 shows the nucleotide sequence of one P23H recognition sequence (RHO-13 / 14). show.

[0061] SEQ ID NO: 5 is the corresponding recognition sequence found in the wild-type RHO allele (i.e., RH The nucleotide sequence of the O3-4 recognition sequence is shown.

[0062] SEQ ID NO: 6 shows the amino acid sequence of the RHO2-L3-59 meganuclease.

[0063] SEQ ID NO: 7 shows the amino acid sequence of the RHO2-L5-14 meganuclease.

[0064] SEQ ID NO: 8 shows the amino acid sequence of the RHO-1 / 2-L2-49 meganuclease.

[0065] SEQ ID NO: 9 shows the amino acid sequence of the RHO1-2x.179 meganuclease.

[0066] SEQ ID NO: 10 shows the amino acid sequence of the RHO1-2x.4 meganuclease.

[0067] SEQ ID NO: 11 shows the amino acid sequence of the RHO1-2x.207 meganuclease.

[0068] SEQ ID NO: 12 shows the amino acid sequence of the RHO1-2x.277 meganuclease.

[0069] SEQ ID NO: 13 shows the amino acid sequence of the RHO1-2x.292 meganuclease.

[0070] SEQ ID NO: 14 shows the amino acid sequence of the RHO1-2x.324 meganuclease.

[0071] SEQ ID NO: 15 shows the amino acid sequence of the RHO1-2x.371 meganuclease.

[0072] SEQ ID NO: 16 shows the amino acid sequence of the RHO1-2x.164 meganuclease.

[0073] SEQ ID NO: 17 shows the amino acid sequence of the RHO1-2x.181 meganuclease.

[0074] SEQ ID NO: 18 shows the amino acid sequence of the RHO1-2x.184 meganuclease.

[0075] SEQ ID NO: 19 shows the amino acid sequence of RHO-1 / 2-L1-21 meganuclease .

[0076] SEQ ID NO: 20 shows the amino acid sequence of RHO-1 / 2-L1-43 meganuclease .

[0077] SEQ ID NO: 21 shows the amino acid sequence of RHO-1 / 2-L1-45 meganuclease .

[0078] SEQ ID NO: 22 shows the amino acid sequence of RHO-1 / 2-L1-60 meganuclease .

[0079] SEQ ID NO: 23 shows the amino acid sequence of RHO-1 / 2-L1-61 meganuclease .

[0080] SEQ ID NO: 24 shows the amino acid sequence of RHO-1 / 2-L1-58 meganuclease .

[0081] SEQ ID NO: 25 shows the amino acid sequence of RHO-1 / 2-L1-7 meganuclease.

[0082] SEQ ID NO: 26 shows the amino acid sequence of RHO-1 / 2-L1-13 meganuclease .

[0083] SEQ ID NO: 27 shows the amino acid sequence of RHO-1 / 2-L1-18 meganuclease .

[0084] SEQ ID NO: 28 shows the amino acid sequence of RHO-1 / 2-L1-70 meganuclease .

[0085] SEQ ID NO: 29 shows the amino acid sequence of RHO-1 / 2-L1-86 meganuclease .

[0086] SEQ ID NO: 30 shows the amino acid sequence of RHO-1 / 2-L2-13 meganuclease .

[0087] SEQ ID NO: 31 shows the amino acid sequence of RHO-1 / 2-L2-24 meganuclease .

[0088] SEQ ID NO: 32 shows the amino acid sequence of RHO-1 / 2-L2-37 meganuclease .

[0089] SEQ ID NO: 33 shows the amino acid sequence of RHO-1 / 2-L2-58 meganuclease .

[0090] SEQ ID NO: 34 shows the amino acid sequence of RHO-1 / 2-L2-31 meganuclease .

[0091] SEQ ID NO: 35 shows the amino acid sequence of RHO-1 / 2-L2-29 meganuclease .

[0092] SEQ ID NO: 36 shows the amino acid sequence of RHO-1 / 2-L2-61 meganuclease .

[0093] SEQ ID NO: 37 shows the amino acid sequence of RHO2-L3-2 meganuclease.

[0094] SEQ ID NO: 38 shows the amino acid sequence of RHO2-L3-3 meganuclease.

[0095] SEQ ID NO: 39 shows the amino acid sequence of RHO2-L3-5 meganuclease.

[0096] SEQ ID NO: 40 shows the amino acid sequence of RHO2-L3-10 meganuclease.

[0097] SEQ ID NO: 41 shows the amino acid sequence of the RHO2-L3-11 meganuclease.

[0098] SEQ ID NO: 42 shows the amino acid sequence of RHO2-L3-12 meganuclease.

[0099] SEQ ID NO: 43 shows the amino acid sequence of the RHO2-L3-13 meganuclease.

[0100] SEQ ID NO: 44 shows the amino acid sequence of RHO2-L3-28 meganuclease.

[0101] SEQ ID NO: 45 shows the amino acid sequence of the RHO2-L3-29 meganuclease.

[0102] SEQ ID NO: 46 shows the amino acid sequence of RHO2-L3-57 meganuclease.

[0103] SEQ ID NO: 47 shows the amino acid sequence of RHO2-L3-80 meganuclease.

[0104] SEQ ID NO: 48 shows the amino acid sequence of RHO2-L3-85 meganuclease.

[0105] SEQ ID NO: 49 shows the amino acid sequence of RHO2-L3-86 meganuclease.

[0106] SEQ ID NO: 50 shows the amino acid sequence of RHO2-L3-92 meganuclease.

[0107] SEQ ID NO: 51 shows the amino acid sequence of the RHO2-L3-4 meganuclease.

[0108] SEQ ID NO: 52 shows the amino acid sequence of the RHO2-L3-20 meganuclease.

[0109] SEQ ID NO: 53 shows the amino acid sequence of RHO2-L3-72 meganuclease.

[0110] SEQ ID NO: 54 shows the amino acid sequence of the RHO1-L1-4 meganuclease.

[0111] SEQ ID NO: 55 shows the amino acid sequence of RHO1-L1-8 meganuclease.

[0112] SEQ ID NO: 56 shows the amino acid sequence of RHO1-L1-13 meganuclease.

[0113] SEQ ID NO: 57 shows the amino acid sequence of the RHO1-L1-19 meganuclease.

[0114] SEQ ID NO: 58 shows the amino acid sequence of RHO1-L1-58 meganuclease.

[0115] SEQ ID NO: 59 shows the amino acid sequence of the RHO1-L1-69 meganuclease.

[0116] SEQ ID NO: 60 shows the amino acid sequence of the RHO1-L1-80 meganuclease.

[0117] SEQ ID NO: 61 shows the amino acid sequence of RHO1-L1-82 meganuclease.

[0118] SEQ ID NO: 62 shows the amino acid sequence of RHO1-L1-73 meganuclease.

[0119] SEQ ID NO: 63 shows the amino acid sequence of RHO1-L1-85 meganuclease.

[0120] SEQ ID NO: 64 shows the amino acid sequence of RHO1-L1-86 meganuclease.

[0121] SEQ ID NO: 65 shows the amino acid sequence of RHO-1 / 2-L4-10 meganuclease .

[0122] SEQ ID NO: 66 shows the amino acid sequence of RHO-1 / 2-L4-29 meganuclease .

[0123] SEQ ID NO: 67 shows the amino acid sequence of RHO-1 / 2-L4-65 meganuclease .

[0124] SEQ ID NO: 68 shows the amino acid sequence of RHO-1 / 2-L4-66 meganuclease .

[0125] SEQ ID NO: 69 shows the amino acid sequence of RHO-1 / 2-L4-85 meganuclease .

[0126] SEQ ID NO: 70 shows the amino acid sequence of RHO1-2x.216 meganuclease.

[0127] SEQ ID NO: 71 shows the amino acid sequence of RHO1-2x.241 meganuclease.

[0128] SEQ ID NO: 72 shows the amino acid sequence of RHO1-2x.94 meganuclease.

[0129] SEQ ID NO: 73 shows the amino acid sequence of RHO1-2x.95 meganuclease.

[0130] SEQ ID NO: 74 shows the amino acid sequence of RHO1-2x.1 meganuclease.

[0131] SEQ ID NO: 75 shows the amino acid sequence of RHO1-2x.60 meganuclease.

[0132] SEQ ID NO: 76 shows the amino acid sequence of the RHO1-2x.74 meganuclease.

[0133] SEQ ID NO: 77 shows the amino acid sequence of RHO1-2x.88 meganuclease.

[0134] SEQ ID NO: 78 shows the amino acid sequence of the RHO1-2x.294 meganuclease.

[0135] SEQ ID NO: 79 shows the amino acid sequence of the RHO1-2x.302 meganuclease.

[0136] SEQ ID NO: 80 shows the amino acid sequence of RHO1-2x.306 meganuclease.

[0137] SEQ ID NO: 81 shows the amino acid sequence of RHO1-2x.338 meganuclease.

[0138] SEQ ID NO: 82 shows the amino acid sequence of RHO1-2x.348 meganuclease.

[0139] SEQ ID NO: 83 shows the amino acid sequence of RHO1-2x.356 meganuclease.

[0140] SEQ ID NO: 84 shows the amino acid sequence of the RHO1-2x.364 meganuclease.

[0141] SEQ ID NO: 85 shows the amino acid sequence of RHO1-2x.142 meganuclease.

[0142] SEQ ID NO: 86 shows the amino acid sequence of RHO1-2x.177 meganuclease.

[0143] SEQ ID NO: 87 shows the amino acid sequence of RHO1-2x.148 meganuclease.

[0144] SEQ ID NO: 88 shows the amino acid sequence of RHO1-2x.20 meganuclease.

[0145] SEQ ID NO: 89 shows the amino acid sequence of RHO1-2x.55 meganuclease.

[0146] SEQ ID NO: 90 shows the amino acid sequence of the RHO1-2x.197 meganuclease.

[0147] SEQ ID NO: 91 shows the amino acid sequence of RHO1-2x.252 meganuclease.

[0148] SEQ ID NO: 92 shows the amino acid sequence of RHO1-2x.372 meganuclease.

[0149] SEQ ID NO: 93 shows the amino acid sequence of RHO1-2x.151 meganuclease.

[0150] SEQ ID NO: 94 shows the amino acid sequence of the wild-type I-CreI meganuclease.

[0151] SEQ ID NO: 95 is the amino acid sequence of the LAGLIDADG motif of I-CreI meganuclease. The amino acid sequence is shown.

[0152] SEQ ID NO: 96 shows the nucleic acid sequence of the coding region of wild-type human rhodopsin.

[0153] SEQ ID NO: 97 shows the nucleic acid sequence of the coding region of mutant P23H rhodopsin.

[0154] SEQ ID NO: 98 shows the nucleic acid sequence of the wild-type human rhodopsin gene.

[0155] SEQ ID NO: 99 contains a C68A mutation that encodes a P23H substitution in rhodopsin. 1 shows the nucleic acid sequence of the human rhodopsin gene.

[0156] SEQ ID NO: 100 shows the amino acid sequence of wild-type human rhodopsin.

[0157] SEQ ID NO: 101 shows the amino acid sequence of mutant P23H rhodopsin.

[0158] SEQ ID NO: 102 represents residues 198 to 344 of the RHO2-L3-59 meganuclease. vinegar.

[0159] SEQ ID NO: 103 represents residues 198 to 344 of the RHO2-L5-14 meganuclease. vinegar.

[0160] SEQ ID NO: 104 identifies residues 198-34 of the RHO-1 / 2-L2-49 meganuclease Shows 4.

[0161] SEQ ID NO: 105 identifies residues 198-344 of the RHO1-2x.179 meganuclease. show.

[0162] SEQ ID NO: 106 shows residues 198 to 344 of the RHO1-2x.4 meganuclease .

[0163] SEQ ID NO: 107 identifies residues 198-344 of the RHO1-2x.207 meganuclease. show.

[0164] SEQ ID NO: 108 identifies residues 198-344 of the RHO1-2x.277 meganuclease. show.

[0165] SEQ ID NO: 109 identifies residues 198-344 of the RHO1-2x.292 meganuclease. show.

[0166] SEQ ID NO: 110 identifies residues 198-344 of the RHO1-2x.324 meganuclease. show.

[0167] SEQ ID NO: 111 identifies residues 198-344 of the RHO1-2x.371 meganuclease. show.

[0168] SEQ ID NO: 112 identifies residues 198-344 of the RHO1-2x.164 meganuclease. show.

[0169] SEQ ID NO: 113 identifies residues 198-344 of the RHO1-2x.181 meganuclease. show.

[0170] SEQ ID NO: 114 identifies residues 198-344 of the RHO1-2x.184 meganuclease. show.

[0171] SEQ ID NO: 115 identifies residues 198-34 of the RHO-1 / 2-L1-21 meganuclease Shows 4.

[0172] SEQ ID NO: 116 identifies residues 198-34 of the RHO-1 / 2-L1-43 meganuclease Shows 4.

[0173] SEQ ID NO: 117 identifies residues 198-34 of the RHO-1 / 2-L1-45 meganuclease Shows 4.

[0174] SEQ ID NO: 118 identifies residues 198-34 of the RHO-1 / 2-L1-60 meganuclease Shows 4.

[0175] SEQ ID NO: 119 identifies residues 198-34 of the RHO-1 / 2-L1-61 meganuclease Shows 4.

[0176] SEQ ID NO: 120 identifies residues 198-34 of the RHO-1 / 2-L1-58 meganuclease Shows 4.

