Genetically engineered meganuclease having specificity to recognition sequence in dystrophin gene

Abstract

To provide a genetically engineered meganuclease that binds and cleaves a recognition site in a dystrophin gene (e.g., human dystrophin gene), compositions comprising such an engineered meganuclease, and use methods thereof.SOLUTION: Disclosed is a genetically engineered meganuclease that binds to a recognition sequence in a dystrophin gene to cleave it, where the engineered meganuclease comprises a first subunit and a second subunit, where the first subunit binds to a first recognition half-site of the recognition site, and comprises a first hyper variable region (HVR1), the second subunit binds to a second recognition half-site of the recognition site and comprises a second hypervariable region (HVR2).SELECTED DRAWING: Figure 11

Description

Technical Field

[0001] This application relates to the fields of genetically engineered meganucleases, molecular biology, and recombinant nucleic acid technology In certain aspects, the invention relates to genetically engineered meganucleases useful for the removal of exons from the dystrophin gene and the treatment of subjects having Duchenne muscular dystrophy

[0002] Reference to the Sequence Listing Submitted as a Text File via EFS-Web This application includes a Sequence Listing submitted in ASCII format via EFS-Web, the entire contents of which are hereby incorporated by reference into this specification. The name of the above-mentioned ASCII copy created on November 12, 2021 is P109070054WO00-SEQ-EPG, and the size is 279,819 bytes

Background Art

[0003] Duchenne muscular dystrophy (DMD) is a rare X-linked muscle degenerative disorder that affects approximately 1 in 3,500 boys worldwide. This disease is caused by mutations in the dystrophin gene, which is the largest known gene The dystrophin gene spans 2.2 Mb on the X chromosome and mainly encodes a 14 kb transcript derived from 79 exons The full-length dystrophin protein, which is expressed in skeletal muscle, smooth muscle, and cardiomyocytes, is 3,685 amino acids and has a molecular weight of 427 kD The severe Duchenne phenotype is generally associated with the loss of full-length dystrophin protein from skeletal and cardiac muscle, which leads to debilitating muscle degeneration and ultimately heart failure. A large number of different dystrophin gene mutations ​​​​​​​are described, many of which result in either severe DMD or milder Becker muscular dystrophy in either case.

[0004] Several therapeutic strategies are being pursued for the treatment of DMD. First, the "gene replacement" strategy is an active area of research (Non-Patent Documents 1 to 4). This approach involves delivering a functional copy of the dystrophin gene to patients using a viral delivery vector, typically an adeno-associated virus (AAV). However, the dystrophin gene is too large to be compatible with the limited carrying capacity of common viral vectors. This requires the use of a "micro dystrophin" gene in which most of the repetitive central portion of the gene has been removed, leaving only a minimal functional protein. However, it is not clear that the expression of "microdystrophin" is sufficient for clinical benefit. Furthermore, this approach is plagued by the possibility of random gene integration into the patient's genome, which can lead to insertional mutagenesis, and the possibility of an immune response to the delivery vector.

[0005] A second approach to treating DMD involves transplanting healthy muscle progenitor cells into the patient's muscle fibers (Non-Patent Documents 5, 6). This approach is plagued by the inefficient migration of transplanted myoblasts and the possibility of immune rejection by the patient.

[0006] A third approach involves suppression of nonsense mutations using PTC124 (Non-Patent Document 7). However, this requires lifelong administration of the drug, and this approach has not yet shown any significant clinical benefit.

[0007] ​​​​​​​​​A fourth approach for treating DMD is what is called "exon skipping" (Non-Patent Literatures 8-14). Generally, the amino (N) and carboxy (C) terminal portions of the dystrophin gene are essential for its role as a "scaffold" protein that maintains the membrane integrity of muscle fibers, but the central "rod domain", which contains 24 spectrin-like repeats, is at least partially non-essential. In fact, the severe Duchenne phenotype is typically associated with mutations in the dystrophin gene that introduce frameshifts and / or premature stop codons, resulting in truncated forms of the dystrophin protein lacking the essential C-terminal domain. Mutations in the central rod domain that include large deletions of entire exons typically result in a

[0008] much milder Becker phenotype when the reading frame is maintained such that the C-terminal domain of the protein is intact. DMD is most frequently caused by deletions of one or more entire exons, resulting in a frameshift. For example, exon 45 is frequently deleted in Duchenne patients. Exon 45 is 176 bp long, which is not divisible by 3, so deleting the exon shifts exons 46-79 into the wrong reading frame. The same is true for exon 44, which is 148 bp long. However, when exons 44 and 45 are deleted, the total size of the deletion is 324 bp, which is divisible by 3. Thus, deletion of both exons does not result in a frameshift. Since these A phenotype is expected to occur. Therefore, patients with the Duchenne phenotype caused by the deletion of one or more exons can potentially be treated by excluding one or more adjacent exons to restore the reading frame. This is the principle behind "exon skipping" which uses modified oligonucleotides to block the splice acceptor sites of dystrophin pre-mRNA so that one or more specific exons are not present in the processed transcript. This approach has been used to restore dystrophin gene expression in the mdx mouse model by skipping exon 23 which has a disease-causing nonsense mutation (Non-Patent Document 15). Oligonucleotide analogs that induce the skipping of exon 51 also show promise in early human clinical trials (Non-Patent Document 16). The main limitations of this approach are as follows: (1) The exon skipping process is inefficient and results in relatively low levels of functional dystrophin expression; (2) Exon skipping oligonucleotides have a relatively short half-life so their effect is transient and repeated and lifelong dosing is required. Therefore, while the exon skipping approach shows some promise in clinical trials, the improvement in disease progression is minimal and variable. Patients having the Duchenne phenotype can potentially be treated by excluding one or more adjacent exons to restore the reading frame. This is the principle behind "exon skipping" which uses modified oligonucleotides to block the splice acceptor sites of dystrophin pre-mRNA so that one or more specific exons are not present in the processed transcript. This approach has been used to restore dystrophin gene expression in the mdx mouse model by skipping exon 23 which has a disease-causing nonsense mutation (Non-Patent Document 15). Oligonucleotide analogs that induce the skipping of exon 51 also show promise in early human clinical trials (Non-Patent Document 16). The main limitations of this approach are as follows: (1) The exon skipping process is inefficient and results in relatively low levels of functional dystrophin expression; (2) Exon skipping oligonucleotides have a relatively short half-life so their effect is transient and repeated and lifelong dosing is required. Therefore, while the exon skipping approach shows some promise in clinical trials, the improvement in disease progression is minimal and variable. Patients having the Duchenne phenotype can potentially be treated by excluding one or more adjacent exons to restore the reading frame. This is the principle behind "exon skipping" which uses modified oligonucleotides to block the splice acceptor sites of dystrophin pre-mRNA so that one or more specific exons are not present in the processed transcript. This approach has been used to restore dystrophin gene expression in the mdx mouse model by skipping exon 23 which has a disease-causing nonsense mutation (Non-Patent Document 15). Oligonucleotide analogs that induce the skipping of exon 51 also show promise in early human clinical trials (Non-Patent Document 16). The main limitations of this approach are as follows: (1) The exon skipping process is inefficient and results in relatively low levels of functional dystrophin expression; (2) Exon skipping oligonucleotides have a relatively short half-life so their effect is transient and repeated and lifelong dosing is required. Therefore, while the exon skipping approach shows some promise in clinical trials, the improvement in disease progression is minimal and variable. Patients having the Duchenne phenotype can potentially be treated by excluding one or more adjacent exons to restore the reading frame. This is the principle behind "exon skipping" which uses modified oligonucleotides to block the splice acceptor sites of dystrophin pre-mRNA so that one or more specific exons are not present in the processed transcript. This approach has been used to restore dystrophin gene expression in the mdx mouse model by skipping exon 23 which has a disease-causing nonsense mutation (Non-Patent Document 15). Oligonucleotide analogs that induce the skipping of exon 51 also show promise in early human clinical trials (Non-Patent Document 16). The main limitations of this approach are as follows: (1) The exon skipping process is inefficient and results in relatively low levels of functional dystrophin expression; (2) Exon skipping oligonucleotides have a relatively short half-life so their effect is transient and repeated and lifelong dosing is required. Therefore, while the exon skipping approach shows some promise in clinical trials, the improvement in disease progression is minimal and variable. reading frame. This is the principle behind "exon skipping" which uses modified oligonucleotides to block the splice acceptor sites of dystrophin pre-mRNA so that one or more specific exons are not present in the processed transcript. This approach has been used to restore dystrophin gene expression in the mdx mouse model by skipping exon 23 which has a disease-causing nonsense mutation (Non-Patent Document 15). Oligonucleotide analogs that induce the skipping of exon 51 also show promise in early human clinical trials (Non-Patent Document 16). The main limitations of this approach are as follows: (1) The exon skipping process is inefficient and results in relatively low levels of functional dystrophin expression; (2) Exon skipping oligonucleotides have a relatively short half-life so their effect is transient and repeated and lifelong dosing is required. Therefore, while the exon skipping approach shows some promise in clinical trials, the improvement in disease progression is minimal and variable. reading frame. This is the principle behind "exon skipping" which uses modified oligonucleotides to block the splice acceptor sites of dystrophin pre-mRNA so that one or more specific exons are not present in the processed transcript. This approach has been used to restore dystrophin gene expression in the mdx mouse model by skipping exon 23 which has a disease-causing nonsense mutation (Non-Patent Document 15). Oligonucleotide analogs that induce the skipping of exon 51 also show promise in early human clinical trials (Non-Patent Document 16). The main limitations of this approach are as follows: (1) The exon skipping process is inefficient and results in relatively low levels of functional dystrophin expression; (2) Exon skipping oligonucleotides have a relatively short half-life so their effect is transient and repeated and lifelong dosing is required. Therefore, while the exon skipping approach shows some promise in clinical trials, the improvement in disease progression is minimal and variable. reading frame. This is the principle behind "exon skipping" which uses modified oligonucleotides to block the splice acceptor sites of dystrophin pre-mRNA so that one or more specific exons are not present in the processed transcript. This approach has been used to restore dystrophin gene expression in the mdx mouse model by skipping exon 23 which has a disease-causing nonsense mutation (Non-Patent Document 15). Oligonucleotide analogs that induce the skipping of exon 51 also show promise in early human clinical trials (Non-Patent Document 16). The main limitations of this approach are as follows: (1) The exon skipping process is inefficient and results in relatively low levels of functional dystrophin expression; (2) Exon skipping oligonucleotides have a relatively short half-life so their effect is transient and repeated and lifelong dosing is required. Therefore, while the exon skipping approach shows some promise in clinical trials, the improvement in disease progression is minimal and variable. The main limitations of this approach are as follows: (1) The exon skipping process is inefficient and results in relatively low levels of functional dystrophin expression; (2) Exon skipping oligonucleotides have a relatively short half-life so their effect is transient and repeated and lifelong dosing is required. Therefore, while the exon skipping approach shows some promise in clinical trials, the improvement in disease progression is minimal and variable. The main limitations of this approach are as follows: (1) The exon skipping process is inefficient and results in relatively low levels of functional dystrophin expression; (2) Exon skipping oligonucleotides have a relatively short half-life so their effect is transient and repeated and lifelong dosing is required. Therefore, while the exon skipping approach shows some promise in clinical trials, the improvement in disease progression is minimal and variable. The main limitations of this approach are as follows: (1) The exon skipping process is inefficient and results in relatively low levels of functional dystrophin expression; (2) Exon skipping oligonucleotides have a relatively short half-life so their effect is transient and repeated and lifelong dosing is required. Therefore, while the exon skipping approach shows some promise in clinical trials, the improvement in disease progression is minimal and variable. The main limitations of this approach are as follows: (1) The exon skipping process is inefficient and results in relatively low levels of functional dystrophin expression; (2) Exon skipping oligonucleotides have a relatively short half-life so their effect is transient and repeated and lifelong dosing is required. Therefore, while the exon skipping approach shows some promise in clinical trials, the improvement in disease progression is minimal and variable. The main limitations of this approach are as follows: (1) The exon skipping process is inefficient and results in relatively low levels of functional dystrophin expression; (2) Exon skipping oligonucleotides have a relatively short half-life so their effect is transient and repeated and lifelong dosing is required. Therefore, while the exon skipping approach shows some promise in clinical trials, the improvement in disease progression is minimal and variable.

[0009] The present disclosure improves the current exon skipping approach by correcting gene expression at the genomic DNA level rather than at the pre-mRNA level. The present invention often uses a pair of engineered site-specific homing endonucleases called meganucleases The present disclosure improves the current exon skipping approach by correcting gene expression at the genomic DNA level rather than at the pre-mRNA level. The present invention often uses a pair of engineered site-specific homing endonucleases called meganucleases The present disclosure improves the current exon skipping approach by correcting gene expression at the genomic DNA level rather than at the pre-mRNA level. The present invention often uses a pair of engineered site-specific homing endonucleases called meganucleases A permanent treatment for DMD that includes excising a specific exon from the dystrophin coding sequence. Such a pair of endonucleases is targeted to a site in the intron region adjacent to the exon within the dystrophin gene, enabling the intervening fragment to be permanently removed from the genome. The resulting cells and their progeny express a modified dystrophin in which a portion of the non-essential spectrin repeat domain has been removed, while the essential N-terminal and C-terminal domains remain intact. Homing endonucleases, i.e., meganucleases, are a group of naturally occurring nucleases that recognize cleavage sites of 15 - 40 base pairs commonly found in plant and fungal genomes. They are often associated with group I self-splicing introns and parasitic DNA elements such as inteins. By causing double-strand breaks in chromosomes,

[0010] they naturally promote homologous recombination or gene insertion at specific positions in the host genome and recruit the cell's DNA repair machinery (Non-Patent Document 17). Homing endonucleases are generally classified into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cys box family, and the HNH family. These families are characterized by structural motifs that affect catalytic activity and recognition sequences. For example, members of the LAGLIDADG family are characterized by having either one or two copies of the conserved LAGLIDADG motif (Non-Patent Document 18). LAGLIDADG homing endonucleases with a single copy of the LAGLIDADG motif form homodimers, while members with two copies of the LAGLIDADG motif are... are generally classified into four families: the LAGLIDADG family, the GIY-YIG family the His-Cys box family, and the HNH family. These families are characterized by structural motifs that affect catalytic activity and recognition sequences. For example, members of the LAGLIDADG family are characterized by having either one or two copies of the conserved LAGLIDADG motif (Non-Patent Document 18). LAGLIDADG homing endonucleases with a single copy of the LAGLIDADG motif form homodimers, while members with two copies of the LAGLIDADG motif are... with a single copy of the LAGLIDADG motif form homodimers, while members with two copies of the LAGLIDADG motif are... - is found as a monomer.

[0011] I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG family of homing endonucleases that recognize and cleave a 22-base pair recognition sequence in the chloroplast chromosome of the alga Chlamydomonas reinhardtii. Using gene selection techniques, the preference of the wild-type I-CreI cleavage site has been modified (Non-Patent Documents 19 to 22). Methods for rationally designing mono-LAGLIDADG homing endonucleases that can target a wide variety of DNA sites, including sites in mammalian, yeast, plant, bacterial, and viral genomes, by comprehensively redesigning I-CreI and other homing endonucleases have been described (Patent Document 1). As first described in Patent Document 2, I-CreI and its genetically engineered derivatives are usually dimers, but can be fused into a single polypeptide using a short peptide linker that binds the C-terminus of the first subunit to the N-terminus of the second subunit (Non-Patent Documents 23, 24). Thus, a functional "single-stranded" meganuclease can be expressed from a single transcript. By delivering genes encoding two different single-stranded meganucleases to the same cell, it is possible to cleave two different sites simultaneously. This, combined with the extremely low frequency of off-target cleavage observed with genetically engineered meganucleases, makes them preferred endonucleases for the present disclosure. - is modified. Including mammalian, yeast, plant, bacterial, and viral genomes. - can be rationally designed. - target a wide variety of DNA sites. - are described.

[0012] As first described in Patent Document 2, I-CreI and its genetically engineered derivatives are usually dimers, but can be fused into a single polypeptide using a short peptide linker that binds the C-terminus of the first subunit to the N-terminus of the second subunit (Non-Patent Documents 23, 24). - can be fused into a single polypeptide. - using a short peptide linker. - can be expressed from a single transcript. - encoding two different single-stranded meganucleases. - can cleave two different sites simultaneously. - is extremely low. - makes them preferred endonucleases for the present disclosure.

Prior Art Documents

Patent Documents

[0013] [Patent Document 1] International Publication WO2007 / 047859 [Patent Document 2] International Publication WO2009 / 059195 [Non-Patent Document]

[0014] [Non-Patent Document 1] Oshima et al. (2009) J. Am. Soc. Gene Ther. 17:73 - 80 [Non-Patent Document 2] Liu et al. (2005) Mol. Ther. 11:245 - 56 [Non-Patent Document 3] Lai et al. (2006) Hum Gene Ther. 17:1036 - 42 [Non-Patent Document 4] Odom et al. (2008) Mol. Ther. 16:1539 - 45 [Non-Patent Document 5] Peault et al. (2007) Mol. Ther. 15:867 - 77 [Non-Patent Document 6] Skuk et al. (2007) Neuromuscul. Disord. 17:38 - 46 [Non-Patent Document 7] Welch et al. (2007) Nature 447:87 - 91 [Non-Patent Document 8] Williams et al. (2008) BMC Biotechnol. 8:35 [Non-Patent Document 9] Jearawiriyapaisarn et al. (2008) Mol Ther. 16:1624 - 29 [Non-Patent Document 10] Yokota et al. (2007) Acta Myol. 26:179 - 84 [Non-Patent Document 11] van Deutekom et al. (2001) Hum. Mol. Gen. 10:1547 - 54 [Non - Patent Document 12] Benedetti et al. (2013) FEBS J. 280:4263 - 80 [Non - Patent Document 13] Rodino - Klapac (2013) Curr Neurol Neurosci Rep. 13:332 [Non - Patent Document 14] Verhaart & Aartsma - Rus (2012) Curr Opin Neurol. 25:588 - 96 [Non - Patent Document 15] Mann et al. (2001) Proc. Nat. Acad. Sci. USA 98:42 - 47 [Non - Patent Document 16] Benedetti et al. (2013) FEBS J. 280:4263 - 80 [Non - Patent Document 17] Stoddard (2006) Q. Rev. Biophys. 38:49 - 95 [Non - Patent Document 18] Chevalier et al. (2001) Nucleic Acids Res. 29:3757 - 74 [Non - Patent Document 19] Sussman et al. (2004) J. Mol. Biol. 342:31 - 41 [Non - Patent Document 20] Chames et al. (2005) Nucleic Acids Res. 33:e178 [Non - Patent Document 21] Seligman et al. (2002) Nucleic Acids Res. 30:3870 - 79 [Non - Patent Document 22] Arnould et al. (2006) J. Mol. Biol. 355:443 - 58 [Non - Patent Document 23] Li et al. (2009) Nucleic Acids Res. 37:1650 - 62 [Non-Patent Document 24] Grizot et al. (2009) Nucleic Acids Res.37:5405-19 Summary of the Invention [Problem to be solved by the invention]

[0015] The present disclosure relates to a recognition sequence within a dystrophin gene (e.g., the human dystrophin gene). and engineered meganucleases that bind to and cleave Compositions comprising the selected meganucleases and methods of using them are provided. In this embodiment, the pair of engineered meganucleases is a pair of exons located upstream of the first exon in an intron. and a second cleavage site in an intron downstream of the second exon. used to remove multiple exons from the dystrophin gene by In certain examples described herein, the first cleavage site is located in the exon of the dystrophin gene. The first cleavage site is generated in the intron 5' upstream of exon 45, and the second cleavage site is generated in the intron 3' downstream of exon 55. This process involves the annealing of two cleavage sites and the creation of an intron in the genome. This allows for the excision and removal of exons 45-55 from the dystrophin gene after repair. The recognition sequences targeted by the disclosed engineered meganucleases are The first and second cleavage sites are complementary 4-base pair 3′s that can ligate perfectly to each other. ´ Selected to have identical 4 base pair core sequences with overhangs (i.e., each base in one overhang pair is identical to its complement on the other overhang) The exons are then paired with the exons of the mutant dystrophin gene, which lacks one or more of these exons. By removing exons 45-55, this approach results in the restoration of the normal (i.e., wild-type) reading frame of the dystrophin gene. Cells treated in this way express a truncated modified form of the dystrophin protein in which part of the central spectrin repeat domain is absent, but the N- terminal and C-terminal domains are intact. This often reduces the severity of the disease. In some cases, it results in a milder Becker phenotype.

[0016] Accordingly, in one aspect, the invention provides a genetically engineered meganuclease that binds to and cleaves a recognition sequence within the dystrophin gene, the genetically engineered meganuclease comprising a first subunit and a second subunit, the first subunit binding to a first recognition half-site of the recognition sequence and comprising a first hypervariable (HVR1) region, and the second

[0017] subunit binding to a second recognition half-site of the recognition

[0018] sequence and comprising a second hypervariable (HVR2) region. In some embodiments, the recognition sequence comprises SEQ ID NO: 6. In some such embodiments, the HVR1 region has at least 80%, 85%, is one of residues 24, 26, 28, 30, 32, 33, 3 8, 40, 42, 44, 46, 68, 70, 75, and 77 corresponding to one or more residues including. In some embodiments, the HVR1 region is any one of residues 36-44 in SEQ ID NO: One of 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77. In some embodiments, the HVR1 region contains Y, R, K, or D in a residue corresponding to residue 66 of any one of residues 36-44 in SEQ ID NO: In some embodiments, the HVR1 region has amino acid substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 and contains any one of residues 24-79 of any one of residues 36-44 in SEQ ID NO: In some embodiments, the HVR1 region contains any one of residues 24-79 of any one of residues 36-44 in SEQ ID NO: .

[0019] In some such embodiments, the first subunit has at least 80%, 85%, 90%, 91%, 92%, 9 3%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any one of residues 7-153 of any one of residues 36-44 in SEQ ID NO: and contains an amino acid sequence. In some embodiments, the first subunit contains G, S, or A in a residue corresponding to residue 19 of any one of residues 36-44 in SEQ ID NO: . In some embodiments, the first subunit contains a residue corresponding to residue 19 of any one of residues 36-44 in SEQ ID NO: . In some embodiments, the first subunit contains E, Q, or K in a residue corresponding to residue 80 of any one of residues 36-44 in SEQ ID NO: . In some embodiments, the first subunit is any one of SEQ ID NOs: 38, 39, or 149 . ~44 corresponding residues contain E, Q, or K. In some embodiments, the first subunit is any one of SEQ ID NOs: 38, 39, or 149 . It includes a residue corresponding to residue 80. In some embodiments, the first subunit is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions and includes any one of residues 7 - 153 of SEQ ID NOs: 36 - 44. In some embodiments, the first subunit includes any one of residues 7 - 153 of SEQ ID NOs: 36 - 44. It includes.

[0020] In some such embodiments, the HVR2 region has at least 80%, 85%, 90% identity with an amino acid sequence corresponding to residues 215 - 270 of any one of SEQ ID NOs: 36 - 44, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. In some embodiments, the HVR2 region includes an amino acid sequence having such sequence identity. In some embodiments, the HVR2 region includes one or more residues corresponding to residues 215, 217, 219, 221, 2 23, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 36 - 44. In some embodiments, the HVR2 region includes residues corresponding to residues 215, 217, 219, 221, 223 of any one of SEQ ID NOs: 36 - 44, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 2 68. In some embodiments, the HVR2 region includes a residue containing Y, R, K, or D at a residue corresponding to residue 257 of any one of SEQ ID NOs: 36 - 44. In some embodiments, the HVR2 region includes a residue corresponding to residue 236 of SEQ ID NO: 39. In some embodiments, the HVR2 region includes a residue corresponding to residue 236 of SEQ ID NO: 39. In one embodiment, the HVR2 region contains a residue corresponding to residue 239 of SEQ ID NO: 37 。In some embodiments, the HVR2 region contains a residue corresponding to residue 241 of any one of SEQ ID NOs: 36-37 。In some embodiments, the HVR2 region contains a residue corresponding to residue 263 of SEQ ID NO: 36 。In some embodiments, the HVR2 region contains a residue corresponding to residue 264 of any one of SEQ ID NOs: 36-44 。In some embodiments, the HVR2 region contains a residue corresponding to residue 215-270 of any one of SEQ ID NOs: 36-44 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions 。In some embodiments, the HVR2 region contains residues 215-270 of any one of SEQ ID NOs: 36-44 。In some such embodiments, the second subunit has at least 80%, 85%, 90%, 91%, 92% 。In some embodiments, the HVR2 region contains residues 215 of any one of SEQ ID NOs: 36-44 ~270

[0021] 。In some such embodiments, the second subunit has at least 80%, 85%, 90%, 91%, 92% identity with any one of residues 198-344 of SEQ ID NOs: 36-44 、93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity 。In some embodiments, the second subunit contains G, S, or A at the residue corresponding to residue 210 of any one of SEQ ID NOs: 36-44 。In some embodiments, the second subunit contains E, Q, or K at the residue corresponding to residue 271 of any one of SEQ ID NOs: 36-44 。In some embodiments, the second subunit contains a residue corresponding to residue 271 of any one of SEQ ID NOs: 36, 39, 40, 43, or 44 。In some embodiments, the second subunit contains a residue corresponding to residue 330 of any one of SEQ ID NOs: 36- 38 or 40-44 。In some embodiments, the second subunit contains a residue corresponding to residue 330 of any one of SEQ ID NOs: 36-38 or 40-44 。In some embodiments, the second subunit contains a residue corresponding to residue 330 of any one of SEQ ID NOs: 36-38 or 40-44 。In some embodiments, the second subunit contains a residue corresponding to residue 330 of any one of SEQ ID NOs: 36-38 or 40-44 In a form, the second subunit has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2 5, 26, 27, 28, 29, or up to 30 amino acid substitutions and includes any one of residues 198 - 344 of SEQ ID NOs: 36 - 4 4. In some embodiments, the second sub - unit includes any one of residues 198 - 344 of SEQ ID NOs: 36 - 44.

[0022] In some such embodiments, the engineered meganuclease is a single - stranded meganuclease that includes a linker that covalently bonds the first subunit and the second subunit. In some embodiments, the engineered meganuclease includes an amino acid sequence having at least 80%, 85%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any one of SEQ ID NOs: 36 - 44. In some embodiments, the engineered meganuclease includes the amino acid sequence of any one of SEQ ID NOs: 36 - 44. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 9 3%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the nucleic acid sequence described in any one of SEQ ID NOs: 60 - 68. In some embodiments, the engineered meganuclease is encoded by the nucleic acid sequence described in any one of SEQ ID NOs: 60 - 68. In some embodiments, the recognition sequence includes SEQ ID NO: 10. In some embodiments, the engineered meganuclease is encoded by the nucleic acid sequence described in any one of SEQ ID NOs: 60 - 68. In some embodiments, the engineered meganuclease

[0023] is encoded by the nucleic acid sequence described in any one of SEQ ID NOs: 60 - 68.

[0024] In some such embodiments, the HVR1 region is any one of SEQ ID NOs: 45-52 and has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with an amino acid sequence corresponding to residues 24-79 of any one of them. In some embodiments, the HVR1 region contains one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 45-52. In some embodiments, the HVR1 region contains residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 45-52. In some embodiments, the HVR1 region contains a residue corresponding to residue 66 of any one of SEQ ID NOs: 45-52 and contains Y, R, K or D. In some embodiments, the HVR1 region has amino acid substitutions of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 in residues 24-79 of any one of SEQ ID NOs: 45-52. In some embodiments, the HVR1 region contains residues 24-79 of any one of SEQ ID NOs: 45-52. In some such embodiments, the first subunit has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with an amino acid sequence corresponding to residues 7-153 of any one of SEQ ID NOs: 45-52. In some embodiments, the first subunit contains residues corresponding to residues 7-153 of any one of SEQ ID NOs: 45-52. contains a residue corresponding to residue 66 of any one of SEQ ID NOs: 45-52 and contains Y, R, K or D. In some embodiments, the HVR1 region has amino acid substitutions of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 in residues 24-79 of any one of SEQ ID NOs: 45-52. In some embodiments, the HVR1 region contains residues 24-79 of any one of SEQ ID NOs: 45-52. In some such embodiments, the first subunit has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with an amino acid sequence corresponding to residues 7-153 of any one of SEQ ID NOs: 45-52. In some embodiments, the first subunit contains residues corresponding to residues 7-153 of any one of SEQ ID NOs: 45-52.

[0025] In some such embodiments, the first subunit has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with an amino acid sequence corresponding to residues 7-153 of any one of SEQ ID NOs: 45-52. In some embodiments, the first subunit contains residues corresponding to residues 7-153 of any one of SEQ ID NOs: 45-52. and has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with an amino acid sequence corresponding to residues 7-153 of any one of SEQ ID NOs: 45-52. In some embodiments, the first subunit contains residues corresponding to residues 7-153 of any one of SEQ ID NOs: 45-52. It contains G, S, or A at a residue corresponding to residue 19 of any one of residues 45 to 52. Some In embodiments, the first subunit contains a residue corresponding to residue 19 of any one of SEQ ID NOs: 45 to 52 In some embodiments, the first subunit contains a residue corresponding to residue 80 of any one of SEQ ID NOs: 45 ~52 and contains E, Q, or K. In some embodiments the first subunit contains a residue corresponding to residue 80 of any one of SEQ ID NOs: 45 to 51 In some embodiments, the first subunit contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or up to 30 amino acid substitutions and contains any one of residues 7 to 153 of SEQ ID NOs: 45 to 52. Some In embodiments, the first subunit contains any one of residues 7 to 153 of SEQ ID NOs: 45 to 52.

[0026] In some such embodiments, the HVR2 region has at least 80%, 85%, 90% amino acid sequence identity with the amino acid sequence corresponding to residues 215 to 270 of any one of SEQ ID NOs: 45 to 52 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher and contains an amino acid sequence having such sequence identity. In some embodiments, HVR2 region contains one or more residues corresponding to residues 215, 217, 219, 221, 2 23, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 45 to 52. In some embodiments, the HVR2 region contains residues 215, 217, 219, 221, 223 , 224, 229, 231, 233, 235, 237, 259, 261, 266, and 2 contains residues corresponding to 68. In some embodiments, the HVR2 region is from SEQ ID NO: 45 to contains Y, R, K, or D in the residue corresponding to residue 257 of any one of 52. In some embodiments, the HVR2 region contains residues corresponding to residues 239, 24 1, and 264 of any one of SEQ ID NOs: 45 to 52. In some embodiments, the HVR2 region contains the residue corresponding to residue 250 of SEQ ID NO: 45. In some embodiments, the HVR2 region contains the residue corresponding to residue 263 of any one of SEQ ID NOs: 45 or 46. In some embodiments, the HVR2 region contains residues 215 - 270 of any one of SEQ ID NOs: 45 - 52 with amino acid substitutions up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 . In some embodiments, the HVR2 region contains residues 215 - 270 of any one of SEQ ID NOs: 45 - 52.

[0027] In some such embodiments, the second subunit has at least 80%, 85%, 90%, 91%, 92% sequence identity with residues 198 - 344 of any one of SEQ ID NOs: 45 - 52. In some embodiments, the second subunit contains G, S, or A in the residue corresponding to residue 210 of any one of SEQ ID NOs: 45 - 52. In some embodiments, the second subunit contains E, Q, or K in the residue corresponding to residue 271 of any one of SEQ ID NOs: 45 - 52. In some embodiments, the second subunit contains the residue corresponding to residue 271 of SEQ ID NO: 52. In some embodiments ​ The second subunit contains a residue corresponding to any one of residues 330 of SEQ ID NOs: 45 to 52. In some embodiments, the second subunit contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 1, 22, 23, 24, 25, 26, 27, 28, 29, or up to 30 amino acid substitutions and contains any one of residues 198 to 344 of SEQ ID NOs: 45 to 52. In some embodiments the second subunit contains any one of residues 198 to 344 of SEQ ID NOs: 45 to 52.

[0028] In some such embodiments, the engineered meganuclease is a single-chain meganuclease containing a linker that covalently links the first subunit and the second subunit. In some embodiments, the engineered meganuclease contains an amino acid sequence having at least 80%, 85%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any one of SEQ ID NOs: 45 to 52. In some embodiments, the engineered meganuclease contains an amino acid sequence of any one of SEQ ID NOs: 45 to 52. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 9 3%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the nucleic acid sequence described in any one of SEQ ID NOs: 69 to 76. In some embodiments, the engineered meganuclease is encoded by the nucleic acid sequence described in any one of SEQ ID NOs: 69 to 76. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 9 3%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the nucleic acid sequence described in any one of SEQ ID NOs: 69 to 76. In some embodiments, the engineered meganuclease is encoded by the nucleic acid sequence described in any one of SEQ ID NOs: 69 to 76. In some embodiments, the engineered meganuclease is encoded by the nucleic acid sequence described in any one of SEQ ID NOs: 69 to 76. is carried out.

[0029] In some embodiments, the recognition sequence includes SEQ ID NO: 12.

[0030] In some such embodiments, the HVR1 region has at least 80%, 85%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with an amino acid sequence corresponding to residues 24 to 79 of any one of SEQ ID NOs: 53 to 59. In some embodiments, the HVR1 region includes one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 3 8, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 53 to 59. In some embodiments, the HVR1 region includes residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 53 to 59. In some embodiments, the HVR1 region includes a residue containing Y, R, K, or D at a residue corresponding to residue 66 of any one of SEQ ID NOs: 53 to 59. In some embodiments, the HVR1 region includes a residue corresponding to residue 64 of SEQ ID NO: 54. In some embodiments, the HVR1 region includes residues 24 to 79 of any one of SEQ ID NOs: 53 to 59 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region includes residues 24 to 79 of any one of SEQ ID NOs: 53 to 59. In some such embodiments, the first subunit is any one of SEQ ID NOs: 53 to 59. In some embodiments, the HVR1 region includes residues 24 to 79 of any one of SEQ ID NOs: 53 to 59 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region includes residues 24 to 79 of any one of SEQ ID NOs: 53 to 59.

[0031] In some such embodiments, the first subunit is any one of SEQ ID NOs: 53 to 59. Any one of residues 7-153 and at least 80%, 85%, 90%, 91%, 92%, 9 3%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity In some embodiments, the first subunit comprises an amino acid sequence having the sequence The residue corresponding to residue 19 in any one of Nos. 53 to 59 contains G, S, or A. In one embodiment, the first subunit comprises residue 19 of any one of SEQ ID NOs: 53-59. In some embodiments, the first subunit comprises a residue corresponding to SEQ ID NO:53. The residue corresponding to residue 80 in any one of the following 59 amino acids contains E, Q, or K. In an embodiment, the first subunit is any one of SEQ ID NOs: 53 to 55, 57, or 58. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 1 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or Residues 7 to 153 of any one of SEQ ID NOs: 53 to 59, having up to 30 amino acid substitutions In some embodiments, the first subunit comprises any one of SEQ ID NOs: 53-59. or one containing residues 7 to 153.

[0032] In some such embodiments, the HVR2 region is any of SEQ ID NOs: 53-59. One amino acid sequence corresponding to residues 215-270 and at least 80%, 85%, or 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more In some embodiments, HVR2 comprises an amino acid sequence having a higher sequence identity than The region is selected from residues 215, 217, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 23, 224, 229, 231, 233, 235, 237, 259, 261, 266, and one or more residues corresponding to 268. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223 of any one of SEQ ID NOs: 53-59 , 224, 229, 231, 233, 235, 237, 259, 261, 266, and 2 68. In some embodiments, the HVR2 region comprises residues corresponding to residue 257 of any one of SEQ ID NOs: 53- 59 and contains Y, R, K, or D. In some embodiments, the HVR2 region comprises residues corresponding to residue 239 of SEQ ID NO: 53 or SEQ ID NO: 55 . In some embodiments, the HVR2 region comprises residues corresponding to residue 241 of any one of SEQ ID NOs: 53-55 . In some embodiments, the HVR2 region comprises residues corresponding to residue 255 of SEQ ID NO: 55 . In some embodiments, the HVR2 region comprises residues corresponding to residue 263 of any one of SEQ ID NOs: 56-59 . In some embodiments, the HVR2 region comprises residues corresponding to residue 264 of any one of SEQ ID NOs: 53-59 that correspond. In some embodiments, the HVR2 region has amino acid substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 and comprises any one of residues 215-270 of SEQ ID NOs: 53-59 . In some embodiments, the HVR2 region comprises any one of residues 215-270 of SEQ ID NOs: 53-59 . In some such embodiments, the second subunit has at least 80%, 85%, 90%, 91%, 92%

[0033] identity with any one of residues 198-344 of SEQ ID NOs: 53-59 and , 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence homology and comprises an amino acid sequence. In some embodiments, the second subunit comprises G, S or A at a residue corresponding to residue 210 of any one of SEQ ID NOs: 53-59. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of any one of SEQ ID NOs: 53-59. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of any one of SEQ ID NOs: 53 or 55-59. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of any one of SEQ ID NOs: 54-59. In some embodiments, the second subunit comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 2 9, or up to 30 amino acid substitutions and comprises residues 198-344 of any one of SEQ ID NOs: 53-59. In some embodiments, the second subunit comprises residues 198-344 of any one of SEQ ID NOs: 53-59. In some such embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker that covalently links the first subunit and the second subunit. In some embodiments, the engineered meganuclease has at least 80%, 85%, 90%, 9

[0034] 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence homology with any one of SEQ ID NOs: 53-59. In some embodiments, the engineered meganuclease has at least 80%, 85%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence homology with any one of SEQ ID NOs: 53-59. 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence homology with any one of SEQ ID NOs: 53-59. 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence homology with any one of SEQ ID NOs: 53-59. It includes an amino acid sequence having high sequence identity. In some embodiments, the engineered meganuclease includes any one of the amino acid sequences of SEQ ID NOs: 53 to 59. In some embodiments, the engineered meganuclease has at least 80%, 85%, 90%, 91%, 92%, 9 3%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the nucleic acid sequence described in any one of SEQ ID NOs: 77 to 83 and is encoded by a nuclear sequence. In some embodiments, the engineered meganuclease is encoded by the nuclear sequence described in any one of SEQ ID NOs: 77 to 83.

[0035] In each of the above embodiments, the engineered meganuclease can include a nuclear localization signal. In some embodiments, the nuclear localization signal is at the N-terminus of the engineered meganuclease. In some embodiments, the nuclear localization signal includes an amino acid sequence having at least 80% or at least 90% sequence identity with SEQ ID NO: 3. In some embodiments, the nuclear localization signal includes SEQ ID NO: 3.

[0036] In another aspect, the present invention provides a polynucleotide comprising a nucleic acid sequence encoding the engineered meganuclease described herein. In some embodiments, the polynucleotide is mRNA.

[0037] In another aspect, the present invention provides a recombinant DNA construct comprising a polynucleotide comprising a nucleic acid sequence encoding the engineered meganuclease described herein.

[0038] In some embodiments, the recombinant DNA construct is a recombinant virus comprising the polynucleotide. In some embodiments, the recombinant virus encodes a recombinant adenovirus. , recombinant lentivirus, recombinant retrovirus, or recombinant AAV. In embodiments, the recombinant virus is a recombinant AAV. The AAV has a rh.74 capsid. In some embodiments, the recombinant AAV is In some embodiments, the rh.74 capsid has a V9 capsid. 182 and at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or more amino acid sequence identity In some embodiments, the rh.74 capsid has the amino acid sequence of SEQ ID NO:182. In some embodiments, the AAV9 capsid comprises at least one of SEQ ID NO: 183 and SEQ ID NO: 193. 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, In some embodiments, the amino acid sequence has 98%, 99% or more identity. In some embodiments, the AAV9 capsid comprises the amino acid sequence of SEQ ID NO: 183. In embodiments, the recombinant AAV has an AAV8 capsid.

[0039] In some embodiments, the nucleic acid sequence is an engineered meganucleotide sequence as described herein. In some embodiments, the nucleic acid sequence encoding the .alpha.-ase is operably linked to a promoter. In some embodiments, the promoter is a muscle-specific promoter. The heterologous promoters are MCK promoter, C5-12 promoter, spc 5-12 promoter, -ta, MHCK7 promoter, CK8 promoter, SK-CRM4 promoter, SP-3 01 promoter, SP-817 promoter, or SP-905 promoter. Some embodiments, the promoter is capable of expressing the engineered meganuclease described herein in muscle progenitor cells (e.g., satellite cells or stem cells).

[0040] In another aspect, the invention provides a recombinant virus comprising a polynucleotide comprising a nucleic acid sequence encoding the engineered meganuclease described herein.

[0041] In some embodiments, the recombinant virus is a recombinant adenovirus, recombinant lentivirus, recombinant retrovirus, or recombinant AAV. In some embodiments, the recombinant virus is recombinant AAV. In some embodiments, the recombinant AAV has an rh.7 4 capsid. In some embodiments, the rh.74 capsid comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 1 82. In some embodiments, the rh.74 capsid comprises the amino acid sequence of SEQ ID NO: 182. In some embodiments, the recombinant AAV has an AAV9 capsid. In some embodiments, the AAV9 capsid comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 183. In some embodiments, the AAV 9 capsid comprises the amino acid sequence of SEQ ID NO: 183. In some embodiments, the recombinant 9 capsid comprises the amino acid sequence of SEQ ID NO: 183. In some embodiments, the recombinant ​​​​​​AAV-e has an AAV8 capsid.

[0042] In some embodiments, the polynucleotide comprises a promoter operably linked to a nucleic acid sequence encoding a gene-edited meganuclease described herein. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the muscle-specific promoter includes an MCK promoter, a C5-12 promoter, an spc 5-1 2 promoter, an MHCK7 promoter, a CK8 promoter, an SK-CRM4 promoter, an SP-301 promoter, an SP-817 promoter, or an SP-905 promoter. In some embodiments, the promoter is capable of expressing the gene-edited meganuclease described herein in muscle progenitor cells (e.g., satellite cells or stem cells).

[0043] In another aspect, the invention provides a lipid nanoparticle composition comprising a lipid nanoparticle comprising a polynucleotide comprising a nucleic acid sequence encoding a gene-edited meganuclease described herein. In some embodiments, the polynucleotide is mRNA.

[0044] In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a gene-edited meganuclease described herein.

[0045] In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polynucleotide described herein.

[0046] In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant DNA construct described herein.

[0047] In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant virus as described herein. and.

[0048] In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a lipid nanoparticle composition as described herein. and.

[0049] In another aspect, the present invention provides a polynucleotide comprising a first nucleic acid sequence encoding a first genetically engineered meganuclease and a second nucleic acid sequence encoding a second genetically engineered meganuclease, wherein the first genetically engineered meganuclease is the genetically engineered meganuclease described herein that binds to and cleaves a recognition sequence comprising SEQ ID NO: 6, and the second genetically engineered meganuclease is the genetically engineered meganuclease described herein that binds to and cleaves a recognition sequence comprising SEQ ID NO: 10, or the second genetically engineered meganuclease is the genetically engineered meganuclease described herein that binds to and cleaves a recognition sequence comprising SEQ ID NO: 12. and. and. and. and. and. and. and.

[0050] In some embodiments, the first genetically engineered meganuclease is the genetically engineered meganuclease described herein that binds to and cleaves a recognition sequence comprising SEQ ID NO: 6, and the second genetically engineered meganuclease is the genetically engineered meganuclease described herein that binds to and cleaves a recognition sequence comprising SEQ ID NO: 10. In some embodiments, the first genetically engineered meganuclease and the second genetically engineered meganuclease are the meganucleases provided in Table 1 (and variants thereof described herein). and. and. and. In some embodiments, the first genetically engineered meganuclease and the second genetically engineered meganuclease are the meganucleases provided in Table 1 (and variants thereof described herein). and. selected from the combination of (Riant).

[0051] [Table 1] TIFF2025098002000003.tif244149

[0052] In some embodiments, the first genetically engineered meganuclease is DMD 19 -20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35-36x.63 (SEQ ID NO: 45) or a variant thereof described herein. In some embodiments, the first genetic ally engineered meganuclease is 19-20x.87 (SEQ ID NO: 37) or as described herein, and the second genetically engineered meganuclease is DMD 35-36x.81 (SEQ ID NO: 46) or a variant thereof described herein . In some embodiments, the first genetically engineered meganuclease is DMD 19 -20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35-36x.81 (SEQ ID NO: 46) , or a variant thereof described herein. In some embodiments, the first genetic ally engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35-36x.63 (SEQ ID NO: 45) or a variant thereof described herein . In some embodiments, the first genetically engineered meganuclease is D MD 19-20L.249 (SEQ ID NO: 38) or a variant thereof described herein and the second genetically engineered meganuclease is DMD 35-36L.195( (SEQ ID NO: 47) or a variant thereof described herein. In some embodiments the first genetically engineered meganuclease is DMD 19-20L.302 (SEQ ID NO: 39) or a variant thereof described herein, and the second genetically engineered me ganuclease is DMD 35-36L.282 (SEQ ID NO: 48), or a variant thereof described herein. In some embodiments, the first genetically engineered me ganuclease is DMD 19-20L.329 (SEQ ID NO: 40) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35- 36L.282 (SEQ ID NO: 48) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20 L.302 (SEQ ID NO: 39) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35-36L.349 (SEQ ID NO: 49) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20L.329 (SEQ ID NO: 40) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35-36L.349 (SEQ ID NO: 49), or a variant thereof described herein.

[0053] In certain embodiments, the first genetically engineered meganuclease comprises SEQ ID NO: 6 A genetically engineered meganuclease described herein that binds to and cleaves a recognition array , the second genetically engineered meganuclease is a genetically engineered meganuclease described herein that binds to and cleaves a recognition array comprising SEQ ID NO: 12 . In some embodiments , the first genetically engineered meganuclease and the second genetically engineered meganuclease are selected from combinations of meganucleases (and variants thereof described herein) provided in Table 2 .

[0054]

Table 2

[0055] In some embodiments, the first genetically engineered meganuclease is DMD 19 -20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x.15 (SEQ ID NO: 53) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein, and the second genetically engineered meganuclease is , DMD 37-38x.15 (SEQ ID NO: 53) or a variant thereof described herein . In some embodiments, the first genetically engineered meganuclease is DM D 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein , and the second genetically engineered meganuclease is DMD 37-38x.66 (SEQ ID NO umber) No. 54) or a variant thereof described herein. In some embodiments, the first engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37 ) or a variant thereof described herein, and the second engineered meganuclease is DMD 37-38x.66 (SEQ ID NO: 54) or a variant thereof described herein. In some embodiments, the first engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second engineered meganuclease is DMD 37-38x.79 (SEQ ID NO: 55) or a variant thereof described herein. In some embodiments , the first engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein, and the second engineered meganuclease is DMD 37-38x.79 (SEQ ID NO: 55) or a variant thereof described herein. In some embodiments , the first engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein, and the second engineered meganuclease is DMD 37-38x.79 (SEQ ID NO: 55) or a variant thereof described herein. In some embodiments , the first engineered meganuclease is DMD 19-20L.249 (SEQ ID NO: 38) or a variant thereof described herein, and the second engineered meganuclease is DMD 37-3 8L.166 (SEQ ID NO: 56), or a variant thereof described herein.

[0056] In some embodiments, the polynucleotide is mRNA. In some embodiments , the first nucleic acid sequence and the second nucleic acid sequence are separated by an IRES or a 2A sequence . In certain embodiments, the 2A sequence is a T2A, P2A, E2A or F2A sequence.

[0057] In another aspect, the present invention provides a recombinant DNA construct comprising the polynucleotides described herein (i.e., a first nucleic acid sequence encoding a first genetically engineered meganuclease and a second nucleic acid sequence encoding a second genetically engineered meganuclease). In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by an IRES or a 2A sequence. In certain embodiments, the 2A sequence is a T2A, P2A, E2A or F2A sequence. In some embodiments, the polynucleotide comprises a promoter operably linked to the first nucleic acid sequence and the second nucleic acid sequence. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the muscle-specific promoter comprises an MCK promoter, a C5-12 promoter, a spc 5-12 promoter, an MHCK7 promoter, a CK8 promoter, a SK-CRM4 promoter, an SP-301 promoter, an SP-817 promoter, or an SP-905 promoter. In some embodiments, the promoter is capable of expressing the first and second genetically engineered meganucleases described herein in muscle progenitor cells (e.g., satellite cells or stem cells).

[0058] In some embodiments, the polynucleotide comprises a first promoter operably linked to the first nucleic acid sequence and a second promoter operably linked to the second nucleic acid sequence. In some embodiments, the first promoter and the second promoter are muscle-specific promoters. In some embodiments, the muscle-specific promoter is an MCK promoter, a C

[0059] In some embodiments, the polynucleotide comprises a promoter operably linked to the first nucleic acid sequence and the second nucleic acid sequence. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the muscle-specific promoter comprises an MCK promoter, a C5-12 promoter, a spc 5-12 promoter, an MHCK7 promoter, a CK8 promoter, a SK-CRM4 promoter, an SP-301 promoter, an SP-817 promoter, or an SP-905 promoter. In some embodiments, the promoter is capable of expressing the first and second genetically engineered meganucleases described herein in muscle progenitor cells (e.g., satellite cells or stem cells). In some embodiments, the polynucleotide comprises a first promoter operably linked to the first nucleic acid sequence and a second promoter operably linked to the second nucleic acid sequence. In some embodiments, the first promoter and the second promoter are muscle-specific promoters. In some embodiments, the muscle-specific promoter is an MCK promoter, a C

[0060] In some embodiments, the polynucleotide comprises a first promoter operably linked to the first nucleic acid sequence and a second promoter operably linked to the second nucleic acid sequence. In some embodiments, the first promoter and the second promoter are muscle-specific promoters. In some embodiments, the muscle-specific promoter is an MCK promoter, a C 5-12 promoter, a spc 5-12 promoter, an MHCK7 promoter, a CK8 promoter, a SK-CRM4 promoter, an SP-301 promoter, an SP-817 promoter, or an SP-905 promoter. In some embodiments, the promoter is capable of expressing the first and second genetically engineered meganucleases described herein in muscle progenitor cells (e.g., satellite cells or stem cells).​​​​​​​​​​​ 5-12 promoter, spc 5-12 promoter, MHCK7 promoter, CK8 promoter , SK-CRM4 promoter, SP-301 promoter, SP-817 promoter , SP-905 promoter, or a combination thereof. In some embodiments, the promoter can express the first and second genetically engineered meganucleases described herein in muscle progenitor cells (e.g., satellite cells or stem cells).

[0061] In some embodiments, the recombinant DNA construct encodes a recombinant virus comprising a polynucleotide. In some embodiments, the recombinant virus is a recombinant adenovirus , recombinant lentivirus, recombinant retrovirus, or recombinant AAV. In some embodiments, the recombinant virus is recombinant AAV. In some embodiments, the recombinant AAV has an rh.74 capsid. In some embodiments, the recombinant AAV has an AAV 9 capsid. In some embodiments, the rh.74 capsid comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 182. In some embodiments, the rh.74 capsid comprises the amino acid sequence of SEQ ID NO: 182. In some embodiments, the AAV9 capsid comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 183. In some embodiments, the AAV9 capsid comprises the amino acid sequence of SEQ ID NO: 183. In some embodiments, In some embodiments, the recombinant AAV has an AAV8 capsid.

[0062] In another aspect, the present invention provides a recombinant virus comprising a polynucleotide described herein (i.e., comprising a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease).

[0063] In some embodiments, the polynucleotide comprises a promoter operably linked to the first nucleic acid sequence and the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by an IRES or a 2A sequence. In certain embodiments, the 2A sequence is a T2A, P2A, E2A or F2A sequence.

[0064] In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the muscle-specific promoter comprises an MCK promoter, a C5-12 promoter, a spc5-12 promoter, an MHCK7 promoter, a CK8 promoter, an SK-CRM4 promoter, an SP-301 promoter, an SP-817 promoter, or an SP-905 promoter. In some embodiments, the promoter can express the engineered meganucleases described herein in muscle precursor cells (e.g., satellite cells or stem cells).