[0177] SEQ ID NO: 121 identifies residues 198 to 344 of the RHO-1 / 2-L1-7 meganuclease. Shows.

[0178] SEQ ID NO: 122 identifies residues 198-34 of the RHO-1 / 2-L1-13 meganuclease Shows 4.

[0179] SEQ ID NO: 123 identifies residues 198-34 of the RHO-1 / 2-L1-18 meganuclease Shows 4.

[0180] SEQ ID NO: 124 identifies residues 198-34 of the RHO-1 / 2-L1-70 meganuclease Shows 4.

[0181] SEQ ID NO: 125 identifies residues 198-34 of the RHO-1 / 2-L1-86 meganuclease Shows 4.

[0182] SEQ ID NO: 126 identifies residues 198-34 of the RHO-1 / 2-L2-13 meganuclease Shows 4.

[0183] SEQ ID NO: 127 identifies residues 198-34 of the RHO-1 / 2-L2-24 meganuclease Shows 4.

[0184] SEQ ID NO: 128 identifies residues 198-34 of the RHO-1 / 2-L2-37 meganuclease Shows 4.

[0185] SEQ ID NO: 129 identifies residues 198-34 of the RHO-1 / 2-L2-58 meganuclease Shows 4.

[0186] SEQ ID NO: 130 identifies residues 198-34 of the RHO-1 / 2-L2-31 meganuclease Shows 4.

[0187] SEQ ID NO: 131 identifies residues 198-34 of the RHO-1 / 2-L2-29 meganuclease Shows 4.

[0188] SEQ ID NO: 132 identifies residues 198-34 of the RHO-1 / 2-L2-61 meganuclease Shows 4.

[0189] SEQ ID NO: 133 represents residues 198 to 344 of the RHO2-L3-2 meganuclease .

[0190] SEQ ID NO: 134 represents residues 198 to 344 of the RHO2-L3-3 meganuclease .

[0191] SEQ ID NO: 135 shows residues 198 to 344 of the RHO2-L3-5 meganuclease .

[0192] SEQ ID NO: 136 represents residues 198 to 344 of the RHO2-L3-10 meganuclease. vinegar.

[0193] SEQ ID NO: 137 represents residues 198 to 344 of the RHO2-L3-11 meganuclease. vinegar.

[0194] SEQ ID NO: 138 represents residues 198 to 344 of the RHO2-L3-12 meganuclease. vinegar.

[0195] SEQ ID NO: 139 represents residues 198 to 344 of the RHO2-L3-13 meganuclease. vinegar.

[0196] SEQ ID NO: 140 represents residues 198 to 344 of the RHO2-L3-28 meganuclease. vinegar.

[0197] SEQ ID NO: 141 represents residues 198 to 344 of the RHO2-L3-29 meganuclease. vinegar.

[0198] SEQ ID NO: 142 represents residues 198 to 344 of the RHO2-L3-57 meganuclease. vinegar.

[0199] SEQ ID NO: 143 represents residues 198 to 344 of the RHO2-L3-80 meganuclease. vinegar.

[0200] SEQ ID NO: 144 represents residues 198 to 344 of the RHO2-L3-85 meganuclease. vinegar.

[0201] SEQ ID NO: 145 represents residues 198 to 344 of the RHO2-L3-86 meganuclease. vinegar.

[0202] SEQ ID NO: 146 represents residues 198 to 344 of the RHO2-L3-92 meganuclease. vinegar.

[0203] SEQ ID NO: 147 shows residues 198 to 344 of the RHO2-L3-4 meganuclease .

[0204] SEQ ID NO: 148 represents residues 198 to 344 of the RHO2-L3-20 meganuclease. vinegar.

[0205] SEQ ID NO: 149 represents residues 198 to 344 of the RHO2-L3-72 meganuclease. vinegar.

[0206] SEQ ID NO: 150 shows residues 198 to 344 of the RHO1-L1-4 meganuclease .

[0207] SEQ ID NO: 151 shows residues 198 to 344 of the RHO1-L1-8 meganuclease .

[0208] SEQ ID NO: 152 represents residues 198 to 344 of the RHO1-L1-13 meganuclease. vinegar.

[0209] SEQ ID NO: 153 represents residues 198 to 344 of the RHO1-L1-19 meganuclease. vinegar.

[0210] SEQ ID NO: 154 represents residues 198 to 344 of the RHO1-L1-58 meganuclease. vinegar.

[0211] SEQ ID NO: 155 represents residues 198 to 344 of the RHO1-L1-69 meganuclease. vinegar.

[0212] SEQ ID NO: 156 represents residues 198 to 344 of the RHO1-L1-80 meganuclease. vinegar.

[0213] SEQ ID NO: 157 represents residues 198 to 344 of the RHO1-L1-82 meganuclease. vinegar.

[0214] SEQ ID NO: 158 represents residues 198 to 344 of the RHO1-L1-73 meganuclease. vinegar.

[0215] SEQ ID NO: 159 represents residues 198 to 344 of the RHO1-L1-85 meganuclease. vinegar.

[0216] SEQ ID NO: 160 represents residues 198 to 344 of the RHO1-L1-86 meganuclease. vinegar.

[0217] SEQ ID NO: 161 identifies residues 198-34 of the RHO-1 / 2-L4-10 meganuclease Shows 4.

[0218] SEQ ID NO: 162 identifies residues 198-34 of the RHO-1 / 2-L4-29 meganuclease Shows 4.

[0219] SEQ ID NO: 163 identifies residues 198-34 of the RHO-1 / 2-L4-65 meganuclease Shows 4.

[0220] SEQ ID NO: 164 identifies residues 198-34 of RHO-1 / 2-L4-66 meganuclease Shows 4.

[0221] SEQ ID NO: 165 identifies residues 198-34 of the RHO-1 / 2-L4-85 meganuclease Shows 4.

[0222] SEQ ID NO: 166 shows residues 7 to 153 of the RHO1-2x.216 meganuclease .

[0223] SEQ ID NO: 167 shows residues 7 to 153 of the RHO1-2x.241 meganuclease .

[0224] SEQ ID NO: 168 shows residues 7 to 153 of the RHO1-2x.94 meganuclease.

[0225] SEQ ID NO: 169 shows residues 7 to 153 of the RHO1-2x.95 meganuclease.

[0226] SEQ ID NO: 170 shows residues 7 to 153 of the RHO1-2x.1 meganuclease.

[0227] SEQ ID NO: 171 shows residues 7 to 153 of the RHO1-2x.60 meganuclease.

[0228] SEQ ID NO: 172 shows residues 7 to 153 of the RHO1-2x.74 meganuclease.

[0229] SEQ ID NO: 173 shows residues 7 to 153 of the RHO1-2x.88 meganuclease.

[0230] SEQ ID NO: 174 shows residues 7 to 153 of the RHO1-2x.294 meganuclease .

[0231] SEQ ID NO: 175 shows residues 7 to 153 of the RHO1-2x.302 meganuclease .

[0232] SEQ ID NO: 176 shows residues 7 to 153 of the RHO1-2x.306 meganuclease .

[0233] SEQ ID NO: 177 shows residues 7 to 153 of the RHO1-2x.338 meganuclease .

[0234] SEQ ID NO: 178 shows residues 7 to 153 of the RHO1-2x.348 meganuclease .

[0235] SEQ ID NO: 179 shows residues 7 to 153 of the RHO1-2x.356 meganuclease .

[0236] SEQ ID NO: 180 shows residues 7 to 153 of the RHO1-2x.364 meganuclease .

[0237] SEQ ID NO: 181 shows residues 7 to 153 of the RHO1-2x.142 meganuclease .

[0238] SEQ ID NO: 182 shows residues 7 to 153 of the RHO1-2x.177 meganuclease .

[0239] SEQ ID NO: 183 represents residues 7 to 153 of the RHO1-2x.148 meganuclease .

[0240] SEQ ID NO: 184 shows residues 7 to 153 of the RHO1-2x.20 meganuclease.

[0241] SEQ ID NO: 185 shows residues 7 to 153 of the RHO1-2x.55 meganuclease.

[0242] SEQ ID NO: 186 shows residues 7 to 153 of the RHO1-2x.197 meganuclease .

[0243] SEQ ID NO: 187 shows residues 7 to 153 of the RHO1-2x.252 meganuclease .

[0244] SEQ ID NO: 188 represents residues 7 to 153 of the RHO1-2x.372 meganuclease .

[0245] SEQ ID NO: 189 shows residues 7 to 153 of the RHO1-2x.151 meganuclease .

[0246] SEQ ID NO: 190 shows residues 7 to 153 of the RHO2-L3-59 meganuclease.

[0247] SEQ ID NO: 191 shows residues 7 to 153 of the RHO2-L5-14 meganuclease.

[0248] SEQ ID NO: 192 identifies residues 7 to 153 of the RHO-1 / 2-L2-49 meganuclease. show.

[0249] SEQ ID NO: 193 shows residues 7 to 153 of the RHO1-2x.179 meganuclease .

[0250] SEQ ID NO: 194 shows residues 7 to 153 of the RHO1-2x.4 meganuclease.

[0251] SEQ ID NO: 195 shows residues 7 to 153 of the RHO1-2x.207 meganuclease .

[0252] SEQ ID NO: 196 shows residues 7 to 153 of the RHO1-2x.277 meganuclease .

[0253] SEQ ID NO: 197 shows residues 7 to 153 of the RHO1-2x.292 meganuclease .

[0254] SEQ ID NO: 198 shows residues 7 to 153 of the RHO1-2x.324 meganuclease .

[0255] SEQ ID NO: 199 shows residues 7 to 153 of the RHO1-2x.371 meganuclease .

[0256] SEQ ID NO: 200 shows residues 7 to 153 of the RHO1-2x.164 meganuclease .

[0257] SEQ ID NO: 201 shows residues 7 to 153 of the RHO1-2x.181 meganuclease .

[0258] SEQ ID NO: 202 shows residues 7 to 153 of the RHO1-2x.184 meganuclease .

[0259] SEQ ID NO: 203 identifies residues 7 to 153 of the RHO-1 / 2-L1-21 meganuclease. show.

[0260] SEQ ID NO: 204 identifies residues 7-153 of the RHO-1 / 2-L1-43 meganuclease. show.

[0261] SEQ ID NO: 205 identifies residues 7-153 of the RHO-1 / 2-L1-45 meganuclease. show.

[0262] SEQ ID NO: 206 identifies residues 7-153 of the RHO-1 / 2-L1-60 meganuclease. show.

[0263] SEQ ID NO: 207 identifies residues 7-153 of the RHO-1 / 2-L1-61 meganuclease. show.

[0264] SEQ ID NO: 208 identifies residues 7 to 153 of RHO-1 / 2-L1-58 meganuclease. show.

[0265] SEQ ID NO: 209 represents residues 7 to 153 of the RHO-1 / 2-L1-7 meganuclease. vinegar.

[0266] SEQ ID NO: 210 identifies residues 7 to 153 of the RHO-1 / 2-L1-13 meganuclease. show.

[0267] SEQ ID NO: 211 identifies residues 7-153 of the RHO-1 / 2-L1-18 meganuclease. show.

[0268] SEQ ID NO: 212 identifies residues 7-153 of the RHO-1 / 2-L1-70 meganuclease. show.

[0269] SEQ ID NO: 213 identifies residues 7-153 of RHO-1 / 2-L1-86 meganuclease. show.

[0270] SEQ ID NO: 214 identifies residues 7-153 of the RHO-1 / 2-L2-13 meganuclease. show.

[0271] SEQ ID NO: 215 identifies residues 7-153 of the RHO-1 / 2-L2-24 meganuclease. show.

[0272] SEQ ID NO: 216 identifies residues 7-153 of the RHO-1 / 2-L2-37 meganuclease. show.

[0273] SEQ ID NO: 217 identifies residues 7-153 of RHO-1 / 2-L2-58 meganuclease. show.

[0274] SEQ ID NO: 218 identifies residues 7-153 of the RHO-1 / 2-L2-31 meganuclease. show.

[0275] SEQ ID NO: 219 identifies residues 7-153 of the RHO-1 / 2-L2-29 meganuclease. show.

[0276] SEQ ID NO: 220 identifies residues 7-153 of the RHO-1 / 2-L2-61 meganuclease. show.

[0277] SEQ ID NO: 221 shows residues 7 to 153 of the RHO2-L3-2 meganuclease.

[0278] SEQ ID NO: 222 shows residues 7 to 153 of the RHO2-L3-3 meganuclease.

[0279] SEQ ID NO: 223 shows residues 7 to 153 of the RHO2-L3-5 meganuclease.

[0280] SEQ ID NO: 224 shows residues 7 to 153 of the RHO2-L3-10 meganuclease.

[0281] SEQ ID NO: 225 shows residues 7 to 153 of the RHO2-L3-11 meganuclease.

[0282] SEQ ID NO: 226 shows residues 7 to 153 of the RHO2-L3-12 meganuclease.

[0283] SEQ ID NO: 227 shows residues 7 to 153 of the RHO2-L3-13 meganuclease.

[0284] SEQ ID NO: 228 shows residues 7 to 153 of the RHO2-L3-28 meganuclease.

[0285] SEQ ID NO: 229 shows residues 7 to 153 of the RHO2-L3-29 meganuclease.

[0286] SEQ ID NO: 230 shows residues 7 to 153 of the RHO2-L3-57 meganuclease.

[0287] SEQ ID NO: 231 shows residues 7 to 153 of the RHO2-L3-80 meganuclease.

[0288] SEQ ID NO: 232 shows residues 7 to 153 of the RHO2-L3-85 meganuclease.