[0065] In some embodiments, the polynucleotide comprises a first promoter operably linked to the first nucleic acid sequence and a second promoter operably linked to the second nucleic acid sequence. In some embodiments, the first promoter and the second promoter are muscle-specific promoters. ​ In some embodiments, the muscle-specific promoter is a MCK promoter, 5-12 promoter, spc 5-12 promoter, MHCK7 promoter, CK8 promoter Motor, SK-CRM4 promoter, SP-301 promoter, SP-817 promoter , SP-905 promoter, or a combination thereof. The promoter is as described herein in muscle precursor cells (e.g., satellite cells or stem cells). The engineered meganucleases described can be expressed. So, recombinant viruses are recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the recombinant AAV has a rh.74 capsid. In some embodiments, the recombinant AAV has an AAV9 capsid. In embodiments, the rh.74 capsid has at least 80%, 85%, 90%, or 100% identity with SEQ ID NO:182. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or In some embodiments, the rh.7 4 capsid comprises the amino acid sequence of SEQ ID NO: 182. The V9 capsid has at least 80%, 85%, 90%, 91%, 92%, or 100% identical sequence to SEQ ID NO:183. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity In some embodiments, the AAV9 capsid comprises an amino acid sequence having the sequence In some embodiments, the recombinant AAV comprises the amino acid sequence of AAV8. Having a capsid.

[0066] In another aspect, the present invention provides a lipid nanoparticle composition comprising a polynucleotide as described herein (i.e., comprising a first nucleic acid sequence encoding a first genetically engineered meganuclease and a second nucleic acid sequence encoding a second genetically engineered meganuclease).

[0067] In some embodiments, the polynucleotide is the mRNA as described herein. In some embodiments, the polynucleotide is the recombinant DNA construct as described herein.

[0068] In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polynucleotide as described herein (i.e., comprising a first nucleic acid sequence encoding a first genetically engineered meganuclease and a second nucleic acid sequence encoding a second genetically engineered meganuclease).

[0069] In some embodiments, the polynucleotide comprises the mRNA as described herein. In some embodiments, the polynucleotide comprises the recombinant DNA construct as described herein. In some embodiments, the pharmaceutical composition comprises the recombinant virus as described herein. In some embodiments, the pharmaceutical composition comprises the lipid nanoparticle composition as described herein.

[0070] In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing a polynucleotide comprising a nucleic acid sequence encoding a genetically engineered meganuclease as described herein into the eukaryotic cell. ​​​​​​​​​​​​comprising introducing into a cell, wherein the engineered meganuclease is expressed in a eukaryotic cell and providing a method for producing a cleavage site in the dystrophin gene at a recognition sequence comprising SEQ ID NO: 6 by the engineered meganuclease. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell . In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, mRNA, or a recombinant virus (e.g., a recombinant AAV).

[0071] In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence in the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell an engineered meganuclease as described herein, wherein the engineered meganuclease produces a cleavage site in the dystrophin gene at a recognition sequence comprising SEQ ID NO: 6. In some embodiments, the eukaryotic cell is a mammalian cell . In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell or a cardiomyocyte . In some embodiments, the mammalian cell is a human cell . In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell or a cardiomyocyte . In some embodiments, the mammalian cell is a human cell

[0072] In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted into the dystrophin gene of the genetically modified eukaryotic cell, the method comprising as described herein ​A first nucleic acid sequence encoding a genetically engineered meganuclease and a second nucleic acid sequence containing a target sequence (wherein the genetically engineered meganuclease is expressed in a eukaryotic cell) are included. A method is provided that includes introducing one or more polynucleotides into a eukaryotic cell, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence containing SEQ ID NO: 6, and the target sequence is inserted into the dystrophin gene at the cleavage site. In some embodiments, the second nucleic acid sequence contains a nucleic acid sequence homologous to the nucleic acid sequence adjacent to the cleavage site, and the target sequence is inserted into the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, one or more polynucleotides are introduced into the eukaryotic cell by lipid nanoparticles, mRNA, or a recombinant virus (e.g., recombinant AAV). In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell containing an exogenous target sequence inserted into the dystrophin gene of the genetically modified eukaryotic cell, the method including introducing into the eukaryotic cell the genetically engineered meganuclease described herein and a polynucleotide containing the target sequence, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence containing SEQ ID NO: 6, and the target sequence is inserted into the dystrophin gene at the cleavage site. In some embodiments, the polynucleotide...

[0073] ​ The polynucleotide comprises a nucleic acid sequence homologous to the nucleic acid sequence adjacent to the cleavage site, and the sequence of interest is , inserted into the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian animal cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, mRNA or a recombinant vi rus (e.g., recombinant AAV).

[0074] In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell a polynucleotide comprising a nucleic acid sequence encoding a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease is expressed in the eukaryotic cell, and the genetically engineered meganuclease produces a cleavage site within the dystroph in gene at a recognition sequence comprising SEQ ID NO: 10. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell) , a skeletal muscle cell or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell . In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, mRNA or a recombinant virus (e.g., recombinant AAV).

[0075] In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease cleaves within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 to produce a cleavage site. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a genetically engineered meganuclease as described herein (wherein the genetically engineered meganuclease is expressed within the eukaryotic cell) and a second nucleic acid sequence comprising the sequence of interest, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 and the sequence of interest is inserted into the dystrophin gene at the cleavage site. In some embodiments, the second nucleic acid sequence comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site and the sequence of interest is inserted at the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease cleaves within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 to produce a cleavage site. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a genetically engineered meganuclease as described herein (wherein the genetically engineered meganuclease is expressed within the eukaryotic cell) and a second nucleic acid sequence comprising the sequence of interest, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 and the sequence of interest is inserted into the dystrophin gene at the cleavage site. In some embodiments, the second nucleic acid sequence comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site and the sequence of interest is inserted at the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease cleaves within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 to produce a cleavage site. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a genetically engineered meganuclease as described herein (wherein the genetically engineered meganuclease is expressed within the eukaryotic cell) and a second nucleic acid sequence comprising the sequence of interest, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 and the sequence of interest is inserted into the dystrophin gene at the cleavage site. In some embodiments, the second nucleic acid sequence comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site and the sequence of interest is inserted at the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease cleaves within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 to produce a cleavage site. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a genetically engineered meganuclease as described herein (wherein the genetically engineered meganuclease is expressed within the eukaryotic cell) and a second nucleic acid sequence comprising the sequence of interest, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 and the sequence of interest is inserted into the dystrophin gene at the cleavage site. In some embodiments, the second nucleic acid sequence comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site and the sequence of interest is inserted at the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments

[0076] In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease cleaves within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 to produce a cleavage site. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a genetically engineered meganuclease as described herein (wherein the genetically engineered meganuclease is expressed within the eukaryotic cell) and a second nucleic acid sequence comprising the sequence of interest, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 and the sequence of interest is inserted into the dystrophin gene at the cleavage site. In some embodiments, the second nucleic acid sequence comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site and the sequence of interest is inserted at the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease cleaves within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 to produce a cleavage site. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a genetically engineered meganuclease as described herein (wherein the genetically engineered meganuclease is expressed within the eukaryotic cell) and a second nucleic acid sequence comprising the sequence of interest, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 and the sequence of interest is inserted into the dystrophin gene at the cleavage site. In some embodiments, the second nucleic acid sequence comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site and the sequence of interest is inserted at the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease cleaves within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 to produce a cleavage site. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a genetically engineered meganuclease as described herein (wherein the genetically engineered meganuclease is expressed within the eukaryotic cell) and a second nucleic acid sequence comprising the sequence of interest, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 and the sequence of interest is inserted into the dystrophin gene at the cleavage site. In some embodiments, the second nucleic acid sequence comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site and the sequence of interest is inserted at the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease cleaves within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 to produce a cleavage site. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a genetically engineered meganuclease as described herein (wherein the genetically engineered meganuclease is expressed within the eukaryotic cell) and a second nucleic acid sequence comprising the sequence of interest, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 and the sequence of interest is inserted into the dystrophin gene at the cleavage site. In some embodiments, the second nucleic acid sequence comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site and the sequence of interest is inserted at the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease cleaves within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 to produce a cleavage site. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a genetically engineered meganuclease as described herein (wherein the genetically engineered meganuclease is expressed within the eukaryotic cell) and a second nucleic acid sequence comprising the sequence of interest, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 10 and the sequence of interest is inserted into the dystrophin gene at the cleavage site. In some embodiments, the second nucleic acid sequence comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site and the sequence of interest is inserted at the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments In this case, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., satellite cell or stem cell), skeletal muscle cell or cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, one or more polynucleotides are introduced into eukaryotic cells by lipid nanoparticles, mRNA or recombinant viruses (e.g., recombinant AAV).

[0077] In another aspect, the present invention is a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted into the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into a eukaryotic cell the engineered meganuclease described herein and a polynucleotide comprising the sequence of interest, wherein the engineered meganuclease produces a cleavage site within the dystrophin gene at the recognition sequence comprising SEQ ID NO: 10, and the sequence of interest is inserted into the dystrophin gene at the cleavage site. In some embodiments, the polynucleotide comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site, and the sequence of interest is inserted into the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., satellite cell or stem cell), skeletal muscle cell or cardiomyocyte. In some embodiments, the mammalian cell is a human cell and in some embodiments, the polynucleotide is introduced into the eukaryotic cell by lipid nanoparticles, mRNA or recombinant virus (e.g., recombinant AAV).

[0078] ​​​​​​​​In another aspect, the invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into a eukaryotic cell a polynucleotide comprising a nucleic acid sequence encoding a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease is expressed within the eukaryotic cell, and wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 12. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., a recombinant AAV). In another aspect, the invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into a eukaryotic cell a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 12. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., a recombinant AAV). In another aspect, the invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into a eukaryotic cell a polynucleotide comprising a nucleic acid sequence encoding a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease is expressed within the eukaryotic cell, and wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 12. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., a recombinant AAV). In another aspect, the invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into a eukaryotic cell a polynucleotide comprising a nucleic acid sequence encoding a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease is expressed within the eukaryotic cell, and wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 12. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., a recombinant AAV). In another aspect, the invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into a eukaryotic cell a polynucleotide comprising a nucleic acid sequence encoding a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease is expressed within the eukaryotic cell, and wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 12. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., a recombinant AAV). In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., a recombinant AAV). In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., a recombinant AAV). In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., a recombinant AAV). In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., a recombinant AAV). In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., a recombinant AAV).

[0079] In another aspect, the invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into a eukaryotic cell a polynucleotide comprising a nucleic acid sequence encoding a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease is expressed within the eukaryotic cell, and wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 12. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., a recombinant AAV). In another aspect, the invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into a eukaryotic cell a polynucleotide comprising a nucleic acid sequence encoding a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease is expressed within the eukaryotic cell, and wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 12. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., a recombinant AAV). In another aspect, the invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into a eukaryotic cell a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 12. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., a recombinant AAV). In another aspect, the invention provides a method for producing a genetically modified eukaryotic cell having a modified target sequence within the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into a eukaryotic cell a genetically engineered meganuclease as described herein, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence comprising SEQ ID NO: 12. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. It is a muscle cell. In some embodiments, the mammalian cell is a human cell.

[0080] In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted into the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a genetically engineered meganuclease as described herein and a second nucleic acid sequence comprising the sequence of interest (wherein the genetically engineered meganuclease is expressed in the eukaryotic cell), wherein the genetically engineered meganuclease produces a cleavage site in the dystrophin gene at a recognition sequence comprising SEQ ID NO: 12, and the sequence of interest is inserted into the dystrophin gene at the cleavage site. In some embodiments, the second nucleic acid sequence comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site, and the sequence of interest is inserted at the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the one or more polynucleotides are introduced into the eukaryotic cell by lipid nanoparticles, mRNA, or a recombinant virus (e.g., recombinant AAV). In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted into the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a genetically engineered meganuclease as described herein and a second nucleic acid sequence comprising the sequence of interest (wherein the genetically engineered meganuclease is expressed in the eukaryotic cell), wherein the genetically engineered meganuclease produces a cleavage site in the dystrophin gene at a recognition sequence comprising SEQ ID NO: 12, and the sequence of interest is inserted into the dystrophin gene at the cleavage site. In some embodiments, the second nucleic acid sequence comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site, and the sequence of interest is inserted at the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the one or more polynucleotides are introduced into the eukaryotic cell by lipid nanoparticles, mRNA, or a recombinant virus (e.g., recombinant AAV).

[0081] In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising an exogenous sequence of interest inserted into the dystrophin gene of the genetically modified eukaryotic cell, the method comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a genetically engineered meganuclease as described herein and a second nucleic acid sequence comprising the sequence of interest (wherein the genetically engineered meganuclease is expressed in the eukaryotic cell), wherein the genetically engineered meganuclease produces a cleavage site in the dystrophin gene at a recognition sequence comprising SEQ ID NO: 12, and the sequence of interest is inserted into the dystrophin gene at the cleavage site. In some embodiments, the second nucleic acid sequence comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site, and the sequence of interest is inserted at the cleavage site by homologous recombination. ​Introducing a genetically engineered meganuclease and a polynucleotide comprising a target sequence into a eukaryotic cell, wherein the genetically engineered meganuclease produces a cleavage site within the dystrophin gene at a recognition sequence, and the target sequence is inserted into the dystrophin gene at the cleavage site. In some embodiments, the polynucleotide comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site, and the target sequence is inserted into the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, mRNA, or a recombinant virus (e.g., a recombinant AAV). wherein the genetically engineered meganuclease comprises SEQ ID NO: 12 and produces a cleavage site within the dystrophin gene at a recognition sequence and the target sequence is inserted into the dystrophin gene at the cleavage site to provide a method. In some embodiments the polynucleotide comprises a nucleic acid sequence homologous to a nucleic acid sequence adjacent to the cleavage site and the target sequence is inserted into the cleavage site by homologous recombination. In some embodiments, the eukaryotic cell is a mammalian cell In some embodiments, the mammalian cell is a muscle cell. In some embodiments the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell or a cardiomyocyte. In some embodiments, the mammalian cell is a human cell . In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle, mRNA or a recombinant virus (e.g., a recombinant AAV).

[0082] In another aspect, the present invention provides a method for producing a genetically modified eukaryotic cell comprising a modified dystrophin gene, the method comprising introducing into a eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a first genetically engineered nuclease and a second nucleic acid sequence encoding a second genetically engineered nuclease wherein the first genetically engineered nuclease binds to and cleaves a recognition sequence in an intron upstream of the 5' end of exon 45 and the second genetically engineered nuclease binds to and cleaves a recognition sequence in an intron downstream of the 3' end of exon 55 and introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a first genetically engineered nuclease and a second nucleic acid sequence encoding a second genetically engineered nuclease wherein the first genetically engineered nuclease binds to and cleaves a recognition sequence in an intron upstream of the 5' end of exon 45 and the second genetically engineered nuclease binds to and cleaves a recognition sequence in an intron downstream of the 3' end of exon 55 and the first genetically engineered nuclease and the second genetically engineered nuclease The nuclease is expressed in eukaryotic cells, and the first genetically engineered nuclease produces a first cleavage site within the dystrophin gene at its recognition sequence, and the second gene engineered nuclease produces a second cleavage site within the dystrophin gene at its recognition sequence, the first cleavage site and the second cleavage site have complementary overhangs, and intervening genomic DNA between the first cleavage site and the second cleavage site is excised from the dystrophin gene, the dystrophin genes are annealed, and a modified dystrophin gene is generated, providing a method.

[0083] In some embodiments, the first genetically engineered nuclease is a genetically engineered meganuclease that binds and cleaves at a recognition sequence comprising SEQ ID NO: 6 as described herein, and the second genetically engineered nuclease is a genetically engineered meganuclease that binds and cleaves at a recognition sequence comprising SEQ ID NO: 10 as described herein. In some embodiments the first genetically engineered meganuclease and the second genetically engineered meganuclease are selected from combinations of meganucleases (and variants thereof described herein) provided in Table 1. In such embodiments, the first cleavage site and the second cleavage site have complementary 3' overhangs. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35-36x.63 (SEQ ID NO: 45) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 35-36x.63 (SEQ ID NO: 45) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is 19 - 20x.87 (SEQ ID NO: 37) or a variant thereof described herein, The second genetically engineered meganuclease is DMD 35 - 36x.81 (SEQ ID NO: 4 6) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19 - 20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35 - 36x.81 (SEQ ID NO: 46), or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is , DMD 19 - 20x.87 (SEQ ID NO: 37) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35 - 36x.63 ( SEQ ID NO: 45) or a variant thereof described herein. In some embodiments the first genetically engineered meganuclease is DMD 19 - 20L.249 (SEQ ID NO: 38) or a variant thereof described herein, and the second genetically engineered me ganuclease is DMD 35 - 36L.195 (SEQ ID NO: 47) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19 - 20L.302 (SEQ ID NO: 39) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35 - 36L.282 (SEQ ID NO: 48), or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19 - 2 0L.329 (array number 40) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35-36L.282 (SEQ ID NO: 48) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20L.302 (SEQ ID NO: 39) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35-36L.349 (SEQ ID NO: 49) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20L.329 (SEQ ID NO: 40) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35-36L.349 ( SEQ ID NO: 49), or a variant thereof described herein.

[0084] In some embodiments, the first genetically engineered nuclease is a genetically engineered meganuclease described herein that binds to and cleaves a recognition sequence containing SEQ ID NO: 6 and the second genetically engineered nuclease is a genetically engineered meganuclease described herein that binds to and cleaves a recognition sequence containing SEQ ID NO: 12 such that in such embodiments, the first cleavage site and the second cleavage site have complementary 3´ overhangs. In some embodiments, the first genetically engineered meganuclease and the second genetically engineered meganuclease are selected from combinations of meganucleases (and variants thereof described herein) provided in Table 2. In some embodiments, the first The genetically engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x.15 (SEQ ID NO: 53) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein and the second genetically engineered meganuclease is DMD 37-38x.15 (SEQ ID NO: 53) or a variant thereof described herein. In some embodiments , the first genetically engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x.66 (SEQ ID NO: 54) or a variant thereof described herein. In some embodiments , the first genetically engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x.66 (SEQ ID NO: 54) or a variant thereof described herein. In some embodiments , the first genetically engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x.79 (SEQ ID NO: 55) or a variant thereof described herein. In some embodiments , the first genetically engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x.79 (SEQ ID NO: 55) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or as described herein and variants thereof, and the second genetically engineered meganuclease is DMD 37- 38x.79 (SEQ ID NO: 55) or variants thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20L .249 (SEQ ID NO: 38) or variants thereof described herein, and the second genetically engineered meganuclease is DMD 37-38L.166 (SEQ ID NO: 56), or variants thereof described herein.

[0085] In some embodiments, the complementary overhangs of the first cleavage site and the second cleavage site (e.g., 3' overhangs) are fully ligated to each other.

[0086] In some embodiments, the dystrophin gene comprises the nucleic acid sequence shown in SEQ ID NO: 32 or 34. In some embodiments, the dystrophin gene comprises the nucleic acid sequence shown in SEQ ID NO: 32. In some embodiments, the dystrophin gene comprises the nucleic acid sequence shown in SEQ ID NO: 3 4.

[0087] In some embodiments, the normal reading frame is restored in the modified dystrophin gene and is restored as compared to the full-length wild-type dystrophin gene.

[0088] In some embodiments, the modified dystrophin gene encodes a modified dystrophin polypeptide that lacks the amino acids encoded by exons 45-55 of the wild-type dystrophin gene. In some embodiments, the modified dystrophin polypeptide comprises the amino acid sequence shown in SEQ ID NO: 5.

[0089] In some embodiments, the method comprises introducing into a eukaryotic cell a first polynucleotide comprising a first nucleic acid sequence encoding a first engineered meganuclease and a second polynucleotide comprising a second nucleic acid sequence encoding a second engineered meganuclease. In some embodiments, the first polynucleotide is a first mRNA. In some embodiments, the second polynucleotide is a second mRNA. In some embodiments, the first mRNA and / or the second mRNA is an mRNA as described herein (i.e., encoding an engineered meganuclease as described herein). In some embodiments, the first polynucleotide is a first recombinant DNA construct. In some embodiments, the second polynucleotide is a second recombinant DNA construct. In some embodiments, the first recombinant DNA construct and / or the second recombinant DNA construct is a recombinant DNA construct as described herein (i.e., comprising a nucleic acid sequence encoding an engineered meganuclease as described herein). In some embodiments, the first polynucleotide and the second polynucleotide are introduced into the eukaryotic cell by one or more lipid nanoparticles. In some embodiments, the first polynucleotide is introduced into the eukaryotic cell by a first lipid nanoparticle. In some embodiments, the second polynucleotide is introduced into the eukaryotic cell by a second lipid nanoparticle. In some embodiments, the first polynucleotide is introduced into the eukaryotic cell by a first recombinant virus. In some embodiments, the second polynucleotide is introduced into the eukaryotic cell by a second recombinant virus. ​​​​​​​​​​​​​​​​​It is introduced into eukaryotic cells by a virus. In some embodiments, the first recombinant virus and / or the second recombinant virus is the recombinant virus described herein (i.e., a polynucleotide containing a nucleic acid sequence encoding a gene-edited meganuclease described herein is included).

[0090] In some embodiments, the method includes introducing into a eukaryotic cell a polynucleotide comprising a first nucleic acid sequence encoding a first gene-edited meganuclease and a second nucleic acid sequence encoding a second gene-edited meganuclease. In some embodiments the polynucleotide is mRNA. In some embodiments, the mRNA is the mRNA described herein (i.e., comprising the first and second nucleic acid sequences encoding the meganucleases described herein respectively ). In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the recombinant DNA construct is the recombinant DNA construct described herein (i.e., comprising the first and second nucleic acid sequences encoding the meganucleases described herein respectively ). In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle. In some embodiments the polynucleotide is introduced into the eukaryotic cell by a recombinant virus. In some embodiments the recombinant virus is the recombinant virus described herein (i.e., a polynucleotide comprising the first and second nucleic acid sequences encoding the meganucleases described herein respectively is included). ). In some embodiments, the recombinant virus is the recombinant virus described herein (i.e., ). In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments ). ).

[0091] In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments The mammalian cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell ( e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments the mammalian cell is a human cell.

[0092] In another aspect, the present invention provides a method for modifying the dystrophin gene in a target cell of a subject, wherein the dystrophin gene is characterized by a mutation that changes the reading frame of the dystrophin gene from wild-type, and delivering to the target cell one or more polynucleotides comprising a first nucleic acid sequence encoding a first engineered nuclease and a second nucleic acid sequence encoding a second engineered nuclease, wherein the first engineered nuclease binds to and cleaves a recognition sequence in an intron upstream of the 5' of exon 45, the second engineered nuclease binds to and cleaves a recognition sequence in an intron downstream of the 3' of exon 55, the first engineered nuclease and the second engineered nuclease are expressed in the target cell, the first engineered nuclease produces a first cleavage site within the dystrophin gene at its recognition sequence, the second engineered nuclease produces a second cleavage site within the dystrophin gene at its recognition sequence, the first cleavage site and the second cleavage site have complementary overhangs, intervening genomic DNA between the first cleavage site and the second cleavage site is excised from the dystrophin gene, the dystrophin gene is annealed, and the normal reading frame of the dystrophin gene is restored as compared to the full-length wild-type dystrophin gene. ​​​​​​​​​​​​

[0093] In some embodiments, the first engineered nuclease is an engineered meganuclease described herein that binds to and cleaves a recognition sequence comprising SEQ ID NO: 6 and the second engineered nuclease is an engineered meganuclease described herein that binds to and cleaves a recognition sequence comprising SEQ ID NO: 10 . In some embodiments , the first engineered meganuclease and the second engineered meganuclease are selected from a combination of meganucleases (and variants thereof described herein) provided in Table 1. In such embodiments, the first cleavage site and the second cleavage site have complementary 3´ overhangs. In some embodiments, the first engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second engineered meganuclease is DMD 35-36x.63 (SEQ ID NO: 45) or a variant thereof described herein. In some embodiments, the first engineered meganuclease is 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein, and the second engineered meganuclease is DMD 35-36x.81 (SEQ ID NO: 4 6) or a variant thereof described herein. In some embodiments, the first engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second engineered meganuclease is DMD 35-36x.81 (SEQ ID NO: 4 6) or a variant thereof described herein. In some embodiments, the first engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second engineered meganuclease is DMD 35-36x.81 (SEQ ID NO: 4 6), or a variant thereof described herein is a reliant. In some embodiments, the first genetically engineered meganuclease is , DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein , and the second genetically engineered meganuclease is DMD 35-36x.63 ( SEQ ID NO: 45) or a variant thereof described herein. In some embodiments , the first genetically engineered meganuclease is DMD 19-20L.249 (SEQ ID NO: 38) or a variant thereof described herein, and the second genetically engineered me ganuclease is DMD 35-36L.195 (SEQ ID NO: 47) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20L.302 (SEQ ID NO: 39) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35- 36L.282 (SEQ ID NO: 48), or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-2 0L.329 (SEQ ID NO: 40) or a variant thereof described herein, and the second genetically engineered me ganuclease is DMD 35-36L.282 (SEQ ID NO: 48) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20L.302 (SEQ ID NO: 39) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 35-36L.349 (SEQ ID NO: 49) or a variant thereof described herein . In some embodiments, the first genetically engineered meganuclease is D , DMD 35-36L.349 (SEQ ID NO: 49) or a variant thereof described herein , and in some embodiments, the first genetically engineered meganuclease is D MD 19-20L.329 (SEQ ID NO: 40) or a variant thereof described herein and the second genetically engineered meganuclease is DMD 35-36L.349( SEQ ID NO: 49), or a variant thereof described herein.

[0094] In some embodiments, the first genetically engineered nuclease is a genetically engineered meganuclease described herein that binds to and cleaves a recognition sequence comprising SEQ ID NO: 6 and the second genetically engineered nuclease is a genetically engineered meganuclease described herein that binds to and cleaves a recognition sequence comprising SEQ ID NO: 12 . In some embodiments , the first genetically engineered meganuclease and the second genetically engineered meganuclease are selected from a combination of meganucleases (and variants thereof described herein ) provided in Table 2. In such embodiments, the first cleavage site and the second cleavage site have complementary 3´ overhangs. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x.15 (SEQ ID NO: 53) or a variant thereof described herein . In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein and the second genetically engineered meganuclease is DMD 37-38x.15 (SEQ ID NO: 53) or a variant thereof described herein. In some embodiments , the first genetically engineered meganuclease and the second genetically engineered meganuclease are selected from a combination of meganucleases (and variants thereof described herein) provided in Table 2. In such embodiments, the first cleavage site and the second cleavage site have complementary 3´ overhangs. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x.15 (SEQ ID NO: 53) or a variant thereof described herein. , The first genetically engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x.66 (SEQ ID NO: 54) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x. 66 (SEQ ID NO: 54) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.13 ( SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x.79 (SEQ ID NO: 55) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.87 ( SEQ ID NO: 37) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x. 66 (SEQ ID NO: 54) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.13 ( SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x.79 (SEQ ID NO: 55) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.87 ( SEQ ID NO: 37) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x. 79 (SEQ ID NO: 55) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20L .249 (SEQ ID NO: 38) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38L.166 (SEQ ID NO: 56), or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37- 38x.79 (SEQ ID NO: 55) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20L .249 (SEQ ID NO: 38) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38L.166 (SEQ ID NO: 56), or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20L .249 (SEQ ID NO: 38) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38L.166 (SEQ ID NO: 56), or a variant thereof described herein. In some embodiments, the complementary overhangs of the first cleavage site and the second cleavage site

[0095] ​ (e.g., 3´ overhangs) are completely ligated to each other.

[0096] In some embodiments, the dystrophin gene comprises the nucleic acid sequence set forth in SEQ ID NO: 32 or 34. In some embodiments, the dystrophin gene comprises the nucleic acid sequence set forth in SEQ ID NO: 32. In some embodiments, the dystrophin gene comprises the nucleic acid sequence set forth in SEQ ID NO: 34.

[0097] In some embodiments, the dystrophin gene encodes a modified dystrophin polypeptide lacking the amino acids encoded by exons 45-55 of the wild-type dystrophin gene. In some embodiments, the modified dystrophin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the subject is converted to a Becker muscular dystrophy phenotype.

[0098] In some embodiments, the method comprises delivering to a target cell a first polynucleotide comprising a first nucleic acid encoding a first engineered meganuclease and a second polynucleotide comprising a second nucleic acid sequence encoding a second engineered meganuclease. In some embodiments, the first polynucleotide is a first mRNA. In some embodiments, the second polynucleotide is a second mRNA. In some embodiments, the first mRNA and / or the second mRNA is as described herein (i.e., encodes an engineered meganuclease as described herein). In some embodiments, the first polynucleotide is a first recombinant DNA construct. In some embodiments, the second polynucleotide is a second recombinant DNA construct . In some embodiments, the first recombinant DNA construct and / or the second recombinant DNA construct is the recombinant DNA construct described herein (i.e., comprising a nucleic acid sequence encoding a genetically engineered meganuclease described herein). In some embodiments , the first polynucleotide and the second polynucleotide are delivered to the target cell by one or more lipid nanoparticles . In some embodiments, the first polynucleotide is delivered to the target cell by a first lipid nanoparticle. In some embodiments, the second polynucleotide is delivered to the target cell by a second lipid nanoparticle. In some embodiments , the first polynucleotide is delivered to the target cell by a first recombinant virus . In some embodiments, the second polynucleotide is delivered to the target cell by a second recombinant virus . In some embodiments, the first recombinant virus and / or the second recombinant virus is the recombinant virus described herein (i.e., comprising a polynucleotide comprising a nucleic acid sequence encoding a genetically engineered meganuclease described herein). In some embodiments, the method comprises delivering to the target cell a polynucleotide comprising a first nucleic acid encoding a first genetically engineered meganuclease and a second nucleic acid sequence encoding a second genetically engineered meganuclease . In some embodiments, the polynucleotide is mRNA. In some embodiments, the mRNA is the mRNA described herein (i.e., encoding the meganucleases described herein respectively ).

[0099] In some embodiments, the method comprises delivering to the target cell a polynucleotide comprising a first nucleic acid encoding a first genetically engineered meganuclease and a second nucleic acid sequence encoding a second genetically engineered meganuclease . In some embodiments, the polynucleotide is mRNA. In some embodiments, the mRNA is the mRNA described herein (i.e., encoding the meganucleases described herein respectively ). In some embodiments, the polynucleotide is delivered to the target cell by one or more lipid nanoparticles . In some embodiments, the mRNA is the mRNA described herein (i.e., encoding the meganucleases described herein respectively ). In some embodiments, the mRNA is the mRNA described herein (i.e., encoding the meganucleases described herein respectively (comprising first and second nucleic acid sequences to be used). In some embodiments, the polynucleotide is , a recombinant DNA construct. In some embodiments, the recombinant DNA construct is as described in this specification (i.e., comprising first and second nucleic acid sequences encoding the meganucleases described in this specification). In some embodiments, the polynucleotide is delivered to the target cells by lipid nanoparticles. In some embodiments, the polynucleotide is delivered to the target cells by a recombinant virus. In some embodiments, the recombinant virus is the recombinant virus described in this specification (i.e., comprising a polynucleotide comprising first and second nucleic acid sequences encoding the meganucleases described in this specification). In some embodiments, the subject is a mammal. In some embodiments, the target cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the subject

[0100] is a human. In another aspect, the present invention is a method for treating DMD in a subject in need of treatment for DMD, wherein DMD is characterized by a mutation in the dystrophin gene that changes the reading frame of the dystrophin gene as compared to the full-length wild-type dystrophin gene, and administering to the subject an effective amount of one or more polynucleotides comprising a first nucleic acid sequence encoding a first engineered nuclease and a second nucleic acid sequence encoding a second engineered nuclease, wherein the first engineered nuclease

[0101] Z binds to and cleaves the recognition sequence in the intron 5' upstream of exon 45 of dystrophin, and the second engineered nuclease binds to and cleaves the recognition sequence in the intron 3' downstream of exon 55 of dystrophin, one or more polynucleotides are delivered to the target cell, the first engineered nuclease and the second engineered nuclease are expressed in the target cell, the first engineered nuclease produces a first cleavage site in the dystrophin gene at its recognition sequence, the second engineered nuclease produces a second cleavage site in the dystrophin gene at its recognition sequence, the first cleavage site and the second cleavage site have complementary overhangs, and the intervening genomic DNA between the first cleavage site and the second cleavage site is excised from the dystrophin gene, the dystrophin gene is annealed, and the normal reading frame of the dystrophin gene is restored as compared to the full-length wild-type dystrophin gene. A method is provided. In some embodiments, the first engineered nuclease is an engineered meganuclease that binds to and cleaves

[0102] the recognition sequence containing SEQ ID NO: 6, and the second engineered nuclease is an engineered meganuclease that binds to and cleaves the recognition sequence containing SEQ ID NO: 10. In some embodiments, the first engineered meganuclease and the second engineered meganuclease are selected from combinations of meganucleases (and variants thereof described herein) provided in Table 1. In such embodiments, the first cleavage site and the second In some embodiments, the first engineered meganuclease and the second engineered meganuclease are selected from combinations of meganucleases (and variants thereof described herein) provided in Table 1. In such The cleavage site has complementary 3´ overhangs. In some embodiments, the first engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second engineered meganuclease is DMD 35-36x.63 (SEQ ID NO: 45) or a variant thereof described herein. In some embodiments, the first engineered meganuclease is 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein, and the second engineered meganuclease is DMD 35-36x.81 (SEQ ID NO: 46) or a variant thereof described herein. In some embodiments, the first engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof described herein, and the second engineered meganuclease is DMD 35-36x.81 (SEQ ID NO: 46), or a variant thereof described herein. In some embodiments, the first engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein, and the second engineered meganuclease is DMD 35-36x.63 ( SEQ ID NO: 45) or a variant thereof described herein. In some embodiments the first engineered meganuclease is DMD 19-20L.249 (SEQ ID NO: 38) or a variant thereof described herein, and the second engineered meganuclease is DMD 35-36L.195 (SEQ ID NO: 47) or a variant thereof described herein. In some embodiments, the first engineered meganuclease The first engineered meganuclease is DMD 19-20L.302 (SEQ ID NO: 39) or a variant thereof described herein, and the second engineered meganuclease is DMD 35- 36L.282 (SEQ ID NO: 48), or a variant thereof described herein. In some embodiments, the first engineered meganuclease is DMD 19-2 0L.329 (SEQ ID NO: 40) or a variant thereof described herein, and the second engineered meganuclease is DMD 35-36L.282 (SEQ ID NO: 48) or a variant thereof described herein. In some embodiments, the first engineered meganuclease is DMD 19-20L.302 (SEQ ID NO: 39) or a variant thereof described herein, and the second engineered meganuclease is DMD 35-36L.349 (SEQ ID NO: 49) or a variant thereof described herein. In some embodiments, the first engineered meganuclease is D MD 19-20L.329 (SEQ ID NO: 40) or a variant thereof described herein, and the second engineered meganuclease is DMD 35-36L.349 ( SEQ ID NO: 49), or a variant thereof described herein. In some embodiments, the first engineered nuclease is an engineered meganuclease described herein that binds to and cleaves a recognition sequence comprising SEQ ID NO: 6, and the second engineered nuclease is an engineered meganuclease described herein that binds to and cleaves a recognition sequence comprising SEQ ID NO: 12. In some embodiments the first engineered meganuclease is DMD 19-20L.329 (SEQ ID NO: 40) or a variant thereof described herein, and the second engineered meganuclease is DMD 35-36L.349 ( SEQ ID NO: 49), or a variant thereof described herein.

[0103] In some embodiments, the first engineered nuclease is an engineered meganuclease described herein that binds to and cleaves a recognition sequence comprising SEQ ID NO: 6, and the second engineered nuclease is an engineered meganuclease described herein that binds to and cleaves a recognition sequence comprising SEQ ID NO: 12. In some embodiments the first engineered nuclease is an engineered meganuclease described herein that binds to and cleaves a recognition sequence ​A first engineered meganuclease and a second engineered meganuclease The meganucleases are those provided in Table 2 (and variants thereof described herein). In such an embodiment, the first cleavage site and the second cleavage site are selected from the combination of The cleavage site of the first has a complementary 3' overhang. The engineered meganuclease is DMD 19-20x.13 (SEQ ID NO: 36) or is a variant thereof described herein, and the second engineered meganuclear The enzyme may be DMD 37-38x.15 (SEQ ID NO:53) or a variant thereof as described herein. In some embodiments, the first engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein The second engineered meganuclease is DMD 37-38x.15 (sequence Sequence no. 53) or a variant thereof described herein. The first engineered meganuclease was identified as DMD 19-20x.13 (SEQ ID NO: 36) or a variant thereof as described herein, The cleavage enzyme may be DMD 37-38x.66 (SEQ ID NO:54) or any of the enzymes described herein. In some embodiments, the first engineered meganuclear The enzyme may be DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein. riant, and the second engineered meganuclease is DMD 37-38x. 66 (SEQ ID NO:54) or a variant thereof as described herein. In terms of morphology, the first engineered meganuclease was DMD 19-20x.13 ( SEQ ID NO: 36) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38x.79 (SEQ ID NO: 55) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37- 38x.79 (SEQ ID NO: 55) or a variant thereof described herein. In some embodiments, the first genetically engineered meganuclease is DMD 19-20L .249 (SEQ ID NO: 38) or a variant thereof described herein, and the second genetically engineered meganuclease is DMD 37-38L.166 (SEQ ID NO: 56), or a variant thereof described herein.

[0104] In some embodiments, the complementary overhangs (e.g., 3´ overhangs) of the first cleavage site and the second cleavage site are completely ligated to each other.

[0105] In some embodiments, the dystrophin gene comprises the nucleic acid sequence shown in SEQ ID NO: 32 or 34. In some embodiments, the dystrophin gene comprises the nucleic acid sequence shown in SEQ ID NO: 32. In some embodiments, the dystrophin gene comprises the nucleic acid sequence shown in SEQ ID NO: 3

[0106] 4. In some embodiments, the dystrophin gene is a modified dystrophin polypeptide lacking the amino acids encoded by exons 45-55 of the wild-type dystrophin gene. In some embodiments, the modified dystrophin polypeptide encodes SEQ ID NO: 5. In some embodiments, the subject has Becker muscular dystrophy. converted to the lophi phenotype.

[0107] In some embodiments, the method comprises encoding a first engineered meganuclease. A first polynucleotide comprising a first nucleic acid encoding a second genetically engineered meganucleotide administering to the subject a second polynucleotide comprising a second nucleic acid sequence encoding an isoform of the . In some embodiments, the first polynucleotide is a first mRNA. In some embodiments, the second polynucleotide is a second mRNA. In embodiments, the first mRNA and / or the second mRNA is an mRNA described herein. (i.e., encoding the engineered meganucleases described herein). In some embodiments, the first polynucleotide is a first recombinant DNA construct. In some embodiments, the second polynucleotide is a second recombinant DNA construct. In some embodiments, the first recombinant DNA construct and / or the second recombinant DNA The construct is a recombinant DNA construct as described herein (i.e., In some embodiments, the nucleic acid sequence encoding the engineered meganuclease is The first polynucleotide and the second polynucleotide are targeted by lipid nanoparticles. In some embodiments, the first polynucleotide is administered to a first lipid nanoparticle. In some embodiments, the second polynucleotide is administered to the subject by a first In some embodiments, the first polynucleotide is administered to the subject via a lipid nanoparticle. The nucleotide is administered to a subject by a first recombinant virus. In some embodiments , the second polynucleotide is administered to a subject by a second recombinant virus. In some embodiments, the first recombinant virus and / or the second recombinant virus is the recombinant virus described herein (i.e., comprising a polynucleotide comprising a nucleic acid sequence encoding a genetically engineered meganuclease described herein).

[0108] In some embodiments, the method comprises administering to a subject a polynucleotide comprising a first nucleic acid encoding a first genetically engineered meganuclease and a second nucleic acid sequence encoding a second genetically engineered meganuclease. In some embodiments, the polynucleotide is mRNA. In some embodiments, the mRNA is the mRNA described herein (i.e., comprising the first and second nucleic acid sequences encoding the meganucleases described herein). In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the recombinant DNA construct is the recombinant DNA construct described herein (i.e., comprising the first and second nucleic acid sequences encoding the meganucleases described herein). In some embodiments, the polynucleotide is administered to a subject by lipid nanoparticles. In some embodiments, the polynucleotide is administered to a subject by a recombinant virus. In some embodiments, the recombinant virus is the recombinant virus described herein (i.e., comprising a polynucleotide comprising the first and second nucleic acid sequences encoding the meganucleases described herein).

[0109] In some embodiments, the subject is a mammal. In some embodiments, the target cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g., a satellite cell or a stem cell), a skeletal muscle cell, or a cardiomyocyte. In some embodiments, the subject is human.

[0110] In another aspect, the present invention provides a polynucleotide comprising the nucleic acid sequence shown in SEQ ID NO: 32 or SEQ ID NO: 34.

[0111] In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 32. In some embodiments, the polynucleotide is the dystrophin gene in the genome of a cell (e.g., a human muscle cell) comprising the nucleic acid sequence shown in SEQ ID NO: 32. In some embodiments, the polynucleotide is a precursor mRNA in a cell (e.g., a human muscle cell) comprising the nucleic acid sequence shown in SEQ ID NO: 32.

[0112] In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 34. In some embodiments, the polynucleotide is the dystrophin gene in the genome of a cell (e.g., a human muscle cell) comprising the nucleic acid sequence shown in SEQ ID NO: 34. In some embodiments, the polynucleotide is a precursor mRNA in a cell (e.g., a human muscle cell) comprising the nucleic acid sequence shown in SEQ ID NO: 34.

[0113] In another aspect, the present invention provides a genetically modified eukaryotic cell comprising a modified dystrophin gene in its genome, wherein the modified dystrophin gene lacks exons 45-55, and the modification The dystrophin gene variant is located within the intron between exon 44 and exon 56 and includes the nucleic acid sequence shown in SEQ ID NO: 32 or the nucleic acid sequence shown in SEQ ID NO: 34.

[0114] In some embodiments, the nucleic acid sequence includes SEQ ID NO: 32. In some embodiments the nucleic acid sequence includes SEQ ID NO: 34.

[0115] In some embodiments, the genetically modified eukaryotic cell is a mammalian cell. In some embodiments the genetically modified eukaryotic cell is a human cell. In some embodiments, the genetically modified eukaryotic cell is a muscle cell. In some embodiments, the muscle cell is a muscle progenitor cell (e.g. satellite cells or stem cells), a skeletal muscle cell or a cardiomyocyte.

[0116] In another aspect, the present invention provides a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 5, wherein the polypeptide is a modified dystrophin protein lacking the amino acids encoded by exons 45-55 of the dystrophin gene, and the polypeptide includes the C-terminal domain of the dystrophin protein. In some embodiments, the polypeptide includes the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the polypeptide includes the amino acid sequence shown in SEQ ID NO: 5.

[0117] In another aspect, the present invention provides a genetically engineered meganuclease described herein for use as a medicament, or a polynucleotide described herein encoding a genetically engineered meganuclease, or a cell described herein expressing a genetically engineered meganuclease. In another aspect, the present invention provides a polynucleotide described herein encoding a genetically engineered meganuclease, or a cell described herein expressing a genetically engineered meganuclease. In another aspect, the present invention provides a cell described herein expressing a genetically engineered meganuclease. In another aspect, the present invention provides a cell described herein expressing a genetically engineered meganuclease.

[0118] In some embodiments, the medicament is useful for producing a modified dystrophin gene in a subject. In some embodiments, the medicament is useful for the treatment of DMD.

[0119] In another aspect, the present invention provides for the use of a genetically engineered meganuclease described herein, or a polynucleotide disclosed herein encoding a genetically engineered meganuclease, or a cell described herein expressing a genetically engineered meganuclease, in the manufacture of a medicament for treating DMD, increasing the level of a modified dystrophin protein (i.e., lacking the amino acids encoded by exons 45-55 of the dystrophin gene), or reducing the symptoms associated with DMD. i.e., lacking the amino acids encoded by exons 45-55 of the dystrophin gene), or reducing the symptoms associated with DMD. In another aspect, the present invention provides for the use of a genetically engineered meganuclease described herein, or a polynucleotide disclosed herein encoding a genetically engineered meganuclease, or a cell described herein expressing a genetically engineered meganuclease, in the manufacture of a medicament for treating DMD, increasing the level of a modified dystrophin protein (i.e., lacking the amino acids encoded by exons 45-55 of the dystrophin gene), or reducing the symptoms associated with DMD. In another aspect, the present invention provides for the use of a genetically engineered meganuclease described herein, or a polynucleotide disclosed herein encoding a genetically engineered meganuclease, or a cell described herein expressing a genetically engineered meganuclease, in the manufacture of a medicament for treating DMD, increasing the level of a modified dystrophin protein (i.e., lacking the amino acids encoded by exons 45-55 of the dystrophin gene), or reducing the symptoms associated with DMD. In another aspect, the present invention provides for the use of a genetically engineered meganuclease described herein, or a polynucleotide disclosed herein encoding a genetically engineered meganuclease, or a cell described herein expressing a genetically engineered meganuclease, in the manufacture of a medicament for treating DMD, increasing the level of a modified dystrophin protein (i.e., lacking the amino acids encoded by exons 45-55 of the dystrophin gene), or reducing the symptoms associated with DMD. In another aspect, the present invention provides for the use of a genetically engineered meganuclease described herein, or a polynucleotide disclosed herein encoding a genetically engineered meganuclease, or a cell described herein expressing a genetically engineered meganuclease, in the manufacture of a medicament for treating DMD, increasing the level of a modified dystrophin protein (i.e., lacking the amino acids encoded by exons 45-55 of the dystrophin gene), or reducing the symptoms associated with DMD. BRIEF DESCRIPTION OF THE DRAWINGS

[0120]

Figure 1

Figure 2

Figure 3

Figure 4A

Figure 4B

Figure 4C

Figure 5

Figure 6A-F

Figure 7A-D

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12A-C

Figure 13

Figure 14

Figure 15

Figure 16A-C

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23A

Figure 23B

Figure 24A

Figure 24B

Figure 25A-E

Figure 26A-E

Figure 27A-E

Figure 28A-C

Figure 29

Figure 30A-C

Figure 31

Figure 32

Figure 33

Figure 34

Figure 35

Figure 36

Figure 37

Figure 38A-D

[0121] Brief Description of the Sequences

[0122] SEQ ID NO: 1 shows the amino acid sequence of the wild-type I-CreI meganuclease from Chlamydomonas re inhardtii. .

[0123] SEQ ID NO: 2 shows the amino acid sequence of the LAGLIDADG motif.

[0124] SEQ ID NO: 3 shows the amino acid sequence of the nuclear localization signal.

[0125] SEQ ID NO: 4 shows the amino acid sequence of the wild-type dystrophin protein CCDS48091.1 (Gene ID 1756).

[0126] SEQ ID NO: 5 shows the amino acid sequence of the wild-type dystrophin protein CCDS48091.1 (Gene ID 1756) lacking the amino acids encoded by exons 45-55.

[0127] SEQ ID NO: 6 shows the nucleic acid sequence of the sense strand of the DMD 19-20 recognition sequence.

[0128] SEQ ID NO: 7 shows the nucleic acid sequence of the antisense strand of the DMD 19-20 recognition sequence.

[0129] SEQ ID NO: 8 shows the nucleic acid sequence of the sense strand of the DMD 29-30 recognition sequence.

[0130] SEQ ID NO: 9 shows the nucleic acid sequence of the antisense strand of the DMD 29-30 recognition sequence.

[0131] SEQ ID NO: 10 shows the nucleic acid sequence of the sense strand of the DMD 35-36 recognition sequence.

[0132] SEQ ID NO: 11 shows the nucleic acid sequence of the antisense strand of the DMD 35-36 recognition sequence.

[0133] SEQ ID NO: 12 shows the nucleic acid sequence of the sense strand of the DMD 37-38 recognition sequence.

[0134] SEQ ID NO: 13 shows the nucleic acid sequence of the antisense strand of the DMD 37-38 recognition sequence.

[0135] SEQ ID NO: 14 shows the nucleic acid sequence of the sense strand of the first half-site of the DMD 19-20 recognition sequence.

[0136] Array number 15 shows the nucleic acid sequence of the first half-site antisense strand of the DMD 19-20 recognition sequence. It shows the nucleic acid sequence of the first half-site antisense strand of the DMD 19-20 recognition sequence.

[0137] Array number 16 shows the nucleic acid sequence of the first half-site sense strand of the DMD 29-30 recognition sequence. It shows the nucleic acid sequence of the first half-site sense strand of the DMD 29-30 recognition sequence.

[0138] Array number 17 shows the nucleic acid sequence of the first half-site antisense strand of the DMD 29-30 recognition sequence. It shows the nucleic acid sequence of the first half-site antisense strand of the DMD 29-30 recognition sequence.

[0139] Array number 18 shows the nucleic acid sequence of the first half-site sense strand of the DMD 35-36 recognition sequence. It shows the nucleic acid sequence of the first half-site sense strand of the DMD 35-36 recognition sequence.

[0140] Array number 19 shows the nucleic acid sequence of the first half-site antisense strand of the DMD 35-36 recognition sequence. It shows the nucleic acid sequence of the first half-site antisense strand of the DMD 35-36 recognition sequence.

[0141] Array number 20 shows the nucleic acid sequence of the first half-site sense strand of the DMD 37-38 recognition sequence. It shows the nucleic acid sequence of the first half-site sense strand of the DMD 37-38 recognition sequence.

[0142] Array number 21 shows the nucleic acid sequence of the first half-site antisense strand of the DMD 37-38 recognition sequence. It shows the nucleic acid sequence of the first half-site antisense strand of the DMD 37-38 recognition sequence.

[0143] Array number 22 shows the nucleic acid sequence of the second half-site sense strand of the DMD 19-20 recognition sequence. It shows the nucleic acid sequence of the second half-site sense strand of the DMD 19-20 recognition sequence.

[0144] Array number 23 shows the nucleic acid sequence of the second half-site antisense strand of the DMD 19-20 recognition sequence. It shows the nucleic acid sequence of the second half-site antisense strand of the DMD 19-20 recognition sequence.

[0145] Array number 24 shows the nucleic acid sequence of the second half-site sense strand of the DMD 29-30 recognition sequence. It shows the nucleic acid sequence of the second half-site sense strand of the DMD 29-30 recognition sequence.

[0146] Sequence number 25 shows the nucleic acid sequence of the second half-site antisense strand of the DMD 29-30 recognition sequence. It shows.

[0147] Sequence number 26 shows the nucleic acid sequence of the second half-site sense strand of the DMD 35-36 recognition sequence. It shows.

[0148] Sequence number 27 shows the nucleic acid sequence of the second half-site antisense strand of the DMD 35-36 recognition sequence. It shows.

[0149] Sequence number 28 shows the nucleic acid sequence of the second half-site sense strand of the DMD 37-38 recognition sequence. It shows.

[0150] Sequence number 29 shows the nucleic acid sequence of the second half-site antisense strand of the DMD 37-38 recognition sequence. It shows.

[0151] Sequence number 30 shows the nucleic acid sequence of the ligated hybrid DMD 19-20 / 29-30 sense strand. It shows.

[0152] Sequence number 31 shows the nucleic acid sequence of the ligated hybrid DMD 19-20 / 29-30 sense strand. It shows.

[0153] Sequence number 32 shows the nucleic acid sequence of the ligated hybrid DMD 19-20 / 35-36 sense strand. It shows.

[0154] Sequence number 33 shows the nucleic acid sequence of the ligated hybrid DMD 19-20 / 35-36 sense strand. It shows.

[0155] Sequence number 34 shows the nucleic acid sequence of the ligated hybrid DMD 19-20 / 37-38 sense strand. It shows.

[0156] Sequence number 35 represents the ligated hybrid DMD 19-20 / 37-38 and shows the nucleic acid sequence of the sense strand.

[0157] Sequence number 36 represents the amino acid sequence of the DMD 19-20x.13 engineered meganuclease.

[0158] Sequence number 37 represents the amino acid sequence of the DMD 19-20x.87 engineered meganuclease.

[0159] Sequence number 38 represents the amino acid sequence of the DMD 19-20L.249 engineered meganuclease.

[0160] Sequence number 39 represents the amino acid sequence of the DMD 19-20L.302 engineered meganuclease.

[0161] Sequence number 40 represents the amino acid sequence of the DMD 19-20L.329 engineered meganuclease.

[0162] Sequence number 41 represents the amino acid sequence of the DMD 19-20L.374 engineered meganuclease.

[0163] Sequence number 42 represents the amino acid sequence of the DMD 19-20L.375 engineered meganuclease.

[0164] Sequence number 43 represents the amino acid sequence of the DMD 19-20L.431 engineered meganuclease.

[0165] Sequence number 44 represents the amino acid sequence of the DMD 19-20L.458 engineered meganuclease.

[0166] SEQ ID NO: 45 shows the amino acid sequence of the engineered meganuclease DMD 35-36x.63. The sequence is shown.