[0289] SEQ ID NO: 233 shows residues 7 to 153 of the RHO2-L3-86 meganuclease.

[0290] SEQ ID NO: 234 shows residues 7 to 153 of the RHO2-L3-92 meganuclease.

[0291] SEQ ID NO: 235 shows residues 7 to 153 of the RHO2-L3-4 meganuclease.

[0292] SEQ ID NO: 236 shows residues 7 to 153 of the RHO2-L3-20 meganuclease.

[0293] SEQ ID NO: 237 shows residues 7 to 153 of the RHO2-L3-72 meganuclease.

[0294] SEQ ID NO: 238 shows residues 7 to 153 of the RHO1-L1-4 meganuclease.

[0295] SEQ ID NO: 239 shows residues 7 to 153 of the RHO1-L1-8 meganuclease.

[0296] SEQ ID NO: 240 shows residues 7 to 153 of the RHO1-L1-13 meganuclease.

[0297] SEQ ID NO: 241 shows residues 7 to 153 of the RHO1-L1-19 meganuclease.

[0298] SEQ ID NO: 242 shows residues 7 to 153 of the RHO1-L1-58 meganuclease.

[0299] SEQ ID NO: 243 shows residues 7 to 153 of the RHO1-L1-69 meganuclease.

[0300] SEQ ID NO: 244 shows residues 7 to 153 of the RHO1-L1-80 meganuclease.

[0301] SEQ ID NO: 245 shows residues 7 to 153 of the RHO1-L1-82 meganuclease.

[0302] SEQ ID NO: 246 shows residues 7 to 153 of the RHO1-L1-73 meganuclease.

[0303] SEQ ID NO: 244 shows residues 7 to 153 of the RHO1-L1-85 meganuclease.

[0304] SEQ ID NO: 248 shows residues 7 to 153 of the RHO1-L1-86 meganuclease.

[0305] SEQ ID NO: 249 identifies residues 7 to 153 of the RHO-1 / 2-L4-10 meganuclease. show.

[0306] SEQ ID NO: 250 identifies residues 7-153 of the RHO-1 / 2-L4-29 meganuclease. show.

[0307] SEQ ID NO: 251 identifies residues 7-153 of the RHO-1 / 2-L4-65 meganuclease. show.

[0308] SEQ ID NO: 252 identifies residues 7-153 of RHO-1 / 2-L4-66 meganuclease. show.

[0309] SEQ ID NO: 253 identifies residues 7 to 153 of the RHO-1 / 2-L4-85 meganuclease. show.

[0310] SEQ ID NO: 254 identifies residues 198-344 of the RHO1-2x.216 meganuclease. show.

[0311] SEQ ID NO: 255 identifies residues 198-344 of the RHO1-2x.241 meganuclease. show.

[0312] SEQ ID NO: 256 represents residues 198 to 344 of the RHO1-2x.94 meganuclease. vinegar.

[0313] SEQ ID NO: 257 represents residues 198 to 344 of the RHO1-2x.95 meganuclease. vinegar.

[0314] SEQ ID NO: 258 represents residues 198 to 344 of the RHO1-2x.1 meganuclease .

[0315] SEQ ID NO: 259 represents residues 198 to 344 of the RHO1-2x.60 meganuclease. vinegar.

[0316] SEQ ID NO: 260 represents residues 198 to 344 of the RHO1-2x.74 meganuclease. vinegar.

[0317] SEQ ID NO: 261 represents residues 198 to 344 of the RHO1-2x.88 meganuclease. vinegar.

[0318] SEQ ID NO: 262 identifies residues 198-344 of the RHO1-2x.294 meganuclease. show.

[0319] SEQ ID NO: 263 identifies residues 198-344 of the RHO1-2x.302 meganuclease. show.

[0320] SEQ ID NO: 264 identifies residues 198-344 of the RHO1-2x.306 meganuclease. show.

[0321] SEQ ID NO: 265 identifies residues 198-344 of the RHO1-2x.338 meganuclease. show.

[0322] SEQ ID NO: 266 identifies residues 198-344 of the RHO1-2x.348 meganuclease. show.

[0323] SEQ ID NO: 267 identifies residues 198-344 of the RHO1-2x.356 meganuclease. show.

[0324] SEQ ID NO: 268 identifies residues 198-344 of the RHO1-2x.364 meganuclease. show.

[0325] SEQ ID NO: 269 identifies residues 198-344 of the RHO1-2x.142 meganuclease. show.

[0326] SEQ ID NO: 270 identifies residues 198-344 of the RHO1-2x.177 meganuclease. show.

[0327] SEQ ID NO: 271 identifies residues 198-344 of the RHO1-2x.148 meganuclease. show.

[0328] SEQ ID NO: 272 represents residues 198 to 344 of the RHO1-2x.20 meganuclease. vinegar.

[0329] SEQ ID NO: 273 represents residues 198 to 344 of the RHO1-2x.55 meganuclease. vinegar.

[0330] SEQ ID NO: 274 identifies residues 198-344 of the RHO1-2x.197 meganuclease. show.

[0331] SEQ ID NO: 275 identifies residues 198-344 of the RHO1-2x.252 meganuclease. show.

[0332] SEQ ID NO: 276 identifies residues 198-344 of the RHO1-2x.372 meganuclease. show.

[0333] SEQ ID NO: 277 identifies residues 198-344 of the RHO1-2x.151 meganuclease. show.

[0334] SEQ ID NO: 278 shows the nucleic acid sequence of the pDS CMV RHO2_L3_59 plasmid vinegar.

[0335] SEQ ID NO: 279 shows the nucleic acid sequence of the pDS CMV RHO2_L5_14 plasmid vinegar.

[0336] SEQ ID NO: 280 sets forth the nucleic acid sequence of the pDS GRK1 RHO2_L3_59 plasmid. show.

[0337] SEQ ID NO: 281 sets forth the nucleic acid sequence of the pDS GRK1 RHO2_L5_14 plasmid. show.

[0338] Detailed Description of the Invention 1.1 References and Definitions The patent and scientific literature referred to herein establishes knowledge that is available to those skilled in the art. Issued U.S. patents, permitted applications, published foreign applications, and references cited in the specification (GenBank k database sequences) are each specifically and individually incorporated by reference. to the same extent as if expressly indicated.

[0339] This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are to be construed as an allegation that this disclosure is thorough and complete. For example, the following description of one embodiment is provided to fully convey the scope of the present invention to those skilled in the art. Features illustrated in one embodiment may be incorporated into other embodiments and may be used interchangeably with the examples provided with respect to a particular embodiment. Features shown may be omitted from the embodiment. For example, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art. and do not depart from the present invention.

[0340] Unless otherwise defined, all technical and scientific terms used herein refer to the The terms "a," "b," and "c" have the same meaning as commonly understood by a person skilled in the art. The terminology used in describing the present invention is for the purpose of describing particular embodiments only. It is not intended to limit the present invention.

[0341] All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. The bodies are incorporated herein by reference.

[0342] As used herein, "a," "an," or "the" refers to "A" cell can mean one or more than one. For example, "a" cell means a single cell. It can mean a cell or a number of cells.

[0343] As used herein, unless specifically indicated otherwise, the word "or" means "either" or "and / or" is used in the inclusive sense rather than the exclusive sense of "either / or."

[0344] As used herein, the term "meganuclease" refers to a nucleic acid having more than 12 base pairs. It refers to an endonuclease that binds to double-stranded DNA at a recognition sequence. The recognition sequence of the meganuclease is 22 base pairs. an endonuclease derived from I-CreI, an engineered mutant of I-CreI, For example, DNA binding specificity, DNA cleavage activity, DNA binding affinity, or dimerization properties It can also refer to engineered mutants that are modified relative to the native I-CreI. Methods for producing such engineered variants of eI are known in the art (e.g., , WO 2007 / 047859). As used herein, meganucleases are Binds to double-stranded DNA as a heterodimer. Meganucleases also bind to double-stranded DNA via peptide linkers. A set of DNA-binding domains was connected to a single polypeptide using a single-chain meganucleotide It can be "crease".

[0345] As used herein, the term "single-chain meganuclease" refers to a single-chain meganuclease that is A single-chain polypeptide refers to a polypeptide comprising a pair of meganuclease subunits connected by a single chain. Meganucleases have the structure: N-terminal subunit-linker-C-terminal subunit. The two meganuclease subunits generally do not have identical amino acid sequences, will recognize non-identical DNA sequences. Thus, single-chain meganucleases are typically , cleaves pseudopalindromic or nonpalindromic recognition sequences. The enzymes are called "single-chain heterodimers" or "single-chain heterodimer meganucleases." For clarity, unless otherwise specified, "meganuclear" refers to a dimer. The term "meganuclease" refers to a dimeric meganuclease or a single-chain meganuclease. Methods for producing single-chain meganuclease variants of I-CreI are known in the art. These methods are well known in the art (e.g., International Publication No. WO 2009 / 059195; Li, et al. 2009) Nucleic Acids Res.37:1650-62;Grizot ,et al.(2009)Nucleic Acids Res.37:5405-1 9) The term "homing endonuclease" is a derivative of the term "meganuclease." It is synonymous with the word.

[0346] As used herein, the term "linker" refers to a linker that connects two meganuclease subunits. Refers to an exogenous peptide sequence used to connect units into a single polypeptide. The anchor may have a sequence found in the native protein or may be a sequence that is unique to any native protein. The linker may be an artificial sequence not found in other known sequences. The linker may be flexible and lack secondary structure. or may have a tendency to form a particular three-dimensional structure under physiological conditions. Examples include, but are not limited to, those covered by U.S. Pat. No. 8,445,251. In some embodiments, the linker may be selected from the group consisting of any one of the residues of SEQ ID NOs: 6-93. It may have an amino acid sequence comprising residues 154-195.

[0347] As used herein with respect to a protein, the term "recombinant" refers to a protein and applying genetic engineering techniques to cells or organisms that express the nucleic acids and proteins encoding the As a result, it means having an altered amino acid sequence. The term "genetic engineering" refers to a gene having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques. transfection, transformation, and other gene transfer techniques; homologous recombination; site-specific By this definition, naturally occurring genes are included in the a protein having the same amino acid sequence as the protein, but which has been cloned and A protein produced in a heterologous host by expression is not considered recombinant.

[0348] As used herein, the term "wild type" refers to an allele of the same type of gene. The most common naturally occurring alleles (i.e., polynucleotide sequences) in a population are identified. A polypeptide encoded by a wild-type allele has its original function. The term "wild-type" also refers to a polypeptide encoded by a wild-type allele. Wild-type alleles (i.e., polynucleotides) and polypeptides are expressed as a sequence that is specific to the wild-type sequence. Mutant or variant alleles and polypeptides all contain one or more mutations and / or substitutions. A wild-type allele or polypeptide is a polypeptide that is distinct from a normal phenotype in an organism. whereas a mutant or variant allele or polypeptide may confer some In some cases, it may confer an altered phenotype. 3H rhodopsin protein is distinguishable from wild-type RHO alleles and rhodopsin protein Additionally, wild-type homing endonucleases may be recombinant or non-naturally occurring. It is distinguishable from other meganucleases.

[0349] As used herein with respect to recombinant proteins, the term "modified" refers to a modification of a reference sequence. of amino acid residues in the recombinant sequence compared to a sequence (e.g., a wild-type or native sequence). Any insertion, deletion, or substitution is meant.

[0350] As used herein, the term "recognition sequence" refers to a sequence that is bound by a meganuclease. In the case of the recombinant meganuclease of the present invention, the recognition sequence is a set of 9 base pair inverted "half sites" or "recognition half sites" separated by 4 base pairs. In the case of single-chain meganucleases, the N-terminal subunit of the protein is The C-terminal subunit of the protein contacts the first half-site and the C-terminal subunit of the protein contacts the second half-site. Cleavage by the meganuclease creates a four base pair 3' "overhang". "Overhangs" or "sticky ends" are the end points that allow meganuclease cleavage of double-stranded DNA sequences. The meganuclease of the present invention is a short single-stranded DNA segment that can be generated by fragmentation. In this case, the overhang comprises bases 10 to 13 of the 22 base pair recognition sequence.

[0351] As used herein, the term "target site" or "target sequence" refers to the staining of a cell. It refers to a region of somatic DNA that contains a recognition sequence for a meganuclease.

[0352] As used herein, the term "DNA binding affinity" or "binding affinity" means The meganuclease binds non-covalently to a reference DNA molecule (e.g., a recognition sequence or an arbitrary sequence). The binding affinity is measured by the dissociation constant, Kd. When used, the meganuclease comprises a sequence of the recombinant meganuclease relative to a reference recognition sequence. The Kd is increased by a statistically significant amount (p<0.05) compared to the reference meganuclease, or If it decreases, it has an "altered" binding affinity.

[0353] As used herein, the term "homologous recombination" or "HR" refers to a sequence that is used as a repair template. refers to the natural cellular process by which double-stranded DNA breaks are repaired using homologous DNA sequences (e.g., Cahill et al. (2006), Front. Biosci. 11 (See, e.g., 1958-1976). The homologous DNA sequence may be an endogenous chromosomal sequence. or it may be an exogenous nucleic acid delivered to the cell.