[0167] SEQ ID NO: 46 shows the amino acid sequence of the engineered meganuclease DMD 35-36x.81. The sequence is shown.

[0168] SEQ ID NO: 47 shows the amino acid sequence of the engineered meganuclease DMD 35-36L.195. The sequence is shown.

[0169] SEQ ID NO: 48 shows the amino acid sequence of the engineered meganuclease DMD 35-36L.282. The sequence is shown.

[0170] SEQ ID NO: 49 shows the amino acid sequence of the engineered meganuclease DMD 35-36L.349. The sequence is shown.

[0171] SEQ ID NO: 50 shows the amino acid sequence of the engineered meganuclease DMD 35-36L.376. The sequence is shown.

[0172] SEQ ID NO: 51 shows the amino acid sequence of the engineered meganuclease DMD 35-36L.457. The sequence is shown.

[0173] SEQ ID NO: 52 shows the amino acid sequence of the engineered meganuclease DMD 35-36L.469. The sequence is shown.

[0174] SEQ ID NO: 53 shows the amino acid sequence of the engineered meganuclease DMD 37-38x.15. The sequence is shown.

[0175] SEQ ID NO: 54 shows the amino acid sequence of the engineered meganuclease DMD 37-38x.66. The sequence is shown.

[0176] SEQ ID NO: 55 shows the amino acid sequence of the DMD 37-38x.79 engineered meganuclease. It shows the sequence.

[0177] SEQ ID NO: 56 shows the amino acid sequence of the DMD 37-38L.166 engineered meganuclease. It shows the sequence.

[0178] SEQ ID NO: 57 shows the amino acid sequence of the DMD 37-38L.478 engineered meganuclease. It shows the sequence.

[0179] SEQ ID NO: 58 shows the amino acid sequence of the DMD 37-38L.512 engineered meganuclease. It shows the sequence.

[0180] SEQ ID NO: 59 shows the amino acid sequence of the DMD 37-38L.528 engineered meganuclease. It shows the sequence.

[0181] SEQ ID NO: 60 shows the nucleic acid sequence encoding the DMD 19-20x.13 engineered meganuclease. It shows the sequence.

[0182] SEQ ID NO: 61 shows the nucleic acid sequence encoding the DMD 19-20x.87 engineered meganuclease. It shows the sequence.

[0183] SEQ ID NO: 62 shows the nucleic acid sequence encoding the DMD 19-20L.249 engineered meganuclease. It shows the sequence.

[0184] SEQ ID NO: 64 shows the nucleic acid sequence encoding the DMD 19-20L.302 engineered meganuclease. It shows the sequence.

[0185] SEQ ID NO: 64 shows the nucleic acid sequence encoding the DMD 19-20L.329 engineered meganuclease. It shows the sequence.

[0186] SEQ ID NO: 65 shows the nucleic acid sequence of the DMD 19-20L.374 engineered meganuclease. It shows the sequence.

[0187] SEQ ID NO: 66 shows the nucleic acid sequence of the DMD 19-20L.375 engineered meganuclease. It shows the sequence.

[0188] SEQ ID NO: 67 shows the nucleic acid sequence of the DMD 19-20L.431 engineered meganuclease. It shows the sequence.

[0189] SEQ ID NO: 68 shows the nucleic acid sequence of the DMD 19-20L.458 engineered meganuclease. It shows the sequence.

[0190] SEQ ID NO: 69 shows the nucleic acid sequence encoding the DMD 35-36x.63 engineered meganuclease. It shows the sequence.

[0191] SEQ ID NO: 70 shows the nucleic acid sequence encoding the DMD 35-36x.81 engineered meganuclease. It shows the sequence.

[0192] SEQ ID NO: 71 shows the nucleic acid sequence encoding the DMD 35-36L.195 engineered meganuclease. It shows the sequence.

[0193] SEQ ID NO: 72 shows the nucleic acid sequence encoding the DMD 35-36L.282 engineered meganuclease. It shows the sequence.

[0194] SEQ ID NO: 73 shows the nucleic acid sequence encoding the DMD 35-36L.349 engineered meganuclease. It shows the sequence.

[0195] SEQ ID NO: 74 shows the nucleic acid sequence encoding the DMD 35-36L.376 engineered meganuclease. It shows the sequence.

[0196] SEQ ID NO: 75 shows the nucleic acid sequence encoding the DMD 35-36L.457 engineered meganuclease.

[0197] SEQ ID NO: 76 shows the nucleic acid sequence encoding the DMD 35-36L.469 engineered meganuclease.

[0198] SEQ ID NO: 77 shows the nucleic acid sequence encoding the DMD 37-38x.15 engineered meganuclease.

[0199] SEQ ID NO: 78 shows the nucleic acid sequence encoding the DMD 37-38x.66 engineered meganuclease.

[0200] SEQ ID NO: 79 shows the nucleic acid sequence encoding the DMD 37-38x.79 engineered meganuclease.

[0201] SEQ ID NO: 80 shows the nucleic acid sequence encoding the DMD 37-38L.166 engineered meganuclease.

[0202] SEQ ID NO: 81 shows the nucleic acid sequence encoding the DMD 37-38L.478 engineered meganuclease.

[0203] SEQ ID NO: 82 shows the nucleic acid sequence encoding the DMD 37-38L.512 engineered meganuclease.

[0204] SEQ ID NO: 83 shows the nucleic acid sequence encoding the DMD 37-38L.528 engineered meganuclease.

[0205] SEQ ID NO: 84 shows the amino acid sequence of the DMD 19-20x.13 engineered meganuclease DMD19 binding subunit.

[0206] ​​​​​​​​​​ SEQ ID NO: 85 shows the amino acid sequence of the binding subunit of the DMD 19-20x.87 engineered meganuclease DMD19

[0207] SEQ ID NO: 86 shows the amino acid sequence of the binding subunit of the DMD 19-20L.249 engineered meganuclease DMD1 9

[0208] SEQ ID NO: 87 shows the amino acid sequence of the binding subunit of the DMD 19-20L.302 engineered meganuclease DMD1 9

[0209] SEQ ID NO: 88 shows the amino acid sequence of the binding subunit of the DMD 19-20L.329 engineered meganuclease DMD1 9

[0210] SEQ ID NO: 89 shows the amino acid sequence of the binding subunit of the DMD 19-20L.374 engineered meganuclease DMD1 9

[0211] SEQ ID NO: 90 shows the amino acid sequence of the binding subunit of the DMD 19-20L.375 engineered meganuclease DMD1 9

[0212] SEQ ID NO: 91 shows the amino acid sequence of the binding subunit of the DMD 19-20L.431 engineered meganuclease DMD1 9

[0213] SEQ ID NO: 92 shows the amino acid sequence of the binding subunit of the DMD 19-20L.458 engineered meganuclease DMD1 9

[0214] SEQ ID NO: 93 shows the amino acid sequence of the binding subunit of the DMD 35-36x.63 engineered meganuclease DMD35

[0215] ​​ SEQ ID NO: 94 shows the amino acid sequence of the binding subunit of the genetically engineered meganuclease DMD35 of DMD 35-36x.81

[0216] SEQ ID NO: 95 shows the amino acid sequence of the binding subunit of the genetically engineered meganuclease DMD35 of DMD 35-36L.195

[0217] SEQ ID NO: 96 shows the amino acid sequence of the binding subunit of the genetically engineered meganuclease DMD35 of DMD 35-36L.282

[0218] SEQ ID NO: 97 shows the amino acid sequence of the binding subunit of the genetically engineered meganuclease DMD35 of DMD 35-36L.349

[0219] SEQ ID NO: 98 shows the amino acid sequence of the binding subunit of the genetically engineered meganuclease DMD35 of DMD 35-36L.376

[0220] SEQ ID NO: 99 shows the amino acid sequence of the binding subunit of the genetically engineered meganuclease DMD35 of DMD 35-36L.457

[0221] SEQ ID NO: 100 shows the amino acid sequence of the binding subunit of the genetically engineered meganuclease DMD35 of DMD 35-36L.469

[0222] SEQ ID NO: 101 shows the amino acid sequence of the binding subunit of the genetically engineered meganuclease DMD37 of DMD 37-38x.15

[0223] SEQ ID NO: 102 shows the amino acid sequence of the binding subunit of the genetically engineered meganuclease DMD37 of DMD 37-38x.66

[0224] ​​​​​​​​​ SEQ ID NO: 103 shows the amino acid sequence of the 7-binding subunit of the DMD 37-38x.79 engineered meganuclease DMD3.

[0225] SEQ ID NO: 104 shows the amino acid sequence of the 37-binding subunit of the DMD 37-38L.166 engineered meganuclease DMD.

[0226] SEQ ID NO: 105 shows the amino acid sequence of the 37-binding subunit of the DMD 37-38L.478 engineered meganuclease DMD.

[0227] SEQ ID NO: 106 shows the amino acid sequence of the 37-binding subunit of the DMD 37-38L.512 engineered meganuclease DMD.

[0228] SEQ ID NO: 107 shows the amino acid sequence of the 37-binding subunit of the DMD 37-38L.528 engineered meganuclease DMD.

[0229] SEQ ID NO: 108 shows the amino acid sequence of the 0-binding subunit of the DMD 19-20x.13 engineered meganuclease DMD2.

[0230] SEQ ID NO: 109 shows the amino acid sequence of the 0-binding subunit of the DMD 19-20x.87 engineered meganuclease DMD2.

[0231] SEQ ID NO: 110 shows the amino acid sequence of the 20-binding subunit of the DMD 19-20L.249 engineered meganuclease DMD.

[0232] SEQ ID NO: 111 shows the amino acid sequence of the 20-binding subunit of the DMD 19-20L.302 engineered meganuclease DMD.

[0233] ​​​​​​​​​ SEQ ID NO: 112 shows the amino acid sequence of the 20-binding subunit of the genetically engineered meganuclease DMD 19-20L.329 of DMD.

[0234] SEQ ID NO: 113 shows the amino acid sequence of the 20-binding subunit of the genetically engineered meganuclease DMD 19-20L.374 of DMD.

[0235] SEQ ID NO: 114 shows the amino acid sequence of the 20-binding subunit of the genetically engineered meganuclease DMD 19-20L.375 of DMD.

[0236] SEQ ID NO: 115 shows the amino acid sequence of the 20-binding subunit of the genetically engineered meganuclease DMD 19-20L.431 of DMD.

[0237] SEQ ID NO: 116 shows the amino acid sequence of the 20-binding subunit of the genetically engineered meganuclease DMD 19-20L.458 of DMD.

[0238] SEQ ID NO: 117 shows the amino acid sequence of the 6-binding subunit of the genetically engineered meganuclease DMD3 35-36x.63 of DMD3.

[0239] SEQ ID NO: 118 shows the amino acid sequence of the 6-binding subunit of the genetically engineered meganuclease DMD3 35-36x.81 of DMD3.

[0240] SEQ ID NO: 119 shows the amino acid sequence of the 36-binding subunit of the genetically engineered meganuclease DMD 35-36L.195 of DMD.

[0241] SEQ ID NO: 120 shows the amino acid sequence of the 36-binding subunit of the genetically engineered meganuclease DMD 35-36L.282 of DMD.

[0242] SEQ ID NO: 121 shows the amino acid sequence of the DMD 35-36L.349 engineered meganuclease DMD 36 binding subunit.

[0243] SEQ ID NO: 122 shows the amino acid sequence of the DMD 35-36L.376 engineered meganuclease DMD 36 binding subunit.

[0244] SEQ ID NO: 123 shows the amino acid sequence of the DMD 35-36L.457 engineered meganuclease DMD 36 binding subunit.

[0245] SEQ ID NO: 124 shows the amino acid sequence of the DMD 35-36L.469 engineered meganuclease DMD 36 binding subunit.

[0246] SEQ ID NO: 125 shows the amino acid sequence of the DMD 37-38x.15 engineered meganuclease DMD3 8 binding subunit.

[0247] SEQ ID NO: 126 shows the amino acid sequence of the DMD 37-38x.66 engineered meganuclease DMD3 8 binding subunit.

[0248] SEQ ID NO: 127 shows the amino acid sequence of the DMD 37-38x.79 engineered meganuclease DMD3 8 binding subunit.

[0249] SEQ ID NO: 128 shows the amino acid sequence of the DMD 37-38L.166 engineered meganuclease DMD 38 binding subunit.

[0250] SEQ ID NO: 129 shows the amino acid sequence of the DMD 37-38L.478 engineered meganuclease DMD 38 binding subunit.

[0251] SEQ ID NO: 130 shows the amino acid sequence of the DMD 37-38L.512 engineered meganuclease DMD 38 binding subunit.

[0252] SEQ ID NO: 131 shows the amino acid sequence of the DMD 37-38L.528 engineered meganuclease DMD 38 binding subunit.

[0253] SEQ ID NO: 132 shows the amino acid sequence of the linker sequence.

[0254] SEQ ID NO: 133 shows the nucleic acid sequence of the probe used in the ddPC R assay for detecting indels in the DMD 19-20 recognition sequence.

[0255] SEQ ID NO: 134 shows the nucleic acid sequence of the forward PCR primer used in the ddPC R assay for detecting indels in the DMD 19-20 recognition sequence.

[0256] SEQ ID NO: 135 shows the nucleic acid sequence of the forward PCR primer used in the ddPC R assay for detecting indels in the DMD 19-20 recognition sequence.

[0257] SEQ ID NO: 136 shows the nucleic acid sequence of the probe used as a reference in the ddPCR assay for detecting indels

[0258] SEQ ID NO: 137 shows the nucleic acid sequence of the forward PCR primer used as a reference in the ddPCR assay for detecting indels

[0259] SEQ ID NO: 138 shows the nucleic acid sequence of the forward PCR primer used as a reference in the ddPCR assay for detecting indels

[0260] SEQ ID NO: 139 shows the nucleic acid sequence of the probe used in the ddPCR assay for detecting indels in the DMD 37-38 recognition sequence.

[0261] SEQ ID NO: 140 shows the nucleic acid sequence of the forward PCR primer used in the ddPCR assay for detecting indels in the DMD 37-38 recognition sequence.

[0262] SEQ ID NO: 141 shows the nucleic acid sequence of the forward PCR primer used in the ddPCR assay for detecting indels in the DMD 37-38 recognition sequence.

[0263] SEQ ID NO: 142 shows the nucleic acid sequence of the probe used in the ddPCR assay for detecting indels in the DMD 35-36 recognition sequence.

[0264] SEQ ID NO: 143 shows the nucleic acid sequence of the forward PCR primer used in the ddPCR assay for detecting indels in the DMD 35-36 recognition sequence.

[0265] SEQ ID NO: 144 shows the nucleic acid sequence of the forward PCR primer used in the ddPCR assay for detecting indels in the DMD 35-36 recognition sequence.

[0266] SEQ ID NO: 145 shows the nucleic acid sequence of the probe used in the ddPCR assay for detecting indels in the DMD 29-30 recognition sequence.

[0267] SEQ ID NO: 146 shows the nucleic acid sequence of the forward PCR primer used in the ddPCR assay for detecting indels in the DMD 29-30 recognition sequence.

[0268] ​​​​​​​​Array number 147 is the ddPC for detecting indels in the DMD 29-30 recognition array It shows the nucleic acid sequence of the forward PCR primer used in the R assay.

[0269] Array number 148 shows the nucleic acid sequence of the forward PCR primer used in the PCR amplification assay of the ligated recognition array of DMD 19-20 to DMD 35-36 Yes.

[0270] Array number 149 shows the nucleic acid sequence of the reverse PCR primer used in the PCR amplification assay of the ligated recognition array of DMD 19-20 to DMD 35-36 .

[0271] Array number 150 shows the nucleic acid sequence of the forward PCR primer used in the PCR amplification assay of the ligated recognition array of DMD 19-20 to DMD 35-36 Yes.

[0272] Array number 151 shows the nucleic acid sequence of the reverse PCR primer used in the PCR amplification assay of the ligated recognition array of DMD 19-20 to DMD 35-36 .

[0273] Array number 152 shows the nucleic acid sequence of the forward PCR primer used in the PCR amplification assay of the ligated recognition array of DMD 19-20 to DMD 29-30 Yes.

[0274] Array number 153 shows the nucleic acid sequence of the reverse PCR primer used in the PCR amplification assay of the ligated recognition array of DMD 19-20 to DMD 29-30 .

[0275] ​​​​​​Array number 154 is the nucleic acid sequence of the forward PCR primer used in the PCR amplification assay of the ligated recognition sequence from DMD 19-20 to DMD 29-30 as shown .

[0276] Array number 155 is the nucleic acid sequence of the reverse PCR primer used in the PCR amplification assay of the ligated recognition sequence from DMD 19-20 to DMD 29-30 as shown .

[0277] Array number 156 is the nucleic acid sequence of the forward PCR primer used in the PCR amplification assay of the ligated recognition sequence from DMD 19-20 to DMD 37-38 as shown .

[0278] Array number 157 is the nucleic acid sequence of the reverse PCR primer used in the PCR amplification assay of the ligated recognition sequence from DMD 19-20 to DMD 37-38 as shown .

[0279] Array number 158 is the nucleic acid sequence of the forward PCR primer used in the PCR amplification assay of the ligated recognition sequence from DMD 19-20 to DMD 37-38 as shown .

[0280] Array number 159 is the nucleic acid sequence of the reverse PCR primer used in the PCR amplification assay of the ligated recognition sequence from DMD 19-20 to DMD 37-38 as shown .

[0281] Array number 160 is the nucleic acid sequence of the probe used in the ddPCR assay for the recognition sequence from DMD 19-20 to DMD 37-38 as shown

[0282] Array number 161 is the nucleic acid sequence of the forward PCR primer used in the ddPCR assay of the ligated recognition sequences of DMD 19-20 to DMD 37-38 as shown here

[0283] Array number 162 is the nucleic acid sequence of the reverse PCR primer used in the ddPCR assay of the ligated recognition sequences of DMD 19-20 to DMD 37-38 as shown here

[0284] Array number 163 is the nucleic acid sequence of the probe used in the ddPCR assay for the ligated recognition sequences of DMD 19-20 to DMD 35-36 as shown

[0285] Array number 164 is the nucleic acid sequence of the forward PCR primer used in the ddPCR assay of the ligated recognition sequences of DMD 19-20 to DMD 35-36 as shown here

[0286] Array number 165 is the nucleic acid sequence of the reverse PCR primer used in the ddPCR assay of the ligated recognition sequences of DMD 19-20 to DMD 35-36 as shown here

[0287] Array number 166 is the nucleic acid sequence of the probe used in the ddPCR assay for the ligated recognition sequences of DMD 19-20 to DMD 29-30 as shown

[0288] Array number 167 is the nucleic acid sequence of the forward PCR primer used in the ddPCR assay of the ligated recognition sequences of DMD 19-20 to DMD 29-30 as shown here

[0289] Array number 168 is for the ligated nucleic acid sequence of the reverse PCR primer used in the ddPCR assay of the recognition array .

[0290] Array number 169 shows the nucleic acid sequence of the C5-12 promoter sequence.

[0291] Array number 170 shows the nucleic acid sequence of the mouse MCK promoter and enhancer sequences.

[0292] Array number 171 shows the nucleic acid sequence of the human MCK promoter sequence.

[0293] Array number 172 shows the nucleic acid sequence of the wild-type MCK enhancer sequence.

[0294] Array number 173 shows the nucleic acid sequence of the modified MCK enhancer sequence.

[0295] Array number 174 shows the nucleic acid sequence of the spc 5-12 promoter sequence.

[0296] Array number 175 shows the nucleic acid sequence of the MHCK7 promoter sequence.

[0297] Array number 176 shows the nucleic acid sequence of the CK8 promoter sequence.

[0298] Array number 177 shows the nucleic acid sequence of the SK-CRM4 promoter sequence.

[0299] Array number 178 shows the nucleic acid sequence of the SP-301 promoter sequence.

[0300] Array number 179 shows the nucleic acid sequence of the SP-817 promoter sequence.

[0301] Array number 180 shows the nucleic acid sequence of the SP-905 promoter sequence.

[0302] SEQ ID NO: 181 shows the nucleic acid sequence of the muscle hybrid promoter sequence.

[0303] SEQ ID NO: 182 shows the amino acid sequence of the rh.74 AAV capsid.

[0304] SEQ ID NO: 183 shows the amino acid sequence of the AAV9 capsid.

[0305] SEQ ID NO: 184 shows the nucleic acid sequence of the forward primer.

[0306] SEQ ID NO: 185 shows the nucleic acid sequence of the reverse primer.

[0307] SEQ ID NO: 186 shows the nucleic acid sequence of the probe.

[0308] SEQ ID NO: 187 shows the nucleic acid sequence of the forward primer.

[0309] SEQ ID NO: 188 shows the nucleic acid sequence of the reverse primer.

[0310] SEQ ID NO: 189 shows the nucleic acid sequence of the probe.

[0311] SEQ ID NO: 190 shows the nucleic acid sequence of the forward primer.

[0312] SEQ ID NO: 191 shows the nucleic acid sequence of the reverse primer.

[0313] SEQ ID NO: 192 shows the nucleic acid sequence of the probe.

[0314] SEQ ID NO: 193 shows the nucleic acid sequence of the reverse primer. DETAILED DESCRIPTION OF THE INVENTION

[0315] 1.1 References and Definitions The patents and scientific literature referred to in this specification establish knowledge available to those skilled in the art. This spec ification incorporates by reference issued U.S. patents, allowed applications, published foreign applications, and Ge References that include the nBank database array are hereby incorporated by reference as if each were specifically and individually set forth. Incorporated herein by reference to the same extent as if each was specifically and individually set forth.

[0316] The present disclosure may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. For example, features shown with respect to one embodiment may be incorporated in other embodiments, and features shown with respect to a particular embodiment may be deleted from that embodiment. Further, numerous modifications and additions to the embodiments proposed herein will be apparent to those skilled in the art in light of the present disclosure and do not depart from the scope of the invention. For example, features shown with respect to one embodiment can be incorporated into other embodiments, and features shown with respect to a particular embodiment can be deleted from that embodiment. Further, numerous modifications and additions to the embodiments proposed herein will be apparent to those skilled in the art in light of the present disclosure and do not depart from the scope of the invention. Features shown with respect to one embodiment can be incorporated into other embodiments, and features shown with respect to a particular embodiment can be deleted from that embodiment. Furthermore, many modifications and additions to the embodiments proposed herein will be apparent to those skilled in the art in light of the present disclosure and do not depart from the scope of the invention. Furthermore, many modifications and additions to the embodiments proposed herein will be apparent to those skilled in the art in light of the present disclosure and do not depart from the scope of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention.

[0317] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety.

[0318] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Incorporated herein by reference in their entirety.

[0319] As used herein, "a," "an," or "the" can mean one or more than one. For example, "a" cell can mean a single cell or a plurality of cells. As used herein, "a," "an," or "the" can mean one or more than one. For example, "a" cell can mean a single cell or a plurality of cells. Can mean.

[0320] As used herein, unless otherwise specified, the word "or" is used in the inclusive sense of "and / or" and not the exclusive sense of "either / or". is not used in the exclusive sense of "either / or".

[0321] As used herein, the terms "nuclease" and "endonuclease" are used interchangeably to refer to naturally occurring or genetically engineered enzymes that cleave phosphodiester bonds within a polynucleotide chain. Genetically engineered nucleases include , genetically engineered meganucleases, zinc finger nucleases, TALENs, compact TALENs, CRISPR system nucleases, and megaTALs, but are not limited to these. Furthermore, any genetically engineered nuclease capable of generating an overhang at its cleavage site is contemplated.

[0322] As used herein, the terms "cleave" or "cleavage" refer to the hydrolysis of a phosphodiester bond within the backbone of a recognition sequence within a target sequence that results in a double-strand break within the target sequence, referred to herein as the "cleavage site".

[0323] As used herein, the term "meganuclease" refers to an endonuclease that binds to double-stranded DNA at a recognition sequence that is greater than 12 base pairs. In some embodiments , the recognition sequence of the meganucleases of the disclosure is 22 base pairs. A meganuclease can be an endonuclease derived from I-CreI (SEQ ID NO: 1) and can refer, for example, to a genetically engineered variant of I-CreI that has been modified with respect to its DNA binding specificity, DNA cleavage activity, DNA binding affinity, or dimerization properties relative to native I-CreI. I-Cr ​​​​​​​Methods for producing such modified variants of eI are known in the art (e.g., International Publication No. 2007 / 047859, which is incorporated by reference in its entirety) . The meganucleases used herein bind to double-stranded DNA as heterodimers . Meganucleases can also be "single-stranded meganucleases" in which a pair of DNA-binding domains are linked to a single polypeptide using a peptide linker . The term "homing endonuclease" is synonymous with the term "meganuclease" . The meganucleases of the present disclosure are substantially non-toxic when expressed in the target cells described herein and can thus be transfected into cells and maintained at 37°C without observing any detrimental effects on cell viability or any significant decrease in meganuclease cleavage activity as measured using the methods described herein .

[0324] As used herein, the term "single-stranded meganuclease" refers to a polypeptide comprising a pair of nuclease subunits linked by a linker . Single-stranded meganucleases have the configuration N-terminal subunit-linker-C-terminal subunit . The two meganuclease subunits generally have non-identical amino acid sequences and bind to non-identical DNA sequences . Thus, single-stranded meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences . Single-stranded meganucleases may be referred to as "single-stranded heterodimers" or "single-stranded heterodimer meganucleases", but are not actually dimers . For clarity, unless otherwise specified, the term "meganuclease" can refer to either a dimer or a single-stranded meganuclease .​​​​​

[0325] As used herein, the term "linker" refers to an exogenous peptide sequence used to link two nuclease subunits into a single polypeptide. The linker may have a sequence found in natural proteins or may be an artificial sequence not found in any natural protein. The linker is flexible and may lack a secondary structure or may tend to form a specific three-dimensional structure under physiological conditions. The linker can include, without limitation, those encompassed by U.S. Patent Nos. 8,445,251, 9,340,777, 9,434,931, and 10,041,053, each of which is incorporated herein by reference in its entirety. In some embodiments, the linker has at least 80%, at least 85%, at least 90%, at least 91% %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence numbered 132, showing residues 154-195 of any one of SEQ ID NOs: 36-59.