[0354] As used herein, the term "non-homologous end joining" or "NHEJ" refers to two A natural gene that repairs double-stranded DNA breaks by direct joining of non-homologous DNA segments. refers to the cellular processes of (See Biosci. 11:1958-1976). DNA by non-homologous end joining A-repair is error-prone and involves the addition or deletion of non-templated DNA sequences at the repair site. In some instances, cleavage at the target recognition sequence results in deletion of the target recognition site. Nuclease-induced cleavage of a target site in the coding sequence of a gene results in NHEJ in the The excision and subsequent DNA repair by NHEJ can correct mutations that disrupt gene function (e.g., A frameshift mutation can be introduced into the coding sequence. The engineered nucleases can be used to effectively knock out genes in populations of cells. It can be used.

[0355] As used herein with respect to both amino acid and nucleic acid sequences, "percent identity" refers to a sequence of amino acids or nucleic acids. Terms such as "sequence identity," "percent similarity," and "sequence similarity" are used to describe alignments. Alignment of sequences to maximize similarity between the amino acid residues or nucleotides involved (This refers to the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and The alignment of two sequences is based on the presence and length of gaps in the sequence alignment. Refers to a measure of the degree of similarity between sequences. Various algorithms and computer programs are available for this purpose. When used, sequence similarity is evaluated using the BLASTp program in the case of amino acid sequences, and the nucleus For nucleic acid sequences, the BLASTn program (both of which are published by the National Center for nter for Biotechnology Information(www.n cbi.nlm.nih.gov / ), e.g., Altschu l et al. (1990), J.Mol.Biol.215:403-410;Gi sh and States(1993),Nature Genet.3:266-2 72;Madden et al.(1996),Meth.Enzymol.266: 131-141;Altschul et al.(1997), Nucleic Ac ids Res.25:33 89-3402);Zhang et al.(2000 ), J. Comput. Biol. 7(1-2):203-14) As used herein, the percentage similarity between two amino acid sequences is measured using a The score is based on the following parameters for the BLASTp algorithm: Gap size=3;Gap opening penalty=-11;Gap extension penalty= Nullity = -1; and scoring matrix = BLOSUM62. If the percent similarity of two nucleic acid sequences is determined using the BLASTn algorithm, The score is based on the following parameters: word size = 1; gap opening pair = 1; Nalty = -5; Gap Extension Penalty = -2; Match Reward = 1; and and mismatch penalty = -3. Similarly, the percent identity is calculated based on the aligned sequences. Alignment of sequences to maximize identity between amino acid residues or nucleotides (this is The number of residues or nucleotides in the larger of the two sequences is The number of people who have died can be established based on the number of people who have died (which is a function of the number of people divided by the total number of people).

[0356] As used herein in reference to modifications of two proteins or amino acid sequences, "corresponding" refers to The term "match" refers to the search for two proteins (e.g., using the BLASTp program). ) When subjected to standard sequence alignment, a particular modification in the first protein is the modification in the second protein is a substitution of the same amino acid residue as the modification in the first protein; The amino acid position of the modification in the first protein corresponds to the amino acid position of the modification in the second protein. It is used to indicate correspondence or agreement. Thus, X and Y can be different numbers. Residues X and Y correspond to each other in the sequence alignment, despite the fact that In this case, the modification of residue "X" to amino acid "A" in the first protein results in the modification of the second protein. This would correspond to the modification of residue "Y" to amino acid "A" in the protein.

[0357] As used herein, the term "recognition half-site," "recognition sequence half-site," or simply The term "half site" refers to a nucleic acid sequence in a double-stranded DNA molecule that forms a homodimer. or by monomers of heterodimeric meganucleases, or by single-chain meganucleases The term "recognized nucleic acid sequence" refers to a nucleic acid sequence recognized by one subunit of the nucleotide sequence.

[0358] As used herein, the term "preferentially" refers to a recognition sequence that is more specific than a second, reference recognition sequence. The characteristics of recombinant meganucleases for recognizing and cleaving specific target recognition sequences in genomes are This refers to isomerism, as known in the art, including, for example, the methods provided in the Examples herein. The recombinant meganucleases of the present invention have a similar ability to recognize the corresponding wild-type nuclease as determined by the method of The P23H recognition sequence (e.g., SEQ ID NO: 5) is recognized and cleaved with higher efficiency than the P23H recognition sequence (e.g., SEQ ID NO: 5). For example, it may preferentially recognize and cleave SEQ ID NO: 1. In some embodiments, the present invention The recombinant meganuclease recognizes and cleaves approximately 5% more efficiently than the corresponding wild-type recognition sequence. , 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% , 90%, or 100% or more efficient. In other embodiments, the recombinant meganuclease of the present invention contains the corresponding wild-type recognition sequence. about 1-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, or 1000-fold greater than recognition and cleavage It can preferentially recognize and cleave the P23H recognition sequence with more than 2-fold higher efficiency.

[0359] As used herein, the term "hypervariable region" refers to a region of a meganuclease monomer or is a localized sequence within a subunit that contains amino acids with relatively high variability. A hypervariable region refers to a sequence of about 50-60 consecutive residues, about 53-57 consecutive residues, or Preferably, it may comprise about 56 residues. In some embodiments, the hypervariable region residues are: It may correspond to positions 24 to 79 or 215 to 270 of any one of SEQ ID NOs: 6 to 93. The variable region may contain one or more residues that contact the DNA bases in the recognition sequence and may be a monomer or subunit. The hypervariable regions can also be modified to change the base preference of the subunits. When the enzyme associates with a double-stranded DNA recognition sequence, it contains one or more residues that bind to the DNA backbone. Such residues may facilitate binding of the meganuclease to the DNA backbone and the target recognition sequence. In a different embodiment of the invention, the hypervariable region may be modified to alter binding affinity. The base preference and / or DNA binding affinity may be affected by the variability of 1 to 20 residues. In certain embodiments, the hypervariable region may be modified to affect the variability of about 1 Contains 5-18 residues and is modified to affect base preference and / or DNA binding affinity In some embodiments, the variable residues within the hypervariable region may be any of the residues of SEQ ID NOS: 6-93. Any one of 24, 26, 28, 29, 30, 32, 33, 38, 39, 40, 42, corresponding to one or more of positions 44, 46, 68, 70, 73, 75, and 77. In SEQ ID NOs: 6 to 93, the variable residues in the hypervariable region are 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 2 219, 220, 221, 223, 224, 229, 230, 231, 233, 235, Corresponding to one or more of positions 237, 259, 261, 264, 266, and 268.

[0360] As used herein, "RHO," "RHO gene," "rhodopsin gene," and "wild-type RHO allele" are used interchangeably and refer to the human rhodopsin gene , preferably identified by the NCBI reference sequence NG_009115.1 (SEQ ID NO: 98) "Mutant RHO allele" and "mutant RHO P23H allele" refer to genes that are involved in the The terms "gene" and "gene" are used interchangeably and refer to a gene that results in a P23H substitution in the encoded protein. "Rhodopsis" refers to the RHO allele sequence containing the C68A mutation (SEQ ID NO: 99). The terms "rhodopsin" and "wild-type rhodopsin" are used interchangeably and refer to the wild-type rhodopsin gene Specifically, the protein encoded by the NCBI reference sequence NP_000530.1 (sequence The term "P23H rhodopsin" refers to the protein identified by the number 100. is a mutant rhodopsin protein containing a P23H substitution, particularly as shown in SEQ ID NO: 101. This refers to proteins that are present in the body.

[0361] "recombinant DNA construct", "recombinant construct", "expression cassette", "expression construct", The terms "chimeric construct," "construct," and "recombinant DNA fragment" are used interchangeably herein. Recombinant constructs include, but are not limited to, nucleic acid fragments that are not found together in nature. This includes the artificial combination of nucleic acid fragments containing regulatory and coding sequences that are not present in the genome. Recombinant DNA constructs may contain regulatory and coding sequences derived from different sources, or from the same source. Regulatory and coding sequences derived from, arranged in a manner different from that found in nature Such constructs may be used by themselves. or may be used in conjunction with a vector.

[0362] As used herein, a "vector" or "recombinant DNA vector" refers to a vector that includes a replication system and and sequences that allow for the transcription and translation of the polypeptide coding sequence in a given host cell. When a vector is used, the selection of the vector is well known to those skilled in the art. The vectors used depend on the method used to transform the host cells. including, but not limited to, plasmid vectors and recombinant AAV vectors, or megagenes of the present invention. Any suitable method known in the art for delivering the gene encoding the nuclease to the target cells can be used. Those skilled in the art will appreciate that the isolated nucleotide sequences or sequences of the present invention can be used in various ways. The vectors are used to successfully transform, select, and grow host cells containing any of the nucleic acid sequences. We have a good understanding of the genetic elements that must be present on the vector.

[0363] As used herein, "vector" can also refer to a viral vector. Examples of vectors include, but are not limited to, retroviral vectors and lentiviral vectors. vectors, adenovirus vectors, and adeno-associated virus vectors (AAV). obtain.

[0364] As used herein, a "target cell" refers to a cell that contains a P23H recognition sequence (e.g., SEQ ID NO:1). These targets refer to cells that contain at least one RHO allele (one of four). Target cells may express the mutant RHO P23H protein. ocular tissue that does not express the P23H recognition sequence in at least one RHO gene allele Cells, preferably cells in the posterior part of the eye, even more preferably cells of the retina (including rod photoreceptors) may be mentioned.

[0365] As used herein, a "control" or "control cell" refers to a control cell that is a genetically modified cell of the same genotype. or refers to cells that provide a reference point for measuring phenotypic changes. (a) i.e., of the same genotype as the starting material for the genetic changes that resulted in the genetically modified cell (b) cells of the same genotype as the genetically modified cells, but containing a null construct cells that have been transformed with a construct (i.e., with a construct that has no known effect on the trait of interest); or (c) Cells that are genetically identical to the genetically modified cells, but with an altered genotype or It may include cells that have not been exposed to conditions or stimuli that induce the expression of a phenotype or to further genetic modifications. do.

[0366] As used herein in reference to modifications of two proteins or amino acid sequences, "corresponding" refers to The term "match" refers to the search for two proteins (e.g., using the BLASTp program). ) When subjected to standard sequence alignment, a particular modification in the first protein is the modification in the second protein is a substitution of the same amino acid residue as the modification in the first protein; The amino acid position of the modification in the first protein corresponds to the amino acid position of the modification in the second protein. It is used to indicate correspondence or agreement. Thus, X and Y can be different numbers. Residues X and Y correspond to each other in the sequence alignment, despite the fact that In this case, the modification of residue "X" to amino acid "A" in the first protein results in the modification of the second protein. This would correspond to the modification of residue "Y" to amino acid "A" in the protein.

[0367] As used herein, the term "treatment" or "treatment of a subject" refers to the treatment of one of the RPs. In order to provide partial or complete relief of the above symptoms, the recombinant meganuclease of the present invention or administering a nucleic acid encoding a recombinant meganuclease of the present invention to a subject with RP. In some embodiments, the recombinant meganuclease of the invention or a gene encoding the same is The nucleic acid is administered during treatment in the form of a pharmaceutical composition of the present invention. P23H mutant allele and functional, normal, or wild-type allele Heterozygous for the gene.

[0368] As used herein, the recitation of a numerical range for a variable includes any one of the values ​​within that range. It is intended to convey that the invention may be practiced with any number of equivalent variables. For a truly discrete variable, the variable can be equal to any integer value within the numeric range, including the end points of the range. Similarly, for a variable that is inherently continuous, the variable can fall within a numerical range, including the end-points of the range. By way of example and not limitation, it can be written as having a value between 0 and 2. The variables listed can take on values ​​of 0, 1, or 2 if the variables are discrete in nature; If the number is continuous in nature, the values ​​0.0, 0.1, 0.01, 0.001, or 0 It can take on any other real value greater than or equal to 2 and less than or equal to 2.

[0369] 2.1 Principles of targeting and inactivation of the mutant RHO P23H allele The present invention provides a mutant RHO P23 gene encoding a pathogenic P23H rhodopsin protein. Correcting autosomal dominant RP by targeting, ablating, and inactivating the H allele Surprisingly, the recombinant megagenes The cleavage enzyme reacts with the P23H recognition sequence (e.g., For example, it can be engineered to recognize and cleave one of SEQ ID NOS: 1 to 4. The meganuclease has the mutation R compared to the corresponding wild-type allele (SEQ ID NO: 96). HO may preferentially target and cleave the P23H allele. NHEJ at the cleavage site results in mutagenesis and disruption of the mutant RHO P23H allele, but is functional The wild-type RHO allele still expresses wild-type rhodopsin in the rod photoreceptors of the retina. Preferential inactivation of the mutant RHO P23H allele and disruption of P23H rhodopsin expression prevents, delays or reduces the progression of RP in patients. It is expected to be reversed.

[0370] 2.2 Meganuclease for recognizing and cleaving the P23H recognition sequence Meganucleases can cause site-specific DNA cleavage in the genome of living cells, Such DNA breaks can be repaired through mutagenesis, NHEJ repair, or transgenic DNA repair. NHEJ can result in permanent genome modification through homologous recombination with the A sequence. NHEJ can cause mutagenesis at the site, resulting in allele inactivation. Related mutagenesis can result in the creation of premature stop codons, leading to abnormal, non-functional proteins. Alleles can be inactivated via a context-shift mutation or by nonsense-mediated mutations. It can trigger mechanisms such as RNA degradation to induce mutagenesis via NHEJ. The use of meganucleases allows targeting of sequences present in specific mutant or wild-type alleles. It can be used for targeting.