[0326] As used herein, the terms "recombinant" or "genetically engineered" with respect to a protein mean having an amino acid sequence that has been changed as a result of the application of genetic engineering techniques to the nucleic acid encoding the protein and to the cell or organism expressing the protein. With respect to a nucleic acid, the terms "recombinant" or "genetically engineered" mean having a nucleic acid sequence that has been altered as a result of the application of genetic engineering techniques. Genetic engineering techniques include PCR and DNA cloning Cloning technology; transfection, transformation and other gene introduction technologies; homologous recombination; Site-directed mutagenesis; and gene fusions are included, but not limited to these. This def inition states that a protein having the same amino acid sequence as a naturally occurring protein, but produced by cloning and expression in a heterologous host, is not considered to be recombinant or genetically engineered.

[0327] As used herein, the term "wild-type" refers to the most common natural allele (i.e., polyn ucleotide sequence) in a population of alleles of the same type of gene, where the polypept ide encoded by the wild-type allele has its original function. The term "wild -type" also refers to the polypeptide encoded by the wild-type allele. A wild -type allele (i.e., polynucleotide) and polypeptide are distinguishable from mutant or var iant alleles and polypeptides that contain one or more mutations and / or substitutions com pared to the wild-type sequence(s). A wild-type allele or polypeptide can confer a norma l phenotype in an organism, while a mutant or variant allele or polypeptide can, in some cases, confer an altered phenotype. A wild-type nuclease is distinguishable from a reco mbinant nuclease or a nuclease not found in nature. The term "wild-type" can also refer to a cell, organism, and / or subject having the wild-type allele of a particular gene, or a cell, organism, and / or subject used for comparison purposes.

[0328] As used herein, the term "genetic modification" means that the genomic DNA sequence is (or ​Refers to a cell or organism that has been intentionally modified by recombinant techniques in its ancestor. As used herein, the term "genetically modified" encompasses the term "transgenic".

[0329] As used herein, the term "modified" with respect to a recombinant protein means any insertion, deletion, or substitution of an amino acid residue within the recombinant sequence relative to a reference sequence (e.g., a wild-type or native sequence).

[0330] As used herein, the term "recognition sequence" or "recognition site" refers to a DNA sequence that is bound and cleaved by a nuclease. In the case of a meganuclease, the recognition sequence contains a pair of inverted 9-base pair "half-sites" separated by 4 base pairs. In the case of a single-stranded meganuclease, the N-terminal domain of the protein contacts the first half-site and the C-terminal domain of the protein contacts the second half-site. Cleavage by a meganuclease produces a 3'-overhang of 4 base pairs. An "overhang" or "sticky end" is a short single-stranded DNA segment that can be produced by endonuclease cleavage of a double-stranded DNA sequence. In the case of a meganuclease from I-CreI and a single-stranded meganuclease, the overhang contains bases 10-13 of the 22-base pair recognition sequence.

[0331] As used herein, the term "target site" or "target sequence" refers to a region of a cell's chromosomal DNA that contains a nuclease recognition sequence.

[0332] As used herein, the term "DNA binding affinity" or "binding affinity" refers to the tendency of a nuclease to non-covalently bind to a reference DNA molecule (e.g., a recognition sequence or any sequence). ​ means direction. Binding affinity is measured by the dissociation constant Kd. As used herein when the Kd of the nuclease for the reference recognition array increases or decreases by only a statistically significant percentage change relative to the reference nuclease, the nuclease has an "altered" binding affinity .

[0333] As used herein, the term "specificity" means the ability of a nuclease to bind and cleave a double-stranded DNA molecule only at a specific sequence of base pairs called the recognition array, or only at a specific set of recognition arrays. The set of recognition arrays shares specific conserved positions or sequence motifs, but can be degenerate at one or more positions. A highly specific nuclease can cleave only one or a very small number of recognition arrays. Specificity can be determined by any method known in the art . .

[0334] As used herein, the term "dystrophin gene" refers to the gene associated with National Center for Biotechnology Information (NCBI) gene ID 1756, as well as its natural variants . The term "dystrophin" refers to the polypeptide encoded by the dystrophin gene. The dystrophin isoform expressed in muscle cells and muscle progenitor cells is known as the Dp 427m dystrophin variant. The amino acid sequence of the full-length wild-type Dp427m dystrophin polypeptide is shown in SEQ ID NO: 4 . NCBI reference numbers NM_004006.3 and NP_003997.2 represent the dystrophin Dp427m mRNA and polypeptide, respectively. Several described herein ​​In one embodiment, the dystrophin gene is edited with a pair of engineered meganucleases to effect excision of exons 45-55 and subsequent full ligation of the dystrophin gene. Removal of exons 45-55 from the wild-type dystrophin gene can result in a dystrophin polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 5.

[0335] As used herein, the term "full ligation" refers to the ligation (i.e., annealing) of all four bases of the 3' overhang of the first cleavage site within the dystrophin gene and all four bases of the complementary 3' overhang of the second cleavage site after cleavage by a pair of the engineered meganucleases of the invention. The recognition sequences targeted by the disclosed engineered meganucleases have the same four-base pair central sequence (e.g., GTAT) such that the first and second cleavage sites have complementary four-base pair 3' overhangs. Thus, each base pair of the first 3' overhang pairs with its complementary base pair on the second 3' overhang and ligation occurs via a DNA ligase enzyme. Examples of sequences resulting from such full ligation are shown in SEQ ID NO: 32 (i.e., full ligation of the DMD 19-20 and DMD 35-36 recognition sequences) and SEQ ID NO: 34 (i.e., full ligation of the DMD 19-20 and DMD 37-38 recognition sequences).

[0336] As used herein, the term "Becker muscular dystrophy phenotype" refers to a form of muscular dystrophy that is less severe compared to DMD. ​​​​​​​​​​​​​​Individuals with this condition still contain a mutation in the dystrophin gene but do not have DMD. These individuals express more functional dystrophin protein in muscle cells (e.g., premuscular These proteins are expressed in progenitor cells, skeletal muscle cells and cardiac muscle cells, and generally result in a better clinical prognosis.

[0337] As used herein, the term "homologous recombination" or "HR" refers to a repair template. A natural cellular process by which double-stranded DNA breaks are repaired using homologous DNA sequences as a template (For example, Cahill et al. (2006) Front. Biosci. 11:1958-76). The homologous DNA sequence may be an endogenous chromosomal sequence or a sequence delivered to the cell. The nucleic acid may be an exogenous nucleic acid.

[0338] As used herein, the term "non-homologous end joining" or "NHEJ" refers to a process for the synthesis of two A natural cell in which strand DNA breaks are repaired by direct joining of two nonhomologous DNA segments This refers to the process of non-homologous termination (see, for example, Cahill et al. (2006)). DNA repair by end-joining is error-prone and involves untemplated mismatches of DNA sequences at the repair site. In some instances, cleavage at the target recognition sequence results in target recognition. nuclease-induced cleavage of a target site in the coding sequence of a gene, resulting in NHEJ at the site and subsequent DNA repair by non-homologous end joining (NHEJ) are key mechanisms for disrupting gene function. Mutations such as Lambda shift mutations can be introduced into the coding sequence. Using engineered nucleases, researchers can effectively knock out genes in cell populations. It can be done.

[0339] As used herein, the term "homologous arm" or "sequence homologous to the sequence adjacent to the nuclease cleavage site" refers to the sequences adjacent to the 5' and 3' ends of a nucleic acid molecule that facilitate the insertion of the nucleic acid molecule into the cleavage site generated by a nuclease. Generally, a homologous arm can be at least 50 base pairs, preferably at least 100 base pairs, up to 2000 base pairs or longer, and can have at least 90%, preferably at least 95% or higher sequence homology with their corresponding sequences in the genome. In some embodiments, the homologous arm is about 500 base pairs. As used herein, for both amino acid sequences and nucleic acid sequences, terms such as "percent identity", "sequence identity", "percentage similarity", "sequence similarity" refer to a measure of the degree of similarity between two sequences based on an alignment of the sequences, which maximizes the similarity between aligned amino acid residues or nucleotides, and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), e.g., Altschul et al. (1990) J. Mol. Bi As used herein, the term "homologous arm" or "sequence homologous to the sequence adjacent to the nuclease cleavage site" refers to the sequences adjacent to the 5' and 3' ends of a nucleic acid molecule that facilitate the insertion of the nucleic acid molecule into the cleavage site generated by a nuclease. Generally, a homologous arm can be at least 50 base pairs, preferably at least 100 base pairs, up to 2000 base pairs or longer, and can have at least 90%, preferably at least 95% or higher sequence homology with their corresponding sequences in the genome. In some embodiments, the homologous arm is about 500 base pairs. As used herein, the term "homologous arm" or "sequence homologous to the sequence adjacent to the nuclease cleavage site" refers to the sequences adjacent to the 5' and 3' ends of a nucleic acid molecule that facilitate the insertion of the nucleic acid molecule into the cleavage site generated by a nuclease. Generally, a homologous arm can be at least 50 base pairs, preferably at least 100 base pairs, up to 2000 base pairs or longer, and can have at least 90%, preferably at least 95% or higher sequence homology with their corresponding sequences in the genome. In some embodiments, the homologous arm is about 500 base pairs. As used herein, the term "homologous arm" or "sequence homologous to the sequence adjacent to the nuclease cleavage site" refers to the sequences adjacent to the 5' and 3' ends of a nucleic acid molecule that facilitate the insertion of the nucleic acid molecule into the cleavage site generated by a nuclease. Generally, a homologous arm can be at least 50 base pairs, preferably at least 100 base pairs, up to 2000 base pairs or longer, and can have at least 90%, preferably at least 95% or higher sequence homology with their corresponding sequences in the genome. In some embodiments, the homologous arm is about 500 base pairs. As used herein, the term "homologous arm" or "sequence homologous to the sequence adjacent to the nuclease cleavage site" refers to the sequences adjacent to the 5' and 3' ends of a nucleic acid molecule that facilitate the insertion of the nucleic acid molecule into the cleavage site generated by a nuclease. Generally, a homologous arm can be at least 50 base pairs, preferably at least 100 base pairs, up to 2000 base pairs or longer, and can have at least 90%, preferably at least 95% or higher sequence homology with their corresponding sequences in the genome. In some embodiments, the homologous arm is about 500 base pairs. As used herein, the term "homologous arm" or "sequence homologous to the sequence adjacent to the nuclease cleavage site" refers to the sequences adjacent to the 5' and 3' ends of a nucleic acid molecule that facilitate the insertion of the nucleic acid molecule into the cleavage site generated by a nuclease. Generally, a homologous arm can be at least 50 base pairs, preferably at least 100 base pairs, up to 2000 base pairs or longer, and can have at least 90%, preferably at least 95% or higher sequence homology with their corresponding sequences in the genome. In some embodiments, the homologous arm is about 500 base pairs.

[0340] As used herein, for both amino acid sequences and nucleic acid sequences, terms such as "percent identity", "sequence identity", "percentage similarity", "sequence similarity" refer to a measure of the degree of similarity between two sequences based on an alignment of the sequences, which maximizes the similarity between aligned amino acid residues or nucleotides, and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), e.g., Altschul et al. (1990) J. Mol. Bi As used herein, for both amino acid sequences and nucleic acid sequences, terms such as "percent identity", "sequence identity", "percentage similarity", "sequence similarity" refer to a measure of the degree of similarity between two sequences based on an alignment of the sequences, which maximizes the similarity between aligned amino acid residues or nucleotides, and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), e.g., Altschul et al. (1990) J. Mol. Bi As used herein, for both amino acid sequences and nucleic acid sequences, terms such as "percent identity", "sequence identity", "percentage similarity", "sequence similarity" refer to a measure of the degree of similarity between two sequences based on an alignment of the sequences, which maximizes the similarity between aligned amino acid residues or nucleotides, and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), e.g., Altschul et al. (1990) J. Mol. Bi As used herein, for both amino acid sequences and nucleic acid sequences, terms such as "percent identity", "sequence identity", "percentage similarity", "sequence similarity" refer to a measure of the degree of similarity between two sequences based on an alignment of the sequences, which maximizes the similarity between aligned amino acid residues or nucleotides, and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), e.g., Altschul et al. (1990) J. Mol. Bi As used herein, for both amino acid sequences and nucleic acid sequences, terms such as "percent identity", "sequence identity", "percentage similarity", "sequence similarity" refer to a measure of the degree of similarity between two sequences based on an alignment of the sequences, which maximizes the similarity between aligned amino acid residues or nucleotides, and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), e.g., Altschul et al. (1990) J. Mol. Bi As used herein, for both amino acid sequences and nucleic acid sequences, terms such as "percent identity", "sequence identity", "percentage similarity", "sequence similarity" refer to a measure of the degree of similarity between two sequences based on an alignment of the sequences, which maximizes the similarity between aligned amino acid residues or nucleotides, and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), e.g., Altschul et al. (1990) J. Mol. Bi As used herein, for both amino acid sequences and nucleic acid sequences, terms such as "percent identity", "sequence identity", "percentage similarity", "sequence similarity" refer to a measure of the degree of similarity between two sequences based on an alignment of the sequences, which maximizes the similarity between aligned amino acid residues or nucleotides, and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), e.g., Altschul et al. (1990) J. Mol. Bi As used herein, for both amino acid sequences and nucleic acid sequences, terms such as "percent identity", "sequence identity", "percentage similarity", "sequence similarity" refer to a measure of the degree of similarity between two sequences based on an alignment of the sequences, which maximizes the similarity between aligned amino acid residues or nucleotides, and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), e.g., Altschul et al. (1990) J. Mol. Bi As used herein, for both amino acid sequences and nucleic acid sequences, terms such as "percent identity", "sequence identity", "percentage similarity", "sequence similarity" refer to a measure of the degree of similarity between two sequences based on an alignment of the sequences, which maximizes the similarity between aligned amino acid residues or nucleotides, and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), e.g., Altschul et al. (1990) J. Mol. Bi As used herein, for both amino acid sequences and nucleic acid sequences, terms such as "percent identity", "sequence identity", "percentage similarity", "sequence similarity" refer to a measure of the degree of similarity between two sequences based on an alignment of the sequences, which maximizes the similarity between aligned amino acid residues or nucleotides, and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), e.g., Altschul et al. (1990) J. Mol. Bi As used herein, for both amino acid sequences and nucleic acid sequences, terms such as "percent identity", "sequence identity", "percentage similarity", "sequence similarity" refer to a measure of the degree of similarity between two sequences based on an alignment of the sequences, which maximizes the similarity between aligned amino acid residues or nucleotides, and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), e.g., Altschul et al. (1990) J. Mol. Bi Vol. 215: 403-10; Gish & States (1993) Nature Ge net. 3: 266-72; Madden et al. (1996) Meth. Enz ymol. 266: 131-41; Altschul et al. (1997) Nuc leic Acids Res. 25: 3389-3402; and Zhang et a l. (2000) J. Comput. Biol. 7: 203-14. As used in this specification, the percent similarity of two amino acid sequences is a score based on the following parameters for the BLASTp al gorithm: word size = 3; gap opening penalty = -11; gap extension penalty = -1; scoring matrix = BLO SUM 62. As used in this specification, the percent similarity of two nucleic acid sequences is a score based on the following parameters for the BL ASTn algorithm: word size = 11; gap opening penalty = -5; gap extension penalty = -2; match reward = 1; mismatch penalty = -3.

[0341] As used in this specification, the term "corresponding to" with respect to a modification of two proteins or amino acid sequences means that a particular modification of the first protein is the same amino acid residue substitution as the modification of the second protein, and that when the two proteins are subjected to a standard sequence alignment (e.g., using the BLASTp program), the amino acid position of the modification of the first protein corresponds to or aligns with the amino acid position of the modification of the second protein. Thus, a modification of residue "X" to amino acid "A" in the first protein means that residues X and Y correspond to each other in the sequence alignment when the first protein is aligned with the second protein. For example, when the first protein is aligned with the second protein using the BLASTp program, the amino acid position of the modification of the first protein corresponds to or aligns with the amino acid position of the modification of the second protein. In the case where residues X and Y correspond to each other in the sequence alignment, the modification of residue "X" to amino acid "A" in the first protein means that the modification of residue "X" to amino acid "A" in the first protein is the same amino acid residue substitution as the modification of residue "Y" to amino acid "A" in the second protein. Notwithstanding the fact that X and Y can be different numbers, it corresponds to the modification of the residue "Y" to the amino acid "A" in the second protein.

[0342] As used herein, the terms "recognition half-site", "recognition sequence half-site", or simply "half-site" refer to a nucleic acid sequence within a double-stranded DNA molecule that is recognized and bound by a monomer of a homodimeric or heterodimeric meganuclease, or by one subunit of a single-stranded meganuclease, or by one subunit of a single-stranded meganuclease.

[0343] As used herein, the term "hypervariable region" refers to a localized sequence within a meganuclease monomer or subunit that contains amino acids with relatively high variability. The hypervariable region can contain about 50 - 60 contiguous residues, about 53 - 57 contiguous residues, or preferably about 56 residues. In some embodiments, the residues of the hypervariable region can correspond to positions 24 - 79 or 215 - 270 of any one of SEQ ID NOs: 36 - 59. The hypervariable region can contain one or more residues that contact the DNA bases in the recognition sequence and can be modified to alter the base selectivity of the monomer or subunit. The hypervariable region can also contain one or more residues that bind to the DNA backbone when the meganuclease associates with the double-stranded DNA recognition sequence. Such residues can be modified to alter the binding affinity of the meganuclease for the DNA backbone and the target recognition sequence. In different embodiments of the present invention, the hypervariable region can contain 1 - 20 residues that can be modified to exhibit variability and affect base selectivity and / or DNA binding affinity. ​ In one form, the hypervariable region contains about 15-20 residues that exhibit variability and can be modified to affect base selectivity and / or DNA binding affinity. In some embodiments, the variable residues within the hypervariable region correspond to one or more of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 36-59. In certain embodiments, the variable residues within the hypervariable region can further correspond to residues 48, 50, and 71-73 of any one of SEQ ID NOs: 36-59. In other embodiments, the variable residues within the hypervariable region correspond to one or more of positions 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 239, 241, 259, 261, 262, 263, 264, 266, and 26 8 of any one of SEQ ID NOs: 36-59. In certain embodiments, the variable residues within the hypervariable region can further correspond to residues 239, 241, and 263-265 of any one of SEQ ID NOs: 36-59. The term "increase" in the context of dystrophin protein or mRNA level refers to any increase in the level of dystrophin protein or mRNA expression compared to a reference level, including at least 1%, 2%, 3%, 4%, 5%, 10 %, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90 %, 95%, 100%, or more increase in dystrophin protein or mRNA expression compared to the reference level. In some embodiments, the increase in dystrophin protein or mRNA

[0344] level is a shortened dystrophin polypeptide or gene compared to the wild-type ​ An increase in dystrophin polypeptide or mRNA transcript, for example, a part of the polypeptide encoded by at least one exon (e.g., the part encoded by exons 45-55) is missing, or a part of the mRNA corresponding to exons 45-55 is missing.

[0345] As used herein, the term "reference level" in the context of dystrophin protein or mRNA level refers to, for example, in a control cell, control cell population or control subject, the level of dystrophin protein or mRNA measured at a previous time point in a control cell, control cell population or subject being treated (e.g., a pre-dose baseline level obtained from a control cell, control cell population or subject), or a predefined threshold level of dystrophin protein or mRNA (e.g., a threshold level identified through previous experiments).

[0346] As used herein, the terms "control" or "control cell" refer to a cell that provides a reference point for measuring changes in the genotype or phenotype of a genetically modified cell. A control cell can be, for example: (a) a wild-type cell, i.e., a wild-type cell of the same genotype as the starting material for the genetic modification that resulted in the genetically modified cell; (b) a cell of the same genotype as the genetically modified cell, but transformed with a null construct (i.e., a construct known not to affect the trait of interest); or (c) a cell that is genetically identical to the genetically modified cell but has not been exposed to conditions or stimuli or further genetic modifications that induce the expression of a changed genotype or phenotype. A control subject can be, for example: a wild-type subject, i.e., a genetically modified subject, or a subject of the same genotype as the genetically modified subject but not exposed to the genetic modification. A wild-type subject (e.g., a wild-type control) of the same genotype as the starting subject for the genetic alteration that resulted in the subject subjects with the same mutation in the strophin gene, have not been exposed to conditions or stimuli that induce the expression of a genetic or phenotypic type or further genetic modification The subject may include wild-type subjects.

[0347] As used herein, "recombinant DNA construct," "recombinant construct," "expression cassette," "expression construct," "chimeric construct," "construct," and "recombinant DNA fragment." The terms are used interchangeably herein and refer to single- or double-stranded polynucleotides. Recombinant constructs include, but are not limited to, regulatory and coding sequences that are not found together in nature. For example, a recombinant DNA construct includes an artificial combination of undefined nucleic acid fragments. The regulatory and coding sequences may be derived from the same source or may be derived from the same source and found in nature. Such constructs may contain regulatory and coding sequences arranged in a manner different from that described above. may be used alone or in combination with a vector.

[0348] As used herein, the term "vector" or "recombinant DNA vector" means A replication system capable of transcription and translation of a polypeptide coding sequence in a given host cell. and a construct comprising the sequence. When a vector is used, the selection of the vector can be determined by those skilled in the art. As is well known to those skilled in the art, the method used to transform host cells will depend on the vector. Examples include, but are not limited to, plasmid vectors and recombinant AAV vectors, or genes Any other vector known in the art suitable for delivering the vector to a target cell may be included. One skilled in the art must have the genetic elements that must be present on the vector to successfully transform, select, and propagate a host cell containing any of the isolated nucleotides or nucleic acid sequences of the

[0349] present invention. In some embodiments, "vector" also refers to a viral vector. Viral vectors can include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, and AAV. As used herein, the term "operably linked" is intended to mean a functional linkage between two or more elements. For

[0350] example, an operable linkage between a nucleic acid sequence encoding a nuclease disclosed herein and a regulatory sequence (e.g., a promoter) is a functional linkage that enables expression of the nucleic acid sequence encoding the nuclease. Operably linked The expression of (with amino acid deficiency) increases. In some embodiments, the expression of a version of the dystrophin protein lacking the amino acids encoded by exons 45- increases. The expression of a version of the dystrophin protein lacking the amino acids encoded by exons 45 - 55 increases. In some embodiments, such treatment shifts the DMD phenotype to the Becker pseudohypertrophic phenotype.

[0351] As used herein, the term "gc / kg" or "gene copy / kilogram" refers to the number of copies of a nucleic acid sequence encoding a genetically engineered meganuclease described herein per kilogram of body weight of a subject to whom a polynucleotide containing the nucleic acid sequence is administered. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity, and the age, weight, physical condition and responsiveness of the subject being treated. In certain embodiments, an effective amount of a genetically engineered meganuclease described herein, or a pair of genetically engineered meganucleases, or a polynucleotide or pair of polynucleotides encoding the same, or a pharmaceutical composition disclosed herein increases the expression level of the dystrophin protein (e.g., a truncated dystrophin protein lacking the amino acids encoded by exons 45 - 55) and ameliorates at least one symptom associated with DMD. As used herein, the term "lipid nanoparticle" refers to a lipid composition having a typically spherical structure with an average diameter of 10 - 1000 nm. In some formulations,

[0352] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity, and the age, weight, physical condition and responsiveness of the subject being treated. In certain embodiments, an effective amount of a genetically engineered meganuclease described herein, or a pair of genetically engineered meganucleases, or a polynucleotide or pair of polynucleotides encoding the same, or a pharmaceutical composition disclosed herein increases the expression level of the dystrophin protein (e.g., a truncated dystrophin protein lacking the amino acids encoded by exons 45 - 55) and ameliorates at least one symptom associated with DMD. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity, and the age, weight, physical condition and responsiveness of the subject being treated. In certain embodiments, an effective amount of a genetically engineered meganuclease described herein, or a pair of genetically engineered meganucleases, or a polynucleotide or pair of polynucleotides encoding the same, or a pharmaceutical composition disclosed herein increases the expression level of the dystrophin protein (e.g., a truncated dystrophin protein lacking the amino acids encoded by exons 45 - 55) and ameliorates at least one symptom associated with DMD. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity, and the age, weight, physical condition and responsiveness of the subject being treated. In certain embodiments, an effective amount of a genetically engineered meganuclease described herein, or a pair of genetically engineered meganucleases, or a polynucleotide or pair of polynucleotides encoding the same, or a pharmaceutical composition disclosed herein increases the expression level of the dystrophin protein (e.g., a truncated dystrophin protein lacking the amino acids encoded by exons 45 - 55) and ameliorates at least one symptom associated with DMD. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity, and the age, weight, physical condition and responsiveness of the subject being treated. In certain embodiments, an effective amount of a genetically engineered meganuclease described herein, or a pair of genetically engineered meganucleases, or a polynucleotide or pair of polynucleotides encoding the same, or a pharmaceutical composition disclosed herein increases the expression level of the dystrophin protein (e.g., a truncated dystrophin protein lacking the amino acids encoded by exons 45 - 55) and ameliorates at least one symptom associated with DMD. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity, and the age, weight, physical condition and responsiveness of the subject being treated. In certain embodiments, an effective amount of a genetically engineered meganuclease described herein, or a pair of genetically engineered meganucleases, or a polynucleotide or pair of polynucleotides encoding the same, or a pharmaceutical composition disclosed herein increases the expression level of the dystrophin protein (e.g., a truncated dystrophin protein lacking the amino acids encoded by exons 45 - 55) and ameliorates at least one symptom associated with DMD. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity, and the age, weight, physical condition and responsiveness of the subject being treated. In certain embodiments, an effective amount of a genetically engineered meganuclease described herein, or a pair of genetically engineered meganucleases, or a polynucleotide or pair of polynucleotides encoding the same, or a pharmaceutical composition disclosed herein increases the expression level of the dystrophin protein (e.g., a truncated dystrophin protein lacking the amino acids encoded by exons 45 - 55) and ameliorates at least one symptom associated with DMD. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity, and the age, weight, physical condition and responsiveness of the subject being treated. In certain embodiments, an effective amount of a genetically engineered meganuclease described herein, or a pair of genetically engineered meganucleases, or a polynucleotide or pair of polynucleotides encoding the same, or a pharmaceutical composition disclosed herein increases the expression level of the dystrophin protein (e.g., a truncated dystrophin protein lacking the amino acids encoded by exons 45 - 55) and ameliorates at least one symptom associated with DMD. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity, and the age, weight, physical condition and responsiveness of the subject being treated. In certain embodiments, an effective amount of a genetically engineered meganuclease described herein, or a pair of genetically engineered meganucleases, or a polynucleotide or pair of polynucleotides encoding the same, or a pharmaceutical composition disclosed herein increases the expression level of the dystrophin protein (e.g., a truncated dystrophin protein lacking the amino acids encoded by exons 45 - 55) and ameliorates at least one symptom associated with DMD. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity, and the age, weight, physical condition and responsiveness of the subject being treated. In certain embodiments, an effective amount of a genetically engineered meganuclease described herein, or a pair of genetically engineered meganucleases, or a polynucleotide or pair of polynucleotides encoding the same, or a pharmaceutical composition disclosed herein increases the expression level of the dystrophin protein (e.g., a truncated dystrophin protein lacking the amino acids encoded by exons 45 - 55) and ameliorates at least one symptom associated with DMD.