[0371] In a preferred embodiment, the nuclease used to practice the present invention is a single-stranded The single-chain meganuclease is a nuclease consisting of two chains connected by a linker peptide. The two domains each contain an N-terminal subunit and a C-terminal subunit. It recognizes half of the sequence (i.e., the recognition half-site), and the site of DNA cleavage is the two subunits. The recognition sequence is located in the center of the nuclease near the interface of the nuclease. DNA strand breaks occur at four base pairs to generate four base pair 3' single-stranded overhangs. And they cancel each other out.

[0372] The recombinant meganuclease of the present invention comprises a P23H recognition sequence (e.g., one of SEQ ID NOs: 1 to 4). Such meganucleases are engineered to recognize and cleave the corresponding The mutant RHO P23H allele is compared to the wild-type RHO recognition sequence (SEQ ID NO: 96) The P23H recognition sequence on the nuclease preferentially cleaves the P23H recognition sequence on the nuclease. are shown in SEQ ID NOs: 6 to 93 (these are referred to herein as "RHO1-2 meganucleic acid"). These are collectively referred to as "RHO1 / 2 meganucleases" or "RHO1 / 2 meganucleases."

[0373] The recombinant meganuclease of the present invention comprises a first hypervariable region (HVR1) region. and a second subunit comprising a second hypervariable region (HVR2). The first subunit binds to the first recognition half-site (e.g., R) in the P23H recognition sequence. The second subunit binds to the second recognition site in the P23H recognition sequence. Recombinant meganuclease that binds to a recognition half-site (e.g., RHO2 half-site) In embodiments where the enzyme is a single-chain meganuclease, the first subunit is an N-terminal subunit. and the second subunit is arranged as the C-terminal subunit. The first and second subunits can be oriented (e.g., SEQ ID NOs: 70-9). 3) In an alternative embodiment, the first subunit is positioned as the C-terminal subunit. The first subunit is arranged so that the second subunit is positioned as the N-terminal subunit. The first and second subunits can be oriented in the same direction (e.g., SEQ ID NOS: 6-69). Representative recombinant meganucleases are shown in Table 1.

[0374] Table 1. Exemplary sequences engineered to recognize and cleave the P23H recognition sequence (SEQ ID NO: 1) Recombinant meganuclease [Table 1] TIFF2025131592000002.tif255164TIFF2025131592000003.tif58169

[0375] *"RHO1 subunit%" and "RHO2 subunit%" are the percentages of each meganuclease. RHO1-binding subunit region and RHO2-binding subunit region of RHO2-L3 The RHO1-binding subunit region and the RHO2-binding subunit region of -59 meganuclease, respectively It represents the amino acid sequence identity between the unit regions.

[0376] 2.3 Methods for delivering and expressing recombinant meganucleases Treatment of RP using the present invention involves the expression of recombinant meganucleases in cells of the appropriate tissue. The target composition for delivery of the recombinant meganuclease of the present invention must be capable of expressing the The tissue is a cell of the eye, preferably a cell in the back of the eye, even more preferably a cell of the retina (rods). Recombinant meganucleases are available as purified proteins or as part of the meganuclease The enzyme may be delivered as RNA or DNA encoding the enzyme. mRNA encoding the meganuclease protein or recombinant meganuclease The vector is delivered to target cells (e.g., cells of the retina) via direct injection into the target tissue. For example, RNA, DNA, or tissue can be delivered to the eye via subretinal or intravitreal injection. Delivery of recombinant AAV vectors has been described in the art (e.g., Martin et al., t al.(2002)Methods.28:267-275;Hauswirth et al.(2008)Human Gene Therapy.19(10):97 9-990;Johnson et al.(2008)Molecular Visi on.14:2211-2226). Alternatively, meganuclease proteins Proteins, mRNA, or DNA can be delivered systemically via the circulatory system.

[0377] In some embodiments, the recombinant meganuclease protein or recombinant meganuclease The DNA / mRNA encoding the enzyme can be prepared using known methods for systemic administration or administration to a target tissue. They are formulated in pharmaceutical carriers according to the art. See, e.g., Remington, The Sci ence And Practice of Pharmacy(21sted.200 In the production of the pharmaceutical preparation of the present invention, the protein / RNA / mRNA is typically Typically, the compound is mixed with a pharmaceutically acceptable carrier. It must be acceptable in the sense of being compatible with any other ingredients of the The carrier may be a solid or a liquid or both. The compound may be obtained and formulated with the compound as a unit dose formulation.

[0378] In some embodiments, the recombinant meganuclease is The DNA / mRNA encoding the protein or recombinant meganuclease is transfected into the cell-permeable peptide. Cell-penetrating peptides known in the art are coupled to a peptide or targeting ligand. Examples of poly-arginine (Jearawiriyapaisarn, et al. (2008) Mol Ther.16:1624-9), TAT derived from HIV virus Peptides (Hudecz et al. (2005), Med. Res. Rev. 25: 679-736), MPG (Simeoni, et al. (2003) Nucleic Acids Res.31:2717-2724), Pep-1(Deshayes et al. (2004) Biochemistry 43:7698-7706 and H SV-1 VP-22 (Deshayes et al. (2005)Cell Mol Life Sci. 62:1839-49. In an alternative embodiment, Nuclease protein / DNA / mRNA binds to target cells and is absorbed by the target cells Recombinant meganuclease or recombinant meganuclease, as ternary The DNA / mRNA encoding recognizes specific cell surface receptors expressed on target cells. Alternatively, the recombinant megagene may be covalently or non-covalently coupled to an antibody that binds the recombinant megagene. The cleavage protein / DNA / mRNA is the natural ligand for such cell surface receptors. (or part of a natural ligand) may be covalently or non-covalently coupled. (McCall,et al.(2014)Tissue Barriers.2(4) :e944449;Dinda,et al.(2013)Curr Pharm Bi otechnol.14:1264-74;Kang,et al.(2014)Cur r Pharm Biotechnol.15(3):220-30;Qian et al. (2014) Expert Opin Drug Metab Toxicol. 10(11):1491-508). Targeting ligases for direct delivery to ocular cells. An example of a bond is RGD (Pollinger et al. (2013) PNAS. 110(15):6115-6120), transferrin (Lajunen et al. l.(2014)Eur J Pharm Sci.62:23-32) and hyaluronic acid Acid (Martens et al. (2015) J Control Release. 202:83-92).

[0379] In some embodiments, the recombinant meganuclease protein or recombinant meganuclease The DNA / mRNA encoding the enzyme is injected or implanted (e.g., intravitreal) into the desired ocular region. The drug is encapsulated in a biodegradable hydrogel for intravenous or subconjunctival injection. sustained and tunable delivery of therapeutic payload to desired ocular regions without the need for frequent injections Stimuli-responsive materials (e.g., temperature-responsive hydrogels and pH-responsive Hydrogels) are designed to release payloads in response to environmental or externally applied cues. (Kang Derwent et al. (2008) Trans Am Op hthalmol Soc.106:206-214).

[0380] In some embodiments, the recombinant meganuclease protein or recombinant meganuclease The DNA / mRNA encoding the enzyme can be attached to nanoparticles using methods known in the art. Covalently, preferably non-covalently coupled to such nanoparticles It is enclosed within the nucleus (Sharma, et al. (2014) Biomed Res I Nanoparticles have a length scale of less than 1 μm, preferably less than 100 nm. Such nanoparticles may be metal, lipid, polymer, or The core may be designed to contain multiple copies of a recombinant meganucleotide. Proteins, mRNA, or DNA may be attached to or encapsulated in the nanoparticle core. This increases the number of copies of the protein / mRNA / DNA delivered to each cell, Increased intracellular expression of each recombinant meganuclease increases the likelihood of cleaving the target recognition sequence. The surface of such nanoparticles is coated with polymers or lipids (e.g., chitosan, carboxymethylcellulose, etc.). The surface can be further functionalized using polymers such as thionic polymers or cationic lipids. Further, core-shell nanoparticles can be formed to enhance cellular delivery and payload uptake. It can be modified (Jian et al. (2012), Biomaterials.33 (30):7621-30). Advantageously, the nanoparticles are targeted to the appropriate cell type, and / or To increase the likelihood of cellular uptake, nanoparticles can be further coupled to targeting molecules. Examples of such targeting molecules include antibodies specific for cell surface receptors and These include the natural ligands (or parts of the natural ligands) of cell surface receptors.

[0381] In some embodiments, a protein encoding a meganuclease or a meganuclease is The DNA / mRNA can be encapsulated in liposomes or transported using cationic lipids. conjugated (e.g., Lipofectamine™, Life Technology ologies Corp., Carlsbad, CA; Zuris et al. (2 015), Nat Biotechnol.33:73-80;Mishra et a (See J Drug Deliv. 2011:863734) Liposome and lipoplex preparations are capable of fusing with and / or transporting the target cell membrane. Through disruption, it protects the payload from degradation and enhances accumulation and retention at the target site; This may enhance cellular uptake and delivery efficiency.

[0382] In some embodiments, the recombinant meganuclease protein or recombinant meganuclease The DNA / mRNA encoding the enzyme is encapsulated within a polymer scaffold (e.g., PLGA). or complexed using cationic polymers (e.g., PEI, PLL) Tamboli et al.(2011),Ther Deliv.2(4):523 -536) Polymeric carriers allow for tunable drug delivery by controlling the erosion of the polymer and the diffusion of the drug. The drug can be designed to provide a drug release rate and high drug encapsulation efficiency to target the desired cell population. It may provide protection of the therapeutic payload until intracellular delivery.

[0383] In some embodiments, the recombinant meganuclease protein or recombinant meganuclease The DNA / mRNA encoding the enzyme is combined with amphiphilic molecules that self-assemble into micelles. (Tong et al. (2007), J Gene Med.9(11):95 6-66) As polymeric micelles, they prevent aggregation, shield charge interactions, and promote absorption of vitreous humor. Hydrophilic polymers (e.g., polyethylene glycol) that can reduce nonspecific interactions within the ) can be mentioned.

[0384] In some embodiments, the recombinant meganuclease protein or recombinant meganuclease The DNA / mRNA encoding the enzyme is then transfected into an E. coli marker for administration and / or delivery to target cells. formulated into emulsions or nanoemulsions (i.e., with an average particle size of less than 1 nm) The term "emulsion" includes, but is not limited to, a water-immiscible phase that, when mixed with an aqueous phase, Hydrophobic forces that move nonpolar residues (e.g., long hydrocarbon chains) from water and polar head groups from water Any oil-in-water, water-in-oil, water-in-oil-in-water, or oil containing lipid structures that may result These other lipid structures include, but are not limited to, oil-in-water dispersions or droplets. These include unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases. Emulsions consist of an aqueous phase and a lipophilic phase (typically containing oil and an organic solvent). Emulsions often also contain one or more surfactants. Nanoemulsion formulations are well known and are described, for example, in U.S. Patent Application Publication No. 2002 / 00456 67 and U.S. Patent Application Publication No. 2004 / 0043041 and U.S. Patent No. 6,015 ,832, U.S. Patent No. 6,506,803, U.S. Patent No. 6,635,676, and No. 6,559,189 (each of which is incorporated herein by reference in its entirety). (incorporated).

[0385] In some embodiments, the recombinant meganuclease protein or recombinant meganuclease The DNA / mRNA encoding the enzyme is conjugated to a multifunctional polymer conjugate, DNA dendrite. Covalently attached to polymers and polymer dendrimers and non-covalently attached to them. (Mastorakos et al. (2015), Nanosca le.7(9):3845-56;Cheng et al.(2008),J Pha rm Sci.97(1):123-43). Dendrimer formation is a key factor in determining payload capacity. The size and size of the nanoparticles can be controlled, providing a high drug payload capacity. The presentation of the group improves stability, reduces non-specific interactions, and allows for cell-specific targeting and drug It can be utilized to enhance release.

[0386] In some embodiments, the gene encoding the recombinant meganuclease is derived from a viral vector. Such vectors are known in the art and are used in lentiviruses. Viral vectors, adenoviral vectors, and adeno-associated viral (AAV) vectors (Vannucci, et al. (2013), New Microbi In some embodiments, viral vectors are used. The drug is injected directly into the target tissue (Bosch, et al. (2000), Mol Ther.1:63-70;Greig et al.(2014),PLoS On e. Nov 13;9(11):e112268). In an alternative embodiment, a viral vector The vector is delivered systemically via the circulatory system. Various AAV vectors are available in the art. It is known that AAVs tend to be localized in various tissues. Efficient transduction of retinal photoreceptors has been demonstrated using immunoglobulins 1, 2, 5, 8, and 9 (P etrs-Silva et al. (2014), Clinical Ophthal (Mology. 8:127-136). AAV vectors also express the second strand in host cells. They can be self-complementary so that DNA synthesis is not required (McCarty et al. (2001), Gene Ther. 8:1248-54).

[0387] In one embodiment, the viral vector used for meganuclease gene delivery is , a self-limiting viral vector. The self-limiting viral vector is a recombinant meganuclear vector. The recognition sequence for the enzyme is present in the vector, allowing it to have a limited lifespan in the cell or organism. Thus, a self-limiting viral vector may have a promoter, Provides coding for the recombinant meganuclease and the meganuclease recognition site within the ITR The meganuclease can be expressed and engineered to bind to an endogenous recognition sequence within the genome. Self-limiting viral vectors are used to create meganucleases so that they can cut into the genome of cells. The meganuclease gene is delivered to a cell, tissue, or organism. The delivered meganuclease can also be used to , finds its target site within the self-limiting viral vector itself, and binds to this target site. Once cut, the vector will be cut at the 5' end of the viral genome and The 3' end is exposed and degraded by exonucleases, killing the virus and preventing recombination. The production of meganucleases stops.

[0388] The recombinant meganuclease gene may be in DNA form (e.g., a plasmid) and / or in a recombinant form. When delivered via a viral vector (e.g., AAV), they are In some embodiments, this is the case with viral promoters. A motor, such as an endogenous promoter from a viral vector (e.g., lentivirus vector) or the well-known cytomegalovirus or SV40 virus early process In a preferred embodiment, the meganuclease gene is The gene is operably linked to a promoter that drives gene expression preferentially in the retina and / or Examples of rod photoreceptor specific promoters include, but are not limited to, human rhodopsin kinase Ze promoter, proximal mouse opsin promoter (mOP), human G protein-coupled Receptor protein kinase 1 promoter (hGRK1) and human interphotoreceptor retinoic acid IRBP promoter (Khani et al. (2007), Invest.Ophthamol.Vis.Sci.48(9):3954-3961 );Beltran et al.(2010),Gene Therapy.17(9 ):1162-1174);Yokoyama et al.(1992),Exp.E Ye Res. 55(2):225-233), and U.S. Patent Application Publication No. 2014 / 0 Examples of such promoters include the rod photoreceptor cell-specific promoter disclosed in US Pat. No. 287510.

[0389] In some embodiments, the present invention provides a method for producing a recombinant DNA molecule of the present invention, comprising administering to a subject a pharmaceutically acceptable carrier and the recombinant DNA molecule of the present invention. A pharmaceutical composition comprising a recombinant ganucleases of the present invention, or a pharmaceutically acceptable carrier. and an isolated polynucleotide comprising a nucleic acid encoding a meganuclease. Such pharmaceutical compositions can be prepared according to known techniques. emington,The Science And Practice of Pha In the manufacture of the pharmaceutical formulation of the present invention, The endonuclease polypeptide (or the DNA / RNA encoding it) is typically is mixed with a pharmaceutically acceptable carrier and the resulting composition is administered to a subject. However, the carrier must be acceptable in the sense of being compatible with any other ingredients in the formulation. The test must be feasible and not harmful to the subject. In embodiments, the pharmaceutical compositions of the present invention comprise one or more compounds useful in treating a disease in a subject. Similarly, the additional drug and / or biological molecule may The molecules may be co-administered as separate compositions.

[0390] A single treatment can reverse the mutation of the P23H RHO allele in a proportion of patient target cells. It is assumed that the P23H allele is permanently inactivated. However, the frequency of P23H allele inactivation If the concentration is low or if a large proportion of target cells needs to be corrected, multiple treatments may be required for each patient. Treatment may be required.

[0391] 2.4 Recombinant meganuclease variants Embodiments of the present invention include the use of the recombinant meganucleases and variants thereof described herein. Further embodiments of the present invention include the recombinant meganucleases described herein. Isolated polynucleotides comprising nucleic acid sequences encoding such polynucleotides This includes variants of the above.

[0392] In particular, the present invention relates to mutants of meganucleases of SEQ ID NOs: 6 to 93, ase has the sequence A mutation in which the hypervariable region is modified to preferentially recognize and cleave one of numbers 2 to 4. Such further mutant meganucleases are described in WO 2007 / 04 No. 7859, U.S. Patent No. 8,021,867, U.S. Patent No. 8,119,361, U.S. Patent No. 8,119,381, U.S. Patent No. 8,124,369, U.S. Patent No. 8,129 ,134, U.S. Patent No. 8,133,697, U.S. Patent No. 8,143,015, U.S. Patent No. 8,143,016, U.S. Patent No. 8,148,098, U.S. Patent No. 8,163 ,514, U.S. Patent No. 8,304,222, U.S. Patent No. 8,377,674, and The methods described in U.S. Pat. No. 8,445,251, as well as those discussed below. It can be produced by routine experimentation according to the method.

[0393] As used herein, "variant" is intended to mean a substantially similar sequence. A "mutated" polypeptide is one that has a mutation at one or more internal sites of the native protein. deletion or addition of one or more amino acids, and / or one or more modifications of the native polypeptide The substitution of one or more amino acids at the above positions results in a "native" polypeptide. As used herein, "native" is intended to mean a polypeptide resulting from a A "variant" polynucleotide or polypeptide includes the parent sequence from which the variant is derived. The variant polypeptides encompassed by the present invention are biologically active, i.e., they are capable of inhibiting the action of the nucleotides in the nucleotide sequence. The desired biological activity of the native protein (i.e., P2 3H recognition sequence (e.g., the ability to preferentially recognize and cleave one of SEQ ID NOs: 1 to 4) Such variants may result, for example, from human manipulation. a biologically active variant of the active polypeptide (e.g., SEQ ID NOs: 6-93), or The biological properties of the RHO1-binding subunit and the RHO2-binding subunit described in the specification The highly active variants (e.g., SEQ ID NOS: 99-274) are described elsewhere herein. The native polynucleotides are aligned as determined by the sequence alignment program and parameters used. a polypeptide or a native subunit whose amino acid sequence is at least about 40%, about 45%, or %, approx. 50%, approx. 55%, approx. 60%, approx. 65%, approx. 70%, approx. 75%, approx. 80%, approx. 85 %, approx. 90%, approx. 91%, approx. 92%, approx. 93%, approx. 94%, approx. 95%, approx. 96%, approx. 97 %, about 98%, or about 99% sequence identity. A biologically active variant of a subunit is a variant of that polypeptide or subunit. or about 1-40 amino acid residues, only about 1-20, only about 1-10, or only about 5 , as few as 4, 3, 2, or even 1 amino acid residue may differ.

[0394] Polypeptides of the embodiments may be modified in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by DNA mutations. Methods for polynucleotide alteration are well known in the art. l(1985), Proc. Natl. Acad. Sci. USA 82:488-49 2;Kunkel et al. (1987), Methods in Enzymol .154:367-382;USPat.No.4,873,192;Walker and Gaastra, eds. (1983), Techniques in Mo. lecular Biology(MacMillan Publishing Com Company, New York) and the references cited therein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest The results are based on the Atlas of Proteins by Dayhoff et al. (1978). n Sequence and Structure(Natl.Biomed.Res Found., Washington, DC) (which is incorporated herein by reference). Conservative substitutions (e.g., one amino acid and a similar replacement with another amino acid with similar properties) may be optimal.

[0395] A significant number of amino acids relative to the DNA recognition domain of wild-type I-CreI meganuclease Modifications that result in a rationally designed meganuclease that differs from the wild-type enzyme. at individual bases within the DNA recognition sequence half-site, so as to have half-site specificity. Amino groups that result in recombinant meganucleases with altered specificities, either alone or in combination. Acid modifications have been previously identified (e.g., U.S. Pat. No. 8,021,867). Table 2 Based on the bases present at each half-site position (-1 to -9) of the recognition half-site, This can be done in recombinant meganuclease monomers or subunits to enhance isomerization. It provides replacement suggestions.

[0396] [Table 2]

[0397] In the case of polynucleotides, a "variant" refers to one or more variations of a native polynucleotide. The deletion and / or addition of one or more nucleotides at the site of Variants of the nucleic acids of the embodiments are constructed so that the open reading frame is maintained. In the case of polynucleotides, conservative variants include those derived from the genetic code. Due to the degeneracy of the code, a sequence encoding one of the amino acid sequences of the polypeptide of the embodiment can be obtained. Mutant polynucleotides include synthetic polynucleotides, for example, site-specific polynucleotides. Recombinant meganucleases of embodiments generated by using selective mutagenesis Generally, certain polynucleotides of the embodiments still encode Variants of the present invention can be identified using sequence alignment programs and parameters described elsewhere herein. and at least about 40%, about 40%, or 5%, approx. 50%, approx. 55%, approx. 60%, approx. 65%, approx. 70%, approx. 75%, approx. 80%, approx. 8 5%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 9 The sequences may have about 7%, about 98%, about 99%, or more sequence identity. A variant of a given polynucleotide (i.e., a reference polynucleotide) also refers to a mutant polynucleotide. The polypeptide encoded by the reference polynucleotide is The degree of sequence identity can be assessed by comparing the percent sequence identity between the polypeptides.

[0398] Deletions, insertions, and substitutions in the protein sequences encompassed herein are intended to be indicative of the characteristics of the polypeptide. However, substitutions, deletions, or modifications may occur before implementation. If it is difficult to predict the exact effect of the insertion, one skilled in the art can easily determine the P23H recognition sequence. The polypeptide is characterized for its ability to preferentially recognize and cleave a sequence (e.g., one of SEQ ID NOS: 1-4). It will be appreciated that efficacy will be assessed by screening the chemotherapeutic agents. [Example]

[0399] This invention is further illustrated by the following examples which should not be construed as limiting. Those skilled in the art will be able to readily identify, using no more than routine experimentation, the features described herein. Many equivalents to certain substances and procedures will be recognized or can be identified. Such equivalents are intended to be encompassed by the claims following the examples below.

[0400] Example 1 Evaluation of meganucleases that recognize and cleave the P23H recognition sequence

[0401] 1. Meganuclease that recognizes and cleaves the P23H recognition sequence One of the P23H recognition sequences present in the mutant RHO P23H allele (i.e., sequence The recombinant meganuclease (SEQ ID NOs: 6 to 93) (the present invention) was engineered to recognize and cleave the nucleotide sequence of the nucleotide sequence of the present invention. Herein, we have engineered RHO1-2 meganucleases (collectively referred to as "RHO1-2 meganucleases") (see Figure 1A). Each RHO1-2 recombinant meganuclease contains an N-terminal nuclease derived from SV40. a meganuclease localization signal, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. and the first meganuclease subunit in each RHO1-2 meganuclease. The first subunit binds to the RHO1 recognition half-site of SEQ ID NO: 1, and the second subunit binds to the RHO1 recognition half-site of SEQ ID NO: 1. It binds to the RHO2 recognition half-site (see Figure 1B).

[0402] As shown in Figures 2 and 3, the RHO1-binding subunit and the RHO2-binding subunit Each unit contains a 56 base pair hypervariable region (HVR1 and HVR2, respectively). The RHO1 binding subunit contains a residue at positions 80 or 271 (containing a Q or E residue). They are identical outside the HVR1 region except for the position 1, and are highly conserved within the HVR1 region. Similarly, RHO2 binding subunits also contain residues at positions 80 or 81 (containing Q or E residues). They are identical outside the HVR2 region except for position 271, and are highly conserved within the HVR2 region. It is being done.

[0403] The RHO1-binding regions of SEQ ID NOs: 6 to 93 are shown in Figures 2A to 2F, and are SEQ ID NOs: Almost all of SEQ ID NOs. 102 to 189 are shown as SEQ ID NOs. 102 (which is the RHO1 binding of meganuclease RHO2-L3-59 (SEQ ID NO: 6) RHO2 of SEQ ID NOs: 6 to 93 shares at least 90% sequence identity with the RHO2 of SEQ ID NOs: 6 to 93. The binding regions are shown in Figures 3A-3F and are designated as SEQ ID NOS: 190-277, respectively. Almost all of SEQ ID NOs: 190 to 277 are SEQ ID NO: 190 (which is a meganuclear RHO2-L3-59 (SEQ ID NO: 6), which is the RHO2-binding region of the enzyme RHO2-L3-59 (SEQ ID NO: 6), and % sequence identity.

[0404] 2. Cleavage of the P23H recognition sequence in CHO cell reporter assays RHO1-2 meganuclease can recognize and cleave the P23H recognition sequence of SEQ ID NO: 1 Use a previously described CHO cell reporter assay to determine whether Each RHO1-2 meganuclease was evaluated (Figure 1 of WO 2012 / 167192). To perform the assay, a non-functional green fluorescent protein integrated into the genome of the cell is used. A set of CHO cell reporter lines carrying a green fluorescent protein (GFP) gene expression cassette Intracellular cleavage of either recognition sequence by meganucleases results in homologous recombination events. Each cell line is engineered with a set of recognition sequences to stimulate expression of the functional GFP gene. The GFP gene was disrupted. In both cell lines, one of the recognition sequences was derived from the RHO gene. The second recognition sequence is "CHO-23 The P of SEQ ID NO: 1 was specifically recognized by a control meganuclease called "P / 24". CHO reporter cells containing the 23H recognition sequence and the CHO-23 / 24 recognition sequence are described herein. These cells are referred to as "RHO1-2 cells" in the literature. 93) or a gene encoding the CHO-23 / 34 meganuclease. Plasmid DNA was transfected into RHO1-2 cells according to the manufacturer's instructions. Using Lipofectamine 2000 (ThermoFisher), In a well plate, 50 ng of plasmid DNA was diluted to approximately 4 x 10 5 CHO cells 48 hours after transfection, the cells were analyzed by flow cytometry. Cells were assessed for GFP positivity compared to the untransfected negative control (RHO1-2bs). The percentage of viable cells was determined. All RHO1-2 meganucleases contain the P23H recognition sequence. In cell lines containing HIV, the frequency was significantly higher than the negative control and similar to the CHO-23 / 24 positive control. It was found that GFP-positive cells were generated at a frequency of 100 or more. Each RHO1-2 meganuclease efficiently recognizes the desired P23H recognition sequence in cells. and cleavage was successfully achieved (see Figure 5).