[0353] As used herein, the term "lipid nanoparticle" refers to a lipid composition having a typically spherical structure with an average diameter of 10 - 1000 nm. In some formulations, the lipid nanoparticle has a typically spherical structure with an average diameter of 10 - 1000 nm. In some formulations, Lipid nanoparticles can comprise at least one cationic lipid, at least one non-cationic lipid , and at least one conjugate lipid. Lipid nanoparticles known in the art suitable for encapsulating nucleic acids such as mRNA are contemplated for use in the present invention. are contemplated.

[0354] As used herein, the recitation of numerical ranges of variables is intended to convey that the disclosure may be practiced with any value within those ranges. Thus, for variables that are essentially discrete, the variable can be equal to any integer value within the numerical range including the endpoints of the range. Similarly, for variables that are essentially continuous, the variable can be equal to any real value within the numerical range including the endpoints of the range. By way of example and not limitation, a variable described as having a value between 0 and 2 can take on the values 0, 1, or 2 if the variable is essentially discrete, and can take on the values 0.0, 0.1, 0.01, 0.001, or any other real value between 0 and 2 if the variable is essentially continuous.

[0355] 2.1 Principle of the Invention The present disclosure is, in part, based on the hypothesis that certain deletions in the dystrophin gene that cause the DMD phenotype can be compensated for by strategically deleting exons within the dystrophin gene using pairs of endonucleases to restore the normal reading frame within the gene. The DMD-Leiden database indicates that most mutations causing DMD are deletions of one or more entire exons that cause a shift in the reading frame. In many cases, the exon immediately before or after the mutation ​​​​​​​​​​​​​By removing it, the reading frame can be restored. As shown in Table 3 As shown, 29 different mutations that cause Duchenne, which account for about 65% of patients can be compensated for by deleting a single exon adjacent to the mutation.

[0356]

Table 3

[0357] For example, patients with a disease caused by a deletion of exon 45 occur in about 7% of patients, and can be treated with a therapeutic agent that deletes exon 46. Therapeutic agents that can delete exon 51 or exon 4 5 can be used to treat 15% and 13% of patients, respectively.

[0358] In particular, more than 50% of all DMD-related mutations within the dystrophin gene are included in exons 45 -55. Therefore, in certain embodiments of the present invention, exons 45-55 of the dystrophin gene are removed to restore the normal reading frame of the gene. As disclosed herein, exon removal generates a pair of cleavage sites in the introns upstream of exon 45 and downstream of exon 55 in genetically engineered muscle cells or muscle progenitor cells (e.g., cardiomyocytes or skeletal muscle cells) by expression of a pair of engineered meganucleases, enabling excision of the intervening genomic region. Following this approach, genetically modified cells (e.g., muscle cells in a treated subject) can produce a certain amount of truncated dystrophin protein from a Becker phenotype similar to the microdystrophin approach without the need to express a microdystrophin transgene. This truncated dystrophin protein​​​ The resulting dystrophin, unlike other therapies that require multiple treatment regimens, may be sufficient to permanently relieve the disease.

[0359] Thus, a single treatment is envisioned to permanently delete an exon from a certain percentage of cells in a subject. In some embodiments, these cells are myoblasts (i.e., muscle cells) or other muscle progenitor cells that can give rise to entire muscle fibers that replicate and express functional (or semi-functional) dystrophin. However, if the frequency of exon deletion is low, multiple treatments may be required for each patient.

[0360] 2.2 Meganucleases that Bind to and Cleave Recognition Sequences within the Dystrophin Gene Recognition Sequences It is possible to perform DNA cleavage within the genome of living cells using site-specific nucleases, and such DNA cleavage can result in permanent modification of the genome via mutagenic NHEJ repair or via homologous recombination with a transgenic DNA sequence, which is known in the art. NHEJ can cause mutagenesis at the cleavage site and can result in inactivation of alleles. NHEJ-related mutagenesis can inactivate alleles via the generation of premature stop codons, frameshift mutations that produce abnormal non-functional proteins, or can induce mechanisms such as nonsense-mediated mRNA decay. The use of nucleases that induce mutagenesis via NHEJ can be used to target specific mutations or sequences present in the wild-type allele. Furthermore, the use of nucleases to induce double-strand cleavage at a target locus, particularly adjacent to sequences homologous to the genomic target It is known to stimulate homologous recombination of transgenic DNA sequences. In this way, an exogenous polynucleotide can be inserted into a target locus. Such an exogenous polynucleotide can encode any sequence or polypeptide of interest.

[0361] In certain embodiments, the engineered meganucleases of the invention bind to and cleave the DMD 19- 20 recognition sequence (SEQ ID NO: 6), the DMD 35-36 recognition sequence (SEQ ID NO: 10), or the DMD 37-38 recognition sequence (SEQ ID NO: 12). Exemplary meganucleases that bind to and cleave the DMD 19-20 recognition sequence are provided in SEQ ID NOs: 36-44. Exemplary meganucleases that bind to and cleave the DMD 35-36 recognition sequence are provided in SEQ ID NOs: 45-52. Exemplary meganucleases that bind to and cleave the DMD 37-38 recognition sequence are provided in SEQ ID NOs: 53-59. The sequences of each recognition sequence, and the four base pair 3´ overhangs that result when cleaved by the engineered meganucleases described herein, are provided in Table 4 below. To modify the dystrophin gene according to the present disclosure, pairs of the engineered meganucleases described herein are utilized together in the same cell. Such pairs of engineered meganucleases are designed to create a first cleavage site in the intron upstream of exon 45 and a second cleavage site in the intron downstream of exon 55, with the intervening genomic

[0362] [Table 4]

[0363] To modify the dystrophin gene according to the present disclosure, pairs of the engineered meganucleases described herein are utilized together in the same cell. Such pairs of engineered meganucleases are designed to create a first cleavage site in the intron upstream of exon 45 and a second cleavage site in the intron downstream of exon 55, with the intervening genomic engineered meganucleases are used together in the same cell. Such engineered meganucleases are designed to create a first cleavage site in the intron upstream of exon 45 and a second cleavage site in the intron downstream of exon 55, with the intervening genomic region ​The removal of the [[mu]] array was enabled. Surprisingly, it was observed that excision of this genomic region from the dystrophin gene could be achieved with high efficiency. Furthermore, the meganuclease recognition sequences were selected to have a 3´ overhang of complementary 4 base pairs after cleavage, and it was observed that the dystrophin gene could be repaired with high frequency by complete ligation of the 3´ overhangs of the two cleavage sites. Such completely ligated recognition sequences contemplated herein are provided in Table 5 below.

[0364]

Table 5

[0365] These recognition sequences are further selected to be within intron sequences that are normally spliced during post-transcriptional modification of the cell process. This reduces the possibility of introducing mutations into the dystrophin gene and the encoded polypeptide.

[0366] Exemplary Genetically Engineered Meganucleases The genetically engineered meganucleases of the present invention include a first subunit containing the HVR1 region and a second subunit containing the HVR2 region. Furthermore, the first subunit binds to the first recognition half-site in the recognition sequence (e.g., the DMD 19 half-site), and the second subunit binds to the second recognition half-site (e.g., the DMD 20 half-site) in the recognition sequence.

[0367] In certain embodiments, the meganuclease used to practice the present invention is a single-stranded meganuclease. The single-stranded meganuclease is linked by a linker peptide. ​ It includes an N-terminal subunit and a C-terminal subunit (i.e., the above-mentioned first and second subunits). Each of the two subunits recognizes and binds to a half-site of the recognition sequence, and the DNA cleavage site is in the center of the recognition sequence near the interface of the two subunits. It is theorized that DNA strand cleavage is offset by four base pairs such that DNA cleavage by the meganuclease generates a pair of 4-base pair 3'-single-stranded overhangs. It includes an N-terminal subunit and a C-terminal subunit (i.e., the above-mentioned first and second subunits). Each of the two subunits recognizes and binds to a half-site of the recognition sequence, and the DNA cleavage site is in the center of the recognition sequence near the interface of the two subunits. It is theorized that DNA strand cleavage is offset by four base pairs such that DNA cleavage by the meganuclease generates a pair of 4-base pair 3'-single-stranded overhangs. It includes an N-terminal subunit and a C-terminal subunit (i.e., the above-mentioned first and second subunits). Each of the two subunits recognizes and binds to a half-site of the recognition sequence, and the DNA cleavage site is in the center of the recognition sequence near the interface of the two subunits. It is theorized that DNA strand cleavage is offset by four base pairs such that DNA cleavage by the meganuclease generates a pair of 4-base pair 3'-single-stranded overhangs. In embodiments where the engineered meganuclease is a single-stranded meganuclease, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the N-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the C-terminal subunit. In an alternative embodiment, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the C-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the N-terminal subunit. In embodiments where the engineered meganuclease is a single-stranded meganuclease, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the N-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the C-terminal subunit. In an alternative embodiment, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the C-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the N-terminal subunit.

[0368] In embodiments where the engineered meganuclease is a single-stranded meganuclease, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the N-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the C-terminal subunit. In an alternative embodiment, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the C-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the N-terminal subunit. In embodiments where the engineered meganuclease is a single-stranded meganuclease, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the N-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the C-terminal subunit. In an alternative embodiment, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the C-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the N-terminal subunit. In embodiments where the engineered meganuclease is a single-stranded meganuclease, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the N-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the C-terminal subunit. In an alternative embodiment, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the C-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the N-terminal subunit. In embodiments where the engineered meganuclease is a single-stranded meganuclease, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the N-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the C-terminal subunit. In an alternative embodiment, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the C-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the N-terminal subunit. In embodiments where the engineered meganuclease is a single-stranded meganuclease, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the N-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the C-terminal subunit. In an alternative embodiment, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the C-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the N-terminal subunit. In embodiments where the engineered meganuclease is a single-stranded meganuclease, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the N-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the C-terminal subunit. In an alternative embodiment, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the C-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the N-terminal subunit. In embodiments where the engineered meganuclease is a single-stranded meganuclease, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the N-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the C-terminal subunit. In an alternative embodiment, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the C-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the N-terminal subunit. In embodiments where the engineered meganuclease is a single-stranded meganuclease, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the N-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the C-terminal subunit. In an alternative embodiment, the first and second subunits can be oriented such that the first subunit, which includes the HVR1 region and binds to the first half-site, is positioned as the C-terminal subunit, and the second subunit, which includes the HVR2 region and binds to the second half-site, is positioned as the N-terminal subunit.

[0369] Exemplary DMD meganucleases of the present invention are provided in SEQ ID NOs: 36-59 and summarized in Tables 6-8 below. Exemplary DMD meganucleases of the present invention are provided in SEQ ID NOs: 36-59 and summarized in Tables 6-8 below.

[0370] [Table 6]

[0371] "DMD19 subunit %" and "DMD20 subunit %" represent the DMD19-binding and DMD20-binding subunit regions of each meganuclease and DMD 19-20x. "DMD19 subunit %" and "DMD20 subunit %" represent the DMD19-binding and DMD20-binding subunit regions of each meganuclease and DMD 19-20x. The amino acid sequence identity between each of the 13 meganucleases and the DMD19-binding and DMD20-binding subunit regions is shown.

[0372] [Table 7]

[0373] "DMD35 subunit %" and "DMD36 subunit %" represent the amino acid sequence identity between the DMD35-binding and DMD36-binding subunit regions of each meganuclease and those of the DMD 35-36x. The DMD35-binding and DMD36-binding subunit regions of the 63 meganucleases and those of the 63 meganucleases. is shown.

[0374] [Table 8]

[0375] "DMD37 subunit %" and "DMD38 subunit %" represent the amino acid sequence identity between the DMD37-binding and DMD38-binding subunit regions of each meganuclease and those of the DMD 37-38x. The DMD37-binding and DMD38-binding subunit regions of the 15 meganucleases and those of the 15 meganucleases. is shown.

[0376] In certain embodiments of the invention, the engineered meganuclease binds to and cleaves a recognition sequence (i.e., the DMD 19-20 recognition sequence) containing SEQ ID NO: 6 within the dystrophin gene, and the engineered meganuclease comprises a first subunit and a second subunit, the first subunit binds to the first recognition half-site of the recognition sequence and comprises an HVR1 region, the second subunit binds to the second recognition half-site of the recognition sequence and comprises an HVR2 region region, and the engineered meganuclease binds to and cleaves a recognition sequence (i.e., the DMD 19-20 recognition sequence) containing SEQ ID NO: 6 within the dystrophin gene. The first subunit binds to the first recognition half-site of the recognition sequence and comprises an HVR1 region. The second subunit binds to the second recognition half-site of the recognition sequence and comprises an HVR2 region. It includes a region. Exemplary DMD 19-20 megonucleases are described below.

[0377] DMD 19-20x.13 (SEQ ID NO: 36)

[0378] In some embodiments, the HVR1 region corresponds to residues 24 to 79 of SEQ ID NO: 36 and has an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity. In some embodiments, the HVR1 region contains residues corresponding to residues 24, 2 6, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 36. In some embodiments, the HVR1 region contains one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 4 6, 68, 70, 75, and 77 of SEQ ID NO: 36. In some embodiments, HV R1 region contains Y, R, K or D in the residue corresponding to residue 66 of SEQ ID NO: 36. In some embodiments, the HVR1 region contains residues 24 to 79 of SEQ ID NO: 36 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or up to 11 amino acid substitutions. In some embodiments, the HVR1 region contains residues 24 to 79 of SEQ ID NO: 36. In some embodiments, the first subunit has an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with residues 7 to 153 of SEQ ID NO: 36. In some

[0379] embodiments, the first subunit contains a residue corresponding to residue 19 of SEQ ID NO: 36 and residues 7 to 153 of SEQ ID NO: 36. In some embodiments, the first subunit contains an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity. In some embodiments, the first subunit contains a residue corresponding to residue 19 of SEQ ID NO: 36 It contains G, S, or A. In some embodiments, the first subunit contains a residue corresponding to residue 19 of SEQ ID NO: 36 In some embodiments, the first subunit contains a residue corresponding to residue 80 of SEQ ID NO: 36 and contains E, Q, or K In some embodiments, the first subunit contains residues 7 - 153 of SEQ ID NO: 36 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 up to 30 amino acid substitutions In some embodiments, the first subunit contains residues 7 - 153 of SEQ ID NO: 36 In some embodiments, the HVR2 region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or higher sequence identity with an amino acid sequence corresponding to residues 215 - 270 of SEQ ID NO: 36

[0380] In some embodiments, the HVR2 region contains one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 23 7, 259, 261, 266, and 268 of SEQ ID NO: 36 In some embodiments, the HVR2 region contains residues corresponding to residues 215, 217, 219, 22 1, 223, 224, 229, 231, 233, 235, 237, 259, 261, 26 6, and 268 of SEQ ID NO: 36 In some embodiments, the HVR2 region contains a residue corresponding to residue 257 of SEQ ID NO: 36 and contains Y, R, K or D In some embodiments, the HVR2 region contains a residue corresponding to residue 241 of SEQ ID NO: 36 In some embodiments, the HVR2 region contains residues corresponding to residues 215, 217, 219, 22 1, 223, 224, 229, 231, 233, 235, 237, 259, 261, 26 6, and 268 of SEQ ID NO: 36 In some embodiments, the HVR2 region contains a residue corresponding to residue 257 of SEQ ID NO: 36 and contains Y, R, K or D In one form, the HVR2 region includes a residue corresponding to residue 263 of SEQ ID NO: 36. In some embodiments, the HVR2 region includes a residue corresponding to residue 264 of SEQ ID NO: 36. In some embodiments, the HVR2 region includes residues 215-270 of SEQ ID NO: 36 having from 1 to 11 amino acid substitutions. In some embodiments, the HVR2 region includes residues 215-270 of SEQ ID NO: 36.

[0381] In some embodiments, the second subunit includes an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to residues 198-344 of SEQ ID NO: 36. In some embodiments, the second subunit includes G, S or A at a residue corresponding to residue 210 of SEQ ID NO: 36. In some embodiments, the second subunit includes E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 36. In some embodiments, the second subunit includes a residue corresponding to residue 271 of SEQ ID NO: 36. In some embodiments, the second subunit includes a residue corresponding to residue 330 of SEQ ID NO: 36. In some embodiments, the second subunit includes residues 198-344 of SEQ ID NO: 36 having from 1 to 30 amino acid substitutions. In some embodiments, the second subunit includes residues 198-344 of SEQ ID NO: 36.

[0382] ​​​In some embodiments, the engineered meganuclease is a single-stranded meganuclease that includes a linker, and the linker co-binds the first subunit and the second subunit. In some embodiments, the engineered meganuclease has an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 36. In some embodiments, the engineered meganuclease includes the amino acid sequence of SEQ ID NO: 36. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 50. In some embodiments,

[0383] DMD 19-20x.87 (SEQ ID NO: 37)

[0384] In some embodiments, the HVR1 region has an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 37. In some embodiments, the HVR1 region includes one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, Residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 4 6, 68, 70, 75, and 77 of SEQ ID NO: 37. In some embodiments, HV The R1 region contains Y, R, K, or D in the residue corresponding to residue 66 of SEQ ID NO: 37. In some embodiments, the HVR1 region contains residues 24-79 of SEQ ID NO: 37 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or up to 11 amino acid substitutions. In some embodiments, the HVR1 region contains residues 24-79 of SEQ ID NO: 37.

[0385] In some embodiments, the first subunit contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with residues 7-153 of SEQ ID NO: 37. In some embodiments, the first subunit contains G, S, or A in the residue corresponding to residue 19 of SEQ ID NO: 37. In some embodiments, the first subunit contains the residue corresponding to residue 19 of SEQ ID NO: 37. In some embodiments, the first subunit contains E, Q, or K in the residue corresponding to residue 80 of SEQ ID NO: 37. In some embodiments the first subunit contains residues 7-153 of SEQ ID NO: 37 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, or up to 30 amino acid substitutions. In some embodiments, the first subunit contains residues 7- 153 of SEQ ID NO: 37. In some embodiments, the first subunit contains residues 7- 153 of SEQ ID NO: 37.

[0386] In some embodiments, the HVR2 region corresponds to residues 215 to 270 of SEQ ID NO: 37 and has at least 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or higher sequence identity with an amino acid sequence comprising. In some embodiments, the HVR2 region is residues 21 5, 217, 219, 221, 223, 224, 229, 231, 233, 235, 23 7, 259, 261, 266, and 268 of SEQ ID NO: 37, including one or more residues corresponding thereto In some embodiments, the HVR2 region is residues 215, 217, 219, 22 1, 223, 224, 229, 231, 233, 235, 237, 259, 261, 26 6, and 268 of SEQ ID NO: 37, including residues corresponding thereto. In some embodiments, the HVR2 region includes a residue corresponding to residue 257 of SEQ ID NO: 37 and contains Y, R, K, or D In some embodiments, the HVR2 region includes a residue corresponding to residue 239 of SEQ ID NO: 37 In some embodiments, the HVR2 region includes a residue corresponding to residue 241 of SEQ ID NO: 37 In some embodiments, the HVR2 region includes a residue corresponding to residue 264 of SEQ ID NO: 37 In some embodiments, the HVR2 region includes residues 215 to 270 of SEQ ID NO: 37 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or up to 11 amino acid substitutions. In some embodiments, the HVR2 region includes residues 215 to 270 of SEQ ID NO: 37 In some embodiments, the second subunit is residues 198 to 344 of SEQ ID NO: 37 and has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96

[0387] % sequence identity with and at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 An amino acid sequence having a sequence identity of 95%, 96%, 97%, 98%, 99% or higher . In some embodiments, the second subunit comprises G, S or A at the residue corresponding to residue 210 of SEQ ID NO: 37 . In some embodiments, the second subunit comprises E, Q, or K at the residue corresponding to residue 271 of SEQ ID NO: 37 . In some embodiments, the second subunit comprises the residue corresponding to residue 330 of SEQ ID NO: 37 . In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 37 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or up to 30 amino acid substitutions . In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 37 . In some embodiments, the engineered meganuclease is a single-chain

[0388] meganuclease comprising a linker, and the linker co-binds the first subunit and the second subunit . In some embodiments, the engineered meganuclease has an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 37 . In some embodiments, the engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 37 . In some embodiments, the engineered meganuclease has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 51 . 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity​ is encoded by a nucleic acid sequence having the property. In some embodiments, the genetically engineered meganuclease is encoded by the nucleic acid sequence set forth in SEQ ID NO: 51.

[0389] DMD 19-20L.249 (SEQ ID NO: 38)

[0390] In some embodiments, the HVR1 region corresponds to residues 24-79 of SEQ ID NO: 38 and has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with an amino acid sequence. In some embodiments, the HVR1 region is residues 24, 2 6, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 38. In some embodiments, the HVR1 region is residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 4 6, 68, 70, 75, and 77 of SEQ ID NO: 38. In some embodiments, HV R1 region contains Y, R, K or D in the residue corresponding to residue 66 of SEQ ID NO: 38. In some embodiments, the HVR1 region contains residues 24-79 of SEQ ID NO: 38 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or up to 11 amino acid substitutions. In some embodiments, the HVR1 region contains residues 24-79 of SEQ ID NO: 38. In some embodiments, the first subunit contains residues 7-153 of SEQ ID NO: 38 and at least

[0391] 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with an amino acid sequence. In some embodiments, the first subunit contains residues 7-153 of SEQ ID NO: 38 and at least In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 38 and containing G, S, or A. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 38. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 38 and containing E, Q, or K. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 38. In some embodiments, the first subunit comprises from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or up to 30 amino acid substitutions of residues 7 - 153 of SEQ ID NO: 38. In some embodiments, the first subunit comprises residues 7 - 153 of SEQ ID NO: 38. In some embodiments, the first subunit comprises residues 7 - 153 of SEQ ID NO: 38.

[0392] In some embodiments, the HVR2 region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with an amino acid sequence corresponding to residues 215 - 270 of SEQ ID NO: 38. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 38. In some embodiments, the HVR2 region comprises residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 38. 5, 217, 219, 221, 223, 224, 229, 231, 233, 235, 23 7, 259, 261, 266, and 268. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 22 1, 223, 224, 229, 231, 233, 235, 237, 259, 261, 26 In some embodiments, the HVR2 region comprises residues corresponding to the sequences In some embodiments, the residue corresponding to residue 257 of sequence number 38 contains Y, R, K, or D. In some embodiments, the HVR2 region contains a residue corresponding to residue 264 of SEQ ID NO: 38. In some embodiments, t...

Claims

1. Engineered meganuclear cells that bind and cleave recognition sequences within the dystrophin gene the engineered meganuclease comprising a first subunit and a second subunit, wherein the first subunit is a first recognition half-site of the recognition sequence. and the second subunit comprises a first hypervariable (HVR1) region and the second subunit binds to the recognition sequence A genetically engineered antibody that binds to a second recognition half-site of the array and contains a second hypervariable (HVR2) region. Meganuclease.

2. The genetically engineered meganuclear of claim 1, wherein the recognition sequence comprises SEQ ID NO:

6. Z.

3. the HVR1 region corresponds to residues 24 to 79 of any one of SEQ ID NOs: 36 to 44; 1 or 2, comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of 2. A genetically engineered meganuclease according to claim 2.

4. The HVR1 region is selected from residues 24, 26, 28, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, Corresponding to 0, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 The genetically engineered nucleotide sequence of any one of claims 1 to 3, comprising one or more residues that Ganuclease.

5. said HVR1 region comprising residues 24-79 of any one of SEQ ID NOs: 36-44. Item 5. The genetically engineered meganuclease according to any one of items 1 to 4.

6. The first subunit comprises at least one amino acid sequence identical to residues 7-153 of any one of SEQ ID NOs: 36-44. Any one of claims 1 to 5, comprising an amino acid sequence having at least 80% sequence identity with 2. A genetically engineered meganuclease as described in claim 1.

7. The first subunit corresponds to residue 19 of any one of SEQ ID NOs: 36 to 44. The engineered meganuclease of any one of claims 1 to 6, comprising a residue.

8. The first subunit is selected from the group consisting of residue 80 of any one of SEQ ID NOs: 38, 39, or 43. The genetically engineered meganucleic acid of any one of claims 1 to 7, comprising residues corresponding to Rease.

9. The first subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 36 to 44. The genetically engineered meganuclease of any one of claims 1 to 8.

10. the HVR2 region corresponds to residues 215 to 270 of any one of SEQ ID NOs: 36 to 44; The amino acid sequence of claim 1 has at least 80% sequence identity with an amino acid sequence 10. The engineered meganuclease of any one of 1 to 9.

11. The HVR2 region is selected from residues 215, 217, 218, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 2 ９、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６ 1, 266, and 268. A genetically engineered meganuclease according to any one of claims 1 to 4.

12. 1 to 3, wherein the HVR2 region comprises a residue corresponding to residue 236 of SEQ ID NO:

39.

1. A genetically engineered meganuclease according to any one of claims 1 to 9.

13. 1 to 3, wherein the HVR2 region comprises a residue corresponding to residue 239 of SEQ ID NO:

37.

3. The genetically engineered meganuclease of any one of claims 2.

14. The HVR2 region comprises a residue corresponding to residue 241 of any one of SEQ ID NOs: 36 to 37.

14. The engineered meganuclease of any one of claims 1 to 13, comprising:

15. 1 to 3, wherein the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO:

36.

5. The engineered meganuclease according to any one of claims 4.

16. The HVR2 region comprises a residue corresponding to residue 264 of any one of SEQ ID NOs: 36 to 44.

16. The engineered meganuclease of any one of claims 1 to 15, comprising:

17. the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 36-44; A genetically engineered meganuclease according to any one of claims 1 to 16.

18. the second subunit is selected from residues 198 to 344 of any one of SEQ ID NOs: 36 to 44 Any of claims 1 to 17, comprising an amino acid sequence having at least 80% sequence identity with A genetically engineered meganuclease according to any one of claims 1 to 4.