[0405] In addition, after the meganuclease was introduced into RHO1-2 cells, 1 day, 4 days, 6 days, and After 1 day and 8 days, the efficacy of the RHO1-2 recombinant meganuclease was determined in a time-dependent manner. In this study, the BioRad Gene Pulser Xce was used according to the manufacturer's instructions. 1 x 10 cells per 100 cells using 111 6 RHO copy meganuclease mRNA 1-2 cells (1.0×10 6 100 cells were electroporated. After 48 hours, cells were assessed by flow cytometry to determine the percentage of GFP-positive cells. At each time point, CHO-23 / 24 meganuclease was also included as a positive control. As shown in Figure 6, the efficacy of RHO1-2 meganuclease was confirmed over the 8-day evaluation period. persisted throughout the evaluation period.

[0406] 3. Conclusion These studies demonstrated that RHO1-2 meganuclease (SEQ ID NO: 1) encompassed by the present invention Nos. 6-93) can target and cleave the P23H recognition sequence of SEQ ID NO: 1 in cells. It has been proven that this is the case.

[0407] Example 2 Specificity of meganucleases for the P23H recognition sequence

[0408] 1. CHO reporter cells containing the corresponding wild-type RHO sequence RHO1-2 meganuclease (SEQ ID NO: 6 to 10) for the P23H recognition sequence of SEQ ID NO: 1 To determine the specificity of the 93 gene, the corresponding wild-type RHO recognition sequence (SEQ ID NO: 5) was used. The RHO3-4 recognition sequence is referred to as the "RHO3-4 recognition sequence" in the specification, and the CHO-23 / 24 recognition sequence is also included. A CHO reporter cell line containing the IgG1 gene was generated as previously described. The resulting cells are referred to herein as "R These cells are called "HO3-4 cells."

[0409] 2. Specificity of RHO1-2 meganuclease RHO1-2 meganuclease is introduced into RHO1-2 cells or RHO3-4 cells, The recombinant meganuclease comprises the P23H recognition sequence of SEQ ID NO: 1 and the corresponding wild-type recognition sequence (SEQ ID NO: 5). By this method, RHO1-2 meganuclease mRNA was transfected into RHO1-2 cells or RHO The percentage of GFP-positive cells was determined 3-5 days after transfection. As shown in Fig. 1, the proportion of GFP-positive RHO1-2 cells was higher than that of RHO3-4 cells. As can be seen from the results, the RHO1-2 meganucleases tested were The P23H recognition sequence was preferentially cleaved compared to the recognition sequence (see Figures 7A-7E). Subsequent experiments demonstrated that the wild-type RHO3-4 recognition sequence (SEQ ID NO: 5) was preferentially targeted and cleaved. It was demonstrated that a recombinant meganuclease capable of cleaving the nucleotide sequence of the CHO cells described above could be developed. When assessed using reporter assays, this set of meganucleases exhibited RHO It was found that the GFP-positive RHO3-4 cells were produced in a higher proportion than the GFP-positive RHO1-2 cells ( See Figure 7F).

[0410] 3. Conclusion The inventors have determined that the RHO1-2 meganuclease encompassed by the present invention is a The P23H recognition sequence of SEQ ID NO: 1 was compared to the wild-type RHO3-4 recognition sequence (SEQ ID NO: 5). Preferential cleavage was demonstrated.

[0411] Example 3 Construction and expression of recombinant AAV vectors for expressing recombinant meganucleases

[0412] 1. Recombinant AAV vector for expressing RHO1-2 meganuclease Recombinant CHO reporter cells were engineered to express RHO1-2 meganuclease. As shown in Figure 8A, the recombinant AAV vector was From 5' to 3', there is the first inverted terminal repeat (ITR) and the nuclease encoding a CMV promoter operably linked to a nucleotide sequence; and a second inverted terminal repeat. In this study, we synthesized the coding sequence of the RHO-1 / 2-L2-49 meganuclease. It was incorporated into a recombinant AAV vector.

[0413] 2. Expression of RHO1-2 Meganuclease after Recombinant AAV Transduction For expression of RHO-1 / 2-L2-49 meganuclease in RHO1-2 cells A standard triple transfection protocol was performed in HEK-293 cells. Recombinant AAV vectors (rAAV-RHO-1 / 2) were prepared using the following methods (Dritt et al., anti et al. (2001), J Gene Med.3:59-71). three The rAAV-RHO-1 / 2 vector was injected into RHO1-2CHO reporter cells at a viral concentration of 100 μg / ml. 24 hours after transduction, cells were lysed and polyclonal meganuclear by Western blot using a β-actin-specific antibody or a β-actin-specific control antibody. As shown in Figure 8B, RHO-1 was inhibited at all three virus concentrations. Expression of / 2-L2-49 was observed.

[0414] 3. Specificity of AAV-delivered RHO1-2 meganuclease rAAV-RHO-1 / 2 vectors were transduced into RHO1-2 or RHO3-4 cells. and the P of SEQ ID NO: 1 compared to the corresponding wild-type RHO3-4 recognition sequence (SEQ ID NO: 5). AAV-delivered RHO-1 / 2-L2-49 meganucleic acid for recognition and cleavage of the 23H recognition sequence As shown in Figure 9, 3 days after transduction, AAV delivery was observed. The RHO-1 / 2-L2-49 meganuclease was effective at all virus concentrations used. induced a higher percentage of GFP-positive RHO1-2 cells than RHO3-4 cells, which , showing that the meganuclease preferentially recognizes and cleaves the mutant sequence.

[0415] 4. Persistence of AAV-delivered RHO1-2 meganuclease expression Recombinant AAV vectors encoding RHO-1 / 2-L2-49 meganuclease or GFP RHO1-2 cells transduced with the vector were treated with cycloheximide to inhibit protein translation. The stability of the expressed protein in the cells was determined. Cells were lysed at various time points after treatment (0, 1.5, 6, and 22 hours). Western blot analysis revealed that RHO-1 / 2-L2-49, GFP, and β-aminobutyric acid The protein levels of RHO-1 / 2- The L2-49 protein persisted significantly longer than GFP, and after 22 hours, No reduction in the enzyme protein was observed.

[0416] 5. Conclusion This study used recombinant AAV vectors to identify RHO1- 2. The ability to express meganucleases in cells and the ability to produce such meganucleases However, the P23H recognition of SEQ ID NO: 1 is significantly different from the corresponding wild-type RHO3-4 sequence (SEQ ID NO: 5). Furthermore, this study demonstrates that recombinant AAV preferentially recognizes and cleaves the recognition sequence. When expressed using a vector, the RHO1-2 meganuclease protein is secreted into cells. It has been demonstrated to be stable at

[0417] Example 4 AAV-delivered RHO1-2 meganuclease in reporter cells Cleavage of the RHO1-2 recognition sequence

[0418] 1. Recombinant AAV Vector Production The aim of this study was to introduce RHO1-2 meganuclease into mammalian cells by viral transduction. The objective of this study was to demonstrate that the RHO1-2 recognition sequence can be cleaved by expressing the RHO1-2 recognition sequence in mammalian cells. The objective of this study was to further demonstrate their ability to

[0419] For this experiment, two recombinant A. The AV2 vector was produced. Two donor plasmids were pDS CMV RHO2_L 3_59 (SEQ ID NO: 248) and pDS CMV RHO2_L5_14 (SEQ ID NO: 24 9) (These are the RHO2-L3-59 and RHO2-L5-14 meganuclei, respectively. These plasmids were designed to encode the nuclease (Fig. 11). The CMV promoter and enhancer that drive expression, the SV40 polyA sequence, and the genomic DNA fragment The self-complementary AAV genome ( These capsids are suitable for the production of scAAVs. The vector was generated using a triple transfection method in HEK293 cells. Recombinant scAAV was produced using the RT-PCR and purified on a CsCl gradient. Viral titers were determined by lot method.

[0420] 2. Analysis of Reporter Cell Transduction and Cleavage Efficiency These AAV particles contain the RHO1-2 recognition sequence but not the RHO3-4 recognition sequence (SEQ ID NO: 5). A functional RHO1-2 megaprotein capable of recognizing and specifically cleaving the recognition sequence (SEQ ID NO: 1) To determine whether nucleases could be delivered to CHO reporters, a previously described They were tested in strains (see WO 2012 / 167192). To perform the assay, a non-functional green fluorescent protein (G) was integrated into the genome of the cells. A set of CHO cell reporter lines carrying the FP gene expression cassette was produced. Intracellular cleavage of the recognition sequence by the enzyme stimulates a homologous recombination event, resulting in the generation of a functional GFP gene. The RHO1-2 or RHO3-4 recognition sequences are used to generate the offspring. The GFP gene of each cell line was disrupted. CHO reporter cells containing the RHO1-2 recognition sequence These cells are referred to as "P23H RHO cells" in this experiment. The HO reporter cells are referred to as "WT RHO cells" in this experiment. Wild-type control cells that do not carry the -cassette are referred to as "CHO ​​K cells" in this experiment.

[0421] The recombinant scAAV was transfected into WT RHO cells, P23H RHO cells, or wild-type CHO cells. K cells (negative control) were infected with 3 MOIs: 1 × 10 8 , 1×10 9 , or 2×1 0 9 ("low," "medium," and "high," respectively). After 48 hours, cells were assessed by flow cytometry and compared to wild-type control CHO cells. The percentage of GFP-positive cells was determined. Control wild-type CHO infected with either -59 or RHO2-L5-14 scAAV The cells showed less than 1% background GFP expression. The cells ("WT RHO cells") did not show significantly higher GFP expression than the control cells. This is because the RHO1-2 meganuclease cleaves the RHO3-4 recognition sequence. In P23H RHO cells, RHO2-L3-59 and RHO2-L3-59 were not expressed. Both HO2-L5-14 meganucleases induced significant GFP expression in a dose-dependent manner. At the highest dose, the level approached 10% GFP+.

[0422] GFP and Meganuclease Expression in P23H RHO Cells Using Western Blot Analysis P23H cells infected with three different doses of scAAV were tested for both cleavage and cleavage. Whole cell lysates were prepared only from RHO cells and WT RHO cells. Duplicate lysates were taken to obtain equal amounts (as determined by protein concentration). Lysates were separated by SDS-PAGE, transferred to a membrane, and transfected with GFP, I-CreI meganuclease, and using antibodies against either cleavage enzyme or β-actin (for loading control). The RHO1-2 meganuclease was probed with a polyclonal antibody against I-CreI. It can be detected using antibodies.

[0423] As shown in Figure 13, Western blot analysis demonstrated dose-dependent detection of GFP. This clearly demonstrates the above flow cytometry data shown in Figure 12. Western blot analysis also demonstrated a dose-dependent RHO1-2 meganuclease This blot shows that RHO2-L5-14 expresses RHO2-L3 This suggests that β-actin was expressed at a higher level than β-59. This indicates proper gel loading.

[0424] 3. Conclusion Taken together, these data demonstrate the expression potential for the RHO1-2 meganuclease of the present invention. The recombinant scAAV carrying the cassette was used to infect the CHO reporter strain and express RHO1-2 megaproteins. This can lead to the expression of a nuclease, which in turn recognizes WT RHO3-4. It was found that the P23H RHO1-2 recognition sequence (SEQ ID NO: 1) can be specifically cleaved without any restriction. This demonstrates that:

[0425] Example 5 In vivo cleavage of the P23H allele in a mouse model of RP

[0426] 1. Production of Recombinant AAV Vectors and Subretinal Delivery to Mouse Eyes The purpose of this study was to investigate whether the RHO1-2 meganuclease of the present invention is effective in the photoreceptor cells of mouse retina. The objective of this study was to determine whether RHO1-2 recognition sequences could be targeted and cleaved in vivo in vivo. It was.

[0427] Prepare recombinant scAAV using the triple transfection method as described above, This was tested in a mouse model of retinitis pigmentosa, in which a transgenic The mice carry a single copy of the human P23H mutation in addition to the endogenous mouse RHO allele. These mice do not exhibit the retinitis pigmentosa phenotype, but they This is useful for molecular analysis of in vivo cleavage of the RHO1-2 recognition sequence. In the O strain, RHO2L5-14 performed better, so RHO2-L5-14 was used. Only the scAAV encoding the vector was used to infect mice (except for the vector shown in SEQ ID NO: 280). RHO2-L3-59 meganuclease, including the production of scAAV using donor plasmids (This experiment could be performed using the scAAV triple transfection.) pDS GRK1 RHO2_L5_14 donor plasmid used in the transplantation protocol The plasmid (SEQ ID NO: 281) is shown in Figure 14. As shown, RHO2 The L5-14 meganuclease was under the control of the rod cell-specific GRK1 promoter. As a control, a recombinant scAAV encoding a GFP expression cassette was also prepared. Capsids from serotype 5 were used to generate these recombinant scAAV vectors.

[0428] One of the P23H transgenic mice was used to transduce the RHO2-L5-14 expression cassette. Subretinal injection of AAV encoding the rho1-2 gene results in cleavage at the RHO1-2 recognition sequence. Briefly, on postnatal day 30, mice were administered 100 mg / kg ketamine and 100 mg / kg ketamine. Mice were placed under general anesthesia by intraperitoneal injection of 10 mg / kg of silazine. Pupils were dilated with 0.5% ethanol and 1% proparacaine. Using a 30-gauge needle, a small incision was made through the cornea adjacent to the limbus. A 33-gauge blunt needle attached to a Luton syringe was inserted through the incision. All injections were performed subretinal. 1 μL of scAAV was administered to one eye and 1 μL of scAAV encoding GFP was administered to the other eye. (both virus preparations contained 7 × 10 particles) 12 The concentration was 1000 / mL. Olescein was added to the vector suspension to aid visualization during injection. Examination and OCT examination were performed to confirm successful subretinal delivery.

[0429] Mice were euthanized using isoflurane, and the eyes were enucleated 30 days after injection. The retina was carefully dissected from other ocular tissues under a microscope. A DNA isolation kit (Qiagen) was used. DNA was isolated from dissected retinas using proteinase K. The retinas were digested for 2 hours at 55°C using a lysis buffer containing HCl. After lysis, the crude extract was The bound DNA was washed several times by passing it through a column. The DNA was eluted and then transferred to NanoDrop (Th Concentrations were estimated by ermo.

[0430] To determine the presence and relative frequency of mutations in the RHO1-2 recognition sequence, The 1-2 locus was PCR amplified and subjected to deep sequencing analysis. PCR primers were designed to amplify a region of approximately 200 bp spanning the RHO1-2 recognition sequence. and subjected to deep sequencing analysis using an Illumina MiSeq instrument. The purified PCR band was provided.

[0431] 2.Results Deep sequencing of PCR bands yielded 5 × 10 per sample. 5 array of items The retinas injected with scAAV encoding GFP showed a strong affinity for the RHO1-2 recognition sequence. Approximately 4 x 10 cells with indels (insertions or deletions) 3 0.54% of the sequences A background was established. The AA In DNA isolated from the retina of mice injected with V, indels were detected in 3.92% of cases. In other experiments (data not shown), the control Mice showed a lower frequency of indels, typically around 0.01%.

[0432] [Table 3]

[0433] 3. Conclusion Deep sequencing data revealed that RHO2-L5-14 encodes a meganuclease Subretinal injection of AAV induces, after cleavage and non-homologous end-joining, the development of retinitis pigmentosa in a mouse model. This demonstrates that the P23H RHO1-2 recognition sequence mutations were introduced in the .

[0434] Example 6: In vivo expression of RHO1-2 meganuclease in retinal cells 1. Western Blot Analysis of Retinal Cells RHO2-L5-14 meganuclear expression in wild-type mouse retinal cells after AAV delivery To confirm that RHO2-expressing rhesus kinase can be expressed in mice, we performed subretinal injection of RHO2- Five wild-type mice were administered scAAV encoding L5-14. In D (right), both eyes were infected. Thirty days after injection, the retinas were dissected and whole cell lysates were prepared. and Western blot analysis was performed using anti-I-CreI antibody as described above.

[0435] Western blot analysis revealed that RHO2L-5-14 expression was predominant in the retina. It was shown that the retina was easily detected (lanes 3, 5, 7, and 10 in FIG. 15). In the case of α-glucanase 1, α-glucanase 2, α-glucanase 3, α-glucanase 4, α-glucanase 5, α-glucanase 6, α-glucanase 7, α-glucanase 8, α-glucanase 9, α-glucanase 10, α-glucanase 11, α-glucanase 12, and 6). In the remaining retina, the level of expression was barely detectable (Layer 1 in Figure 15). Subretinal injections rely on precise delivery to a very small area behind the eye. This procedure is fairly common in adult patients, but in mouse models it is not a common laboratory procedure. Therefore, the difference in expression was attributed to the difficulty of subretinal injection in mice.

[0436] 2. Conclusion Western blot analysis demonstrated that subretinal delivery of RHO2L-5-14 to mice resulted in increased cellular responses to steroids. It was demonstrated that the scAAV delivered RHO1-2 meganuclease expression. Combined with indel data from mouse models of the human P23H RHO gene These data therefore suggest that AAV delivery of RHO1-2 meganuclease significantly improves the survival of P23H This suggests that it is effective in causing deletions in RHO alleles.

Claims

1. A recombinant meganuclease that recognizes and cleaves the P23H recognition sequence, a first subunit and a second subunit, wherein the first subunit contains the P23H recognition sequence Recognizing a first recognition half site of (a) residues 198 to 344 of any one of SEQ ID NOs: 6 to 69 or SEQ ID NOs: 70 to 93 an amino acid sequence having at least 80% sequence identity with residues 7 to 153 of any one of and (b) the first hypervariable (HVR1) region Including, the second subunit recognizes a second recognition half-site of the P23H recognition sequence; (a) residues 7 to 153 of any one of SEQ ID NOs: 6 to 69 or any one of SEQ ID NOs: 70 to 93 an amino acid sequence having at least 80% sequence identity with any one of residues 198-344 and (b) the second hypervariable (HVR2) region A recombinant meganuclease comprising:

2. The first subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 6 to 69 or has at least 85% sequence identity with residues 7-153 of any one of SEQ ID NOs: 70-93 and wherein the second subunit comprises an amino acid sequence having the sequence of any one of SEQ ID NOs: 6 to 69. residues 7-153 of one of SEQ ID NOs: 70-93 or residues 198-344 of any one of SEQ ID NOs: 70-93 and at least 2. The recombinant megakaryon of claim 1, comprising an amino acid sequence having at least 85% sequence identity. Nuclease.

3. The first subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 6 to 69 or has at least 90% sequence identity with residues 7-153 of any one of SEQ ID NOs: 70-93 and wherein the second subunit comprises an amino acid sequence having the sequence of any one of SEQ ID NOs: 6 to 69. residues 7-153 of one of SEQ ID NOs: 70-93 or residues 198-344 of any one of SEQ ID NOs: 70-93 and at least 3. The recombinant vector of claim 1 or 2, comprising an amino acid sequence having at least 90% sequence identity. Meganuclease.

4. The first subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 6 to 69 or has at least 95% sequence identity with residues 7-153 of any one of SEQ ID NOs: 70-93 and wherein the second subunit comprises an amino acid sequence having the sequence of any one of SEQ ID NOs: 6 to 69. residues 7-153 of one of SEQ ID NOs: 70-93 or residues 198-344 of any one of SEQ ID NOs: 70-93 and at least Any one of claims 1 to 3, comprising an amino acid sequence having at least 95% sequence identity with said A recombinant meganuclease according to claim 1.

5. the P23H recognition sequence comprises SEQ ID NO: 1, and the HVR1 region comprises: (A) position 215 of any one of SEQ ID NOs: 6 to 69; or (B) position 24 of any one of SEQ ID NOs: 70 to 93 The recombinant vector according to any one of claims 1 to 4, comprising W or Y at the position corresponding to Ganuclease.

6. The P23H recognition sequence comprises SEQ ID NO: 1, and the HVR1 region comprises any of SEQ ID NOs: 6 to 69. Residues 215-270 of any one of SEQ ID NOs: 70-93 or residues 24- 79. The recombinant meganuclease of any one of claims 1 to 5.

7. The P23H recognition sequence comprises SEQ ID NO: 1, and the HVR2 region comprises any of SEQ ID NOs: 6 to 69. Residues 24-79 of any one of SEQ ID NOs: 70-93 or residues 215-215 of any one of SEQ ID NOs: 70-93 70. The recombinant meganuclease of any one of claims 1 to 6.

8. a linker covalently connecting the first subunit and the second subunit; The recombinant meganuclease according to any one of claims 1 to 7, which is a single-chain meganuclease comprising Nuclease.

9. The first subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 6 to 69 or any one of claims 1 to 8, comprising residues 7 to 153 of any one of SEQ ID NOs: 70 to 93.

2. A recombinant meganuclease according to claim 1.

10. The second subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 6 to 69 or Any of claims 1 to 9, comprising residues 198 to 344 of any one of sequences 70 to 93 2. A recombinant meganuclease according to claim 1.

11. Any one of claims 1 to 10, comprising an amino acid sequence of any one of SEQ ID NOs: 6 to 93.

2. A recombinant meganuclease according to claim 1.

12. 12. Any one of claims 1 to 11, wherein the P23H recognition sequence comprises one of SEQ ID NOs: 1 to 4. A recombinant meganuclease according to claim 1.

13. The combination of any one of claims 1 to 12, wherein the P23H recognition sequence comprises SEQ ID NO:

1. Recombinant meganucleases.

14. P23H recognition sequences containing SEQ ID NO: 1 are preferentially recognized compared to recognition sequences containing SEQ ID NO:

5. The recombinant meganuclease according to any one of claims 1 to 13, which recognizes and cleaves a target gene.

15. A nucleic acid sequence encoding a recombinant meganuclease according to any one of claims 1 to 14. An isolated polynucleotide comprising:

16. A recombinant DNA construct comprising the isolated polynucleotide of claim 15.

17. The recombinant adeno-associated virus (AAV) vector of claim 16 . DNA constructs.

18. A recombinant AAV vector comprising the isolated polynucleotide of claim 15.

19. 1. A method for treating retinitis pigmentosa in a subject in need thereof, comprising: A recombinant meganuclear cell that recognizes and cleaves the DNA of a target cell of a subject at the P23H recognition sequence. contacting the enzyme with the target cell contains the P23H recognition sequence in an RHO gene allele; cleavage of the P23H recognition sequence inhibits expression of the RHO gene allele; the recombinant meganuclease comprises a first subunit and a second subunit; the first subunit recognizes a first recognition half-site of the P23H recognition sequence; (a) residues 198 to 344 of any one of SEQ ID NOs: 6 to 69 or SEQ ID NOs: 70 to 93 an amino acid sequence having at least 80% sequence identity with residues 7 to 153 of any one of and (b) the first hypervariable (HVR1) region Including, the second subunit recognizes a second recognition half-site of the P23H recognition sequence; (i) residues 7 to 153 of any one of SEQ ID NOs: 6 to 69 or any one of SEQ ID NOs: 70 to 93 an amino acid sequence having at least 80% sequence identity with any one of residues 198-344 and (ii) the second hypervariable (HVR2) region A method comprising:

20. 20. The method of claim 19 for treating autosomal dominant retinitis pigmentosa.

21. The first subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 6 to 69 or has at least 85% sequence identity with residues 7-153 of any one of SEQ ID NOs: 70-93 and wherein the second subunit comprises an amino acid sequence having the sequence of any one of SEQ ID NOs: 6 to 69. residues 7-153 of one of SEQ ID NOs: 70-93 or residues 198-344 of any one of SEQ ID NOs: 70-93 and at least 21. The method of claim 19 or 20, comprising an amino acid sequence having at least 85% sequence identity with the method.

22. The first subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 6 to 69 or has at least 90% sequence identity with residues 7-153 of any one of SEQ ID NOs: 70-93 and wherein the second subunit comprises an amino acid sequence having the sequence of any one of SEQ ID NOs: 6 to 69. residues 7-153 of one of SEQ ID NOs: 70-93 or residues 198-344 of any one of SEQ ID NOs: 70-93 and at least Any of claims 19 to 21, comprising an amino acid sequence having at least 90% sequence identity.

2. The method according to claim 1.

23. The first subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 6 to 69 or has at least 95% sequence identity with residues 7-153 of any one of SEQ ID NOs: 70-93 and wherein the second subunit comprises an amino acid sequence having the sequence of any one of SEQ ID NOs: 6 to 69. residues 7-153 of one of SEQ ID NOs: 70-93 or residues 198-344 of any one of SEQ ID NOs: 70-93 and at least Any of claims 19 to 22, comprising an amino acid sequence having at least 95% sequence identity.

2. The method according to claim 1.

24. the P23H recognition sequence comprises SEQ ID NO: 1, and the HVR1 region comprises: (A) position 215 of any one of SEQ ID NOs: 6 to 69; or (B) position 24 of any one of SEQ ID NOs: 70 to 93 The method of any one of claims 19 to 23, comprising W or Y at a position corresponding to 。

25. The P23H recognition sequence comprises SEQ ID NO: 1, and the HVR1 region comprises any of SEQ ID NOs: 6 to 69. Residues 215-270 of any one of SEQ ID NOs: 70-93 or residues 24- 79. The method of any one of claims 19 to 24, including

26. The P23H recognition sequence comprises SEQ ID NO: 1, and the HVR2 region comprises any of SEQ ID NOs: 6 to 69. Residues 24-79 of any one of SEQ ID NOs: 70-93 or residues 215-215 of any one of SEQ ID NOs: 70-93 70. The method of any one of claims 19 to 25.

27. the recombinant meganuclease comprises the first subunit and the second subunit 19 to 2, which is a single-chain meganuclease comprising a linker covalently connecting 7. The method according to any one of claims 6 to 6.

28. The first subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 6 to 69 or any one of claims 19 to 27, comprising residues 7 to 153 of any one of SEQ ID NOs: 70 to 93. The method according to any one of claims 1 to 10.

29. The second subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 6 to 69 or Any of claims 19 to 28, comprising residues 198 to 344 of any one of sequences 70 to 93 The method according to any one of claims 1 to 10.

30. The recombinant meganuclease comprises any one of the amino acid sequences set forth in SEQ ID NOs: 6 to 93. The method according to any one of claims 19 to 29.

31. Any one of claims 19 to 30, wherein the P23H recognition sequence comprises one of SEQ ID NOs: 1 to 4.

2. A recombinant meganuclease according to claim 1.

32. 32. The method of claim 19, wherein the P23H recognition sequence comprises SEQ ID NO:

1. Recombinant meganucleases.

33. The recombinant meganuclease has a recognition sequence comprising SEQ ID NO: 1 compared to a recognition sequence comprising SEQ ID NO:

5. The method according to any one of claims 19 to 32, which preferentially recognizes and cleaves a P23H recognition sequence containing the P23H recognition sequence. How to post.

34. 34. Any one of claims 19 to 33, wherein the target cells in the subject are retinal cells. The method described below.

35. A recombinant AAV vector is used to express the gene encoding the recombinant meganuclease. The method according to any one of claims 19 to 34, wherein the delivery is to the target cells.

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