19. The second subunit is any one of SEQ ID NOs: 36, 39, 40, 43, or 44. The gene recombinant according to any one of claims 1 to 18, comprising a residue corresponding to residue 271 of The meganuclease created.

20. The second subunit comprises any one of the residues of SEQ ID NOs: 36-38 or 40-44.

20. The genetically engineered peptide of claim 1, comprising a residue corresponding to 330. Meganucleases.

21. the second subunit is selected from residues 198 to 344 of any one of SEQ ID NOs: 36 to 44 21. The engineered meganuclease of any one of claims 1 to 20, comprising:

22. The engineered meganuclease is a single-chain meganuclease containing a linker. and the linker covalently bonds the first subunit and the second subunit.

22. The genetically engineered meganuclease according to any one of claims 1 to 21.

23. An amino acid having at least 80% sequence identity with any one of SEQ ID NOs: 36 to 44. The engineered meganuclease according to any one of claims 1 to 22, comprising the sequence 。

24. Any one of claims 1 to 23, comprising an amino acid sequence of any one of SEQ ID NOs: 36 to 44.

13. The genetically engineered meganuclease of claim 1.

25. At least 80% sequence identity with the nucleic acid sequence set forth in any one of SEQ ID NOs: 60 to 68 The genetic manipulation according to any one of claims 1 to 24, The meganuclease created.

26. 1, encoded by a nucleic acid sequence according to any one of SEQ ID NOs: 60 to 68.

26. The genetically engineered meganuclease of any one of claims 1 to 25.

27. The genetically engineered meganuclear molecule of claim 1, wherein the recognition sequence comprises SEQ ID NO:

10. -ze.

28. the HVR1 region corresponds to residues 24 to 79 of any one of SEQ ID NOs: 45 to 52; 27. The amino acid sequence of claim 26, which has at least 80% sequence identity with the amino acid sequence of 2. A genetically engineered meganuclease as described in claim 1.

29. the HVR1 region comprising residues 24, 26, 28, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 Corresponding to 0, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 29. The engineered meganucleic acid of claim 27 or 28, comprising one or more residues Aze.

30. said HVR1 region comprising residues 24-79 of any one of SEQ ID NOs: 45-52.

30. The genetically engineered meganuclease of any one of claims 27 to 29.

31. The first subunit comprises at least one amino acid sequence identical to residues 7-153 of any one of SEQ ID NOs: 45-52. Any of claims 27 to 30, comprising an amino acid sequence having at least 80% sequence identity.

13. The genetically engineered meganuclease of claim 1.

32. The first subunit corresponds to residue 19 of any one of SEQ ID NOs: 45 to 52. The engineered meganuclear of any one of claims 27 to 31, comprising the residue Z.

33. The first subunit corresponds to residue 80 of any one of SEQ ID NOs: 45 to 51. The engineered meganuclear of any one of claims 27 to 32, comprising the residue Z.

34. The first subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 45 to 52.

34. The genetically engineered meganuclease of any one of claims 27 to 33.

35. the HVR2 region corresponds to residues 215 to 270 of any one of SEQ ID NOs: 45 to 52; The amino acid sequence of claim 1 has at least 80% sequence identity with an amino acid sequence 35. The engineered meganuclease of any one of claims 27 to 34.

36. The HVR2 region is selected from residues 215, 217, 218, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 2 ９、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６ 1, 266, and 268. A genetically engineered meganuclease according to any one of claims 1 to 4.

37. the HVR2 region comprising residues 239, 241, and 264. Engineered meganucleases.

38. 27 to 29, wherein the HVR2 region comprises a residue corresponding to residue 250 of SEQ ID NO:

45.

38. The engineered meganuclease of any one of claims 37.

39. The HVR2 region comprises a residue corresponding to residue 263 of any one of SEQ ID NOs: 45 or 46. The engineered meganuclease according to any one of claims 27 to 38, comprising a group. 。

40. the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 45-52; 40. The engineered meganuclease of any one of claims 27 to 39.

41. the second subunit comprising residues 198 to 344 of any one of SEQ ID NOs: 45 to 52 Any of claims 27 to 40, comprising an amino acid sequence having at least 80% sequence identity with A genetically engineered meganuclease according to any one of claims 1 to 4.

42. The second subunit comprises a residue corresponding to residue 271 of SEQ ID NO:

52.

42. The engineered meganuclease of any one of claims 27 to 41.

43. The second subunit corresponds to residue 330 of any one of SEQ ID NOs: 45 to 52. The engineered meganuclear molecule of any one of claims 27 to 42, comprising a residue -ze.

44. the second subunit comprising residues 198 to 344 of any one of SEQ ID NOs: 45 to 52 44. The engineered meganuclease of any one of claims 27 to 43, comprising:

45. The engineered meganuclease is a single-chain meganuclease containing a linker. and the linker covalently bonds the first subunit and the second subunit.

45. The genetically engineered meganuclease of any one of claims 27 to 44.

46. An amino acid having at least 80% sequence identity with any one of SEQ ID NOs: 45 to 52 The engineered meganuclear of any one of claims 27 to 45, comprising the sequence Z.

47. Any one of claims 27 to 46, comprising an amino acid sequence of any one of SEQ ID NOs: 45 to 52. A genetically engineered meganuclease according to any one of claims 1 to 4.

48. At least 80% sequence identity with the nucleic acid sequence set forth in any one of SEQ ID NOs: 69-76 The gene according to any one of claims 27 to 47, encoded by a nucleic sequence having the formula: Engineered meganucleases.

49. 2. The method according to claim 1, wherein the nucleic acid sequence is encoded by any one of SEQ ID NOs: 69 to 76. 7-48. The engineered meganuclease of any one of claims 7 to 48.

50. The genetically engineered meganuclear molecule of claim 1, wherein the recognition sequence comprises SEQ ID NO:

12. -ze.

51. the HVR1 region corresponds to residues 24 to 79 of any one of SEQ ID NOs: 53 to 59; 50. The amino acid sequence of claim 50, which has at least 80% sequence identity with the amino acid sequence of 2. A genetically engineered meganuclease as described in claim 1.

52. The HVR1 region is selected from residues 24, 26, 28, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, Corresponding to 0, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 52. The genetically engineered megakaryon of claim 50 or claim 51, comprising one or more residues that Nuclease.

53. 5. The method of claim 4, wherein the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO:

54.

3. The genetically engineered meganuclease of any one of claims 2.

54. said HVR1 region comprising residues 24-79 of any one of SEQ ID NOs:53-59.

54. The engineered meganuclease of any one of paragraphs 50 to 53.

55. The first subunit comprises at least one amino acid sequence identical to residues 7-153 of any one of SEQ ID NOs: 53-59. Any of claims 50 to 54, comprising an amino acid sequence having at least 80% sequence identity.

13. The genetically engineered meganuclease of claim 1.

56. The first subunit corresponds to residue 19 of any one of SEQ ID NOs: 53 to 59. The engineered meganuclear of any one of claims 50 to 55, comprising the residue Z.

57. The first subunit corresponds to residue 80 of SEQ ID NO:53-55, 57, or 58. The engineered meganuclear molecule of any one of claims 50 to 56, comprising a residue -ze.

58. The first subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 53 to 59.

58. The genetically engineered meganuclease of any one of claims 50 to 57.

59. the HVR2 region corresponds to residues 215 to 270 of any one of SEQ ID NOs: 53 to 59; The amino acid sequence of claim 1 has at least 80% sequence identity with an amino acid sequence 59. The engineered meganuclease of any one of claims 50 to 58.

60. The HVR2 region is selected from residues 215, 217, 21 ９、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６ 60. Any of claims 50 to 59, comprising one or more residues corresponding to 1, 266, and 268. A genetically engineered meganuclease according to any one of claims 1 to 4.

61. the HVR2 region includes a residue corresponding to residue 239 of SEQ ID NO:53 or SEQ ID NO:55; 61. The genetically engineered meganuclease of any one of claims 50 to 60.

62. The HVR2 region comprises a residue corresponding to residue 241 of any one of SEQ ID NOs: 53 to 55.

62. The engineered meganuclease of any one of claims 50 to 61, comprising:

63. 50 to 55, wherein the HVR2 region comprises a residue corresponding to residue 255 of SEQ ID NO:

55.

63. The engineered meganuclease of any one of claims 62.

64. The HVR2 region comprises a residue corresponding to residue 263 of any one of SEQ ID NOs: 56 to 59.

64. The engineered meganuclease of any one of claims 50 to 63, comprising:

65. The HVR2 region comprises a residue corresponding to residue 264 of any one of SEQ ID NOs: 53 to 59.

65. The engineered meganuclease of any one of claims 50 to 64, comprising:

66. the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 53-59; 66. The engineered meganuclease of any one of claims 50 to 65.

67. the second subunit comprising residues 198 to 344 of any one of SEQ ID NOs:53 to 59 Any of claims 60 to 66, comprising an amino acid sequence having at least 80% sequence identity with A genetically engineered meganuclease according to any one of claims 1 to 4.

68. The second subunit comprises residue 271 of any one of SEQ ID NOs: 53, or 55-59. The genetically engineered megakaryon according to any one of claims 50 to 67, comprising a residue corresponding to Nuclease.

69. The second subunit corresponds to residue 330 of any one of SEQ ID NOs: 54 to 59. The engineered meganuclear molecule of any one of claims 50 to 68, comprising a residue -ze.

70. the second subunit comprising residues 198 to 344 of any one of SEQ ID NOs:53 to 59 70. The engineered meganuclease of any one of claims 50 to 69, comprising:

71. The engineered meganuclease is a single-chain meganuclease containing a linker. and the linker covalently bonds the first subunit and the second subunit.

71. The genetically engineered meganuclease of any one of claims 50 to 70.

72. An amino acid having at least 80% sequence identity with any one of SEQ ID NOs: 53 to 59. The engineered meganuclear of any one of claims 50 to 71, comprising the sequence Z.

73. Any one of claims 50 to 72, comprising an amino acid sequence of any one of SEQ ID NOs: 53 to 59. A genetically engineered meganuclease according to any one of claims 1 to 4.

74. At least 80% sequence identity with the nucleic acid sequence set forth in any one of SEQ ID NOs: 77 to 83 The gene according to any one of claims 50 to 73, encoded by a nucleic sequence having the formula: Engineered meganucleases.

75. 5. The method according to claim 5, wherein the nucleic acid sequence is encoded by any one of SEQ ID NOs: 77 to 83.

75. The engineered meganuclease of any one of claims 0-74.

76. The engineered meganuclease according to any one of claims 1 to 75 is A polynucleotide comprising a nucleic acid sequence that encodes ...

77. 77. The polynucleotide of claim 76, wherein the polynucleotide is an mRNA.

78. The engineered meganuclease according to any one of claims 1 to 75 is A recombinant DNA construct comprising a polynucleotide comprising a nucleic acid sequence encoding a polypeptide of the present invention.

79. The recombinant DNA construct encodes a recombinant virus comprising the polynucleotide.

79. The recombinant DNA construct of claim 78.

80. The recombinant virus may be a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, 80. The recombinant vector of claim 79, which is a virus or a recombinant adeno-associated virus (AAV). DNA constructs.

81. The recombinant virus of claim 79 or claim 80 is a recombinant AAV. DNA constructs.

82. 9. The recombinant AAV of claim 8, wherein the recombinant AAV has a rh.74 capsid or an AAV9 capsid.

1. The recombinant DNA construct according to claim 1.

83. The polynucleotide is a nucleic acid encoding the engineered meganuclease.

83. The method according to any one of claims 78 to 82, comprising a promoter operably linked to the nucleic acid sequence. The recombinant DNA constructs described above.

84. 84. The recombinant DNA construct of claim 83, wherein the promoter is a muscle-specific promoter. thing.

85. The muscle-specific promoter is an MCK promoter, a C5-12 promoter, a spc5- 12 promoter, MHCK7 promoter, CK8 promoter, SK-CRM4 promoter , SP-301 promoter, SP-817 promoter, or SP-905 promoter.

85. The recombinant DNA construct of claim 84.

86. The engineered meganuclease according to any one of claims 1 to 75 is A recombinant virus comprising a polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising the polypeptide of claim 1.

87. The recombinant virus may be a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, The recombinant virus of claim 86, which is a virus or a recombinant AAV.

88. 88. The recombinant virus of claim 87, wherein the recombinant virus is a recombinant AAV.

89. 9. The recombinant AAV of claim 8, wherein the recombinant AAV has a rh.74 capsid or an AAV9 capsid. 7 or 88. A recombinant virus as described in claim 88.

90. The polynucleotide is a nucleic acid encoding the engineered meganuclease.

90. The method according to any one of claims 86 to 89, comprising a promoter operably linked to the nucleic acid sequence. The recombinant virus described above.

91. 91. The recombinant virus of claim 90, wherein the promoter is a muscle-specific promoter.

92. The muscle-specific promoter is an MCK promoter, a C5-12 promoter, a spc5- 12 promoter, MHCK7 promoter, CK8 promoter, SK-CRM4 promoter , SP-301 promoter, SP-817 promoter, or SP-905 promoter. The recombinant virus of claim 91.

93. A lipid nanoparticle composition comprising lipid nanoparticles containing a polynucleotide, the polynucleotide comprising The nucleotide is the genetically engineered meganucleotide of any one of claims 1 to 75. A lipid nanoparticle composition comprising a nucleic acid sequence encoding an ase.

94. The lipid nanoparticle composition of claim 93, wherein the polynucleotide is mRNA.

95. A pharma- ceutically acceptable carrier and the genetically engineered and a pharmaceutical composition comprising the meganuclease.

96. 78. The polynucleotide of claim 76 or claim 77, together with a pharma- ceutically acceptable carrier. and a pharmaceutical composition comprising:

97. A pharma- ceutically acceptable carrier and the recombinant DNA of any one of claims 78 to 85. A pharmaceutical composition comprising the A construct.

98. A pharma- ceutically acceptable carrier and the recombinant virus according to any one of claims 86 to 92. A pharmaceutical composition comprising:

99. 95. The lipid nanoparticle composition of claim 93 or 94, together with a pharma- ceutically acceptable carrier. and a pharmaceutical composition comprising the product.

100. A first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease. a polynucleotide comprising a second nucleic acid sequence encoding an engineered meganuclease. Thus, the first engineered meganuclease is according to any one of claims 2 to 26. The genetically engineered meganuclease according to claim 1, wherein the second genetically engineered meganuclease The nuclease is a nuclease comprising the genetically engineered meganuclease according to any one of claims 27 to 49. or the engineered meganucleotide according to any one of claims 50 to 75. A polynucleotide that is an ase.

101. The polynucleotide of any one of claims 100 to 120, wherein the polynucleotide is an mRNA. Cleotide.

102. The first and second nucleic acid sequences are separated by an IRES or 2A sequence.

102. The polynucleotide of claim 100 or claim 101,

103. A recombinant DNA construct comprising the polynucleotide of claim 100.

104. The first nucleic acid sequence and the second nucleic acid sequence are separated by an IRES or 2A sequence. The recombinant DNA construct of claim 103.

105. The polynucleotide is operably linked to the first nucleic acid sequence and the second nucleic acid sequence.

104. The recombinant DNA construct of claim 103, comprising an attached promoter.

106. 106. The recombinant DNA construct of claim 105, wherein the promoter is a muscle-specific promoter. building.

107. The muscle-specific promoter is an MCK promoter, a C5-12 promoter, a spc5- 12 promoter, MHCK7 promoter, CK8 promoter, SK-CRM4 promoter , SP-301 promoter, SP-817 promoter, or SP-905 promoter. The recombinant DNA construct of claim 106.

108. The polynucleotide comprises a first promoter operably linked to the first nucleic acid sequence. and a second promoter operably linked to the second nucleic acid sequence.

3. A recombinant DNA construct according to claim 3.

109. The first promoter and the second promoter are muscle-specific promoters.

109. The recombinant DNA construct of paragraph 108.

110. The muscle-specific promoter is an MCK promoter, a C5-12 promoter, a spc5- 12 promoter, MHCK7 promoter, CK8 promoter, SK-CRM4 promoter , SP-301 promoter, SP-817 promoter, SP-905 promoter, or the like The recombinant DNA construct of claim 109, comprising any of these combinations.

111. The recombinant DNA construct encodes a recombinant virus comprising the polynucleotide. A recombinant DNA construct according to any one of claims 103 to 110.

112. The recombinant virus may be a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, 112. The recombinant vector according to claim 111, which is a virus or a recombinant adeno-associated virus (AAV). DNA constructs.

113. The method of claim 111 or claim 112, wherein the recombinant virus is a recombinant AAV. Recombinant DNA constructs.

114. 1 , wherein the recombinant AAV has a rh.74 capsid or an AAV9 capsid.

14. The recombinant DNA construct according to claim 13.

115. A recombinant virus comprising the polynucleotide of claim 100.

116. The polynucleotide is operably linked to the first nucleic acid sequence and the second nucleic acid sequence.

116. The recombinant virus of claim 115, comprising an ligated promoter.

117. The first nucleic acid sequence and the second nucleic acid sequence are separated by an IRES or 2A sequence.

117. The recombinant virus of claim 115 or claim 116.

118. 118. The method of claim 116 or claim 117, wherein the promoter is a muscle-specific promoter. Recombinant viruses.

119. The muscle-specific promoter is an MCK promoter, a C5-12 promoter, a spc5- 12 promoter, MHCK7 promoter, CK8 promoter, SK-CRM4 promoter , SP-301 promoter, SP-817 promoter, or SP-905 promoter. The recombinant virus of claim 118.

120. The polynucleotide comprises a first promoter operably linked to the first nucleic acid sequence. and a second promoter operably linked to the second nucleic acid sequence.

5. A recombinant virus according to claim 5.

121. The first promoter and the second promoter are muscle-specific promoters.

121. The recombinant virus of claim 120.

122. The muscle-specific promoter is an MCK promoter, a C5-12 promoter, a spc5- 12 promoter, MHCK7 promoter, CK8 promoter, SK-CRM4 promoter , SP-301 promoter, SP-817 promoter, SP-905 promoter, or the like The recombinant virus of claim 121, comprising a combination thereof.

123. The recombinant virus may be a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, Any of claims 115 to 122, which is a virus or a recombinant adeno-associated virus (AAV). A recombinant virus described in any one of claims 1 to 4.

124. 124. The method according to any one of claims 115 to 123, wherein the recombinant virus is a recombinant AAV. The recombinant virus described.

125. 1 , wherein the recombinant AAV has a rh.74 capsid or an AAV9 capsid. 23 or 125. A recombinant virus as described in claim 124.

126. A lipid nanoparticle composition comprising lipid nanoparticles comprising the polynucleotide of claim 100. Composition.

127. The polynucleotide is the mRNA of claim 101 or claim 102.

127. The lipid nanoparticle composition of claim 126.

128. The polynucleotide is the recombinant D according to any one of claims 103 to 114. The lipid nanoparticle composition of claim 126, which is a NA construct.

129. A pharma- ceutically acceptable carrier, a first engineered meganuclease, and a second gene A pharmaceutical composition comprising a genetically engineered meganuclease, the first genetically engineered meganuclease comprising: The meganuclease is a gene encoding the engineered meganuclease according to any one of claims 2 to 26. nuclease, and the second engineered meganuclease is any one of claims 27 to 49. The engineered meganuclease according to any one of claims 50 to 75 or A pharmaceutical composition, which is the engineered meganuclease described in any one of claims 1 to 4.

130. A pharma- ceutically acceptable carrier and the polynucleotide according to any one of claims 100 to 102. A pharmaceutical composition comprising:

131. The polynucleotide comprises the mRNA of claim 101 or claim 102.

131. The pharmaceutical composition of claim 130.

132. The polynucleotide is the recombinant D according to any one of claims 103 to 114. The pharmaceutical composition of claim 130, comprising a NA construct.

133. 13. The recombinant virus of claim 115, comprising the recombinant virus of claim 125.

10. The pharmaceutical composition according to claim 0.

134. The lipid nanoparticle composition according to any one of claims 126 to 128, 130. The pharmaceutical composition according to claim 130.

135. 1. A method for producing a genetically modified eukaryotic cell comprising a modified dystrophin gene, comprising: A first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease. One or more polynucleotides comprising a second nucleic acid sequence encoding an engineered meganuclease. introducing the nucleotide into a eukaryotic cell; The first engineered meganuclease is according to any one of claims 2 to 26. and the second engineered meganuclease. The engineered meganuclear enzyme according to any one of claims 27 to 49 is or said second engineered meganuclease is a meganuclease according to any one of claims 50 to 75.

2. The engineered meganuclease according to claim 1 , The first engineered meganuclease and the second engineered meganuclease a nuclease is expressed in said eukaryotic cell; The first engineered meganuclease has a recognition sequence comprising SEQ ID NO:6 A first cleavage site is generated in the dystrophin gene, and the second genetically engineered The ganucleases recognize the dystrophy at a recognition sequence comprising SEQ ID NO: 10 or SEQ ID NO:

12. creating a second cleavage site in the fin gene; the first cleavage site and the second cleavage site have complementary 3' overhangs; The intervening genomic DNA between the first and second cleavage sites is Cut out from the fin gene, The dystrophin gene is annealed to generate the modified dystrophin gene. The way it is done.

136. the complementary 3' overhangs of the first cleavage site and the second cleavage site are The method of claim 135, wherein the ligation is directly re-ligated to

137. The dystrophin gene comprises the nucleic acid sequence set forth in SEQ ID NO: 32 or 34. The method of claim 135 or claim 136.

138. The normal reading frame is similar to that of the full-length wild-type dystrophin gene. Any one of claims 135 to 137, which is restored in a modified dystrophin gene. The method described above.

139. The modified dystrophin gene is a mutant of exons 45 to 55 of a wild-type dystrophin gene. A modified dystrophin polypeptide encoding a modified dystrophin polypeptide lacking the amino acid encoded by The method according to any one of claims 135 to 138.

140. The method further comprises: providing a first nucleic acid sequence encoding the first engineered meganuclease; A first polynucleotide comprising a sequence and said second engineered meganuclease. and introducing into said eukaryotic cell a second polynucleotide comprising a second nucleic acid sequence that encodes said The method of any one of claims 135 to 139, comprising:

141. 141. The method of claim 140, wherein the first polynucleotide is a first mRNA.

142. 140 or 141, wherein the second polynucleotide is a second mRNA. The method described above.

143. The first mRNA and / or the second mRNA is the mRNA of claim 77. The method of claim 141 or claim 142,

144. 141. The method of claim 140, wherein the first polynucleotide is a first recombinant DNA construct. How to.

145. 140. The method of claim 140, wherein the second polynucleotide is a second recombinant DNA construct.

44. The method according to claim 44.

146. The first recombinant DNA construct and / or the second recombinant DNA construct are selected from the group consisting of: 144 or claim 145, which is the recombinant DNA construct according to any one of claims 8 to 85. Item 146. The method according to item 145.

147. The first polynucleotide and the second polynucleotide are linked to one or more lipids. The method according to any one of claims 140 to 146, which is introduced into the eukaryotic cell by nanoparticles. Method of posting.

148. The first polynucleotide is introduced into the eukaryotic cell by a first lipid nanoparticle. The method of any one of claims 140 to 147,

149. The second polynucleotide is introduced into the eukaryotic cell by a second lipid nanoparticle. The method according to any one of claims 140 to 148,

150. The first polynucleotide is introduced into the eukaryotic cell by a first recombinant virus. The method according to any one of claims 140 to 143,

151. The second polynucleotide is introduced into the eukaryotic cell by a second recombinant virus. The method according to any one of claims 140 to 143 and 150,

152. The first recombinant virus and / or the second recombinant virus is a recombinant virus of any one of claims 86 to 92.

152. The recombinant virus of claim 150 or claim 151. Method of posting.

153. The method further comprises: providing a first nucleic acid encoding the first engineered meganuclease; and a second nucleic acid sequence encoding the second engineered meganuclease.

136. The method of claim 135, comprising introducing a nucleotide into the eukaryotic cell.

154. 154. The method of claim 153, wherein the polynucleotide is mRNA.

155. 1, wherein the mRNA is the mRNA of claim 101 or claim 102.

54. The method according to claim 54.

156. 154. The method of claim 153, wherein the polynucleotide is a recombinant DNA construct.

157. The recombinant DNA construct according to any one of claims 103 to 114. The method of claim 156, which is a DNA construct.

158. 13. The method of claim 1, wherein the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle. 53-157. A method according to any one of claims 53 to 157.

159. The polynucleotide is introduced into the eukaryotic cell by a recombinant virus.

153. The method according to claim 153.

160. The recombinant virus according to any one of claims 115 to 125. The method of claim 159, wherein the

161. The method according to any one of claims 135 to 160, wherein the eukaryotic cell is a mammalian cell. Law.

162. 162. The method of claim 161, wherein the mammalian cell is a muscle cell.

163. 163. The method of claim 162, wherein the muscle cell is a muscle progenitor cell, a skeletal muscle cell, or a cardiac muscle cell. How to.

164. The method according to any one of claims 161 to 163, wherein the mammalian cell is a human cell. Law.

165. A method for modifying a dystrophin gene in a target cell of a subject, comprising: The gene alters the reading frame of the dystrophin gene from the wild type. The method is characterized by a mutation, A first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease. One or more polynucleotides comprising a second nucleic acid sequence encoding an engineered meganuclease. delivering a nucleotide to said target cell; The first engineered meganuclease is according to any one of claims 2 to 26. and the second engineered meganuclease. The engineered meganuclear enzyme according to any one of claims 27 to 49 is or said second engineered meganuclease is a meganuclease according to any one of claims 50 to 75.

2. The engineered meganuclease according to claim 1 , The first engineered meganuclease and the second engineered meganuclease a nuclease is expressed in the target cell; The first engineered meganuclease has a recognition sequence comprising SEQ ID NO:6 A first cleavage site is generated in the dystrophin gene, and the second genetically engineered The ganucleases recognize the dystrophy at a recognition sequence comprising SEQ ID NO: 10 or SEQ ID NO:

12. creating a second cleavage site in the fin gene; the first cleavage site and the second cleavage site have complementary 3' overhangs; The intervening genomic DNA between the first and second cleavage sites is Cut out from the fin gene, the dystrophin gene is annealed; The normal reading frame of the dystrophin gene is a full-length, wild-type dystrophin gene. The method is as follows:

166. the complementary 3' overhangs of the first cleavage site and the second cleavage site are The method of claim 165, wherein the ligation is directly re-ligated to

167. The dystrophin gene comprises the nucleic acid sequence set forth in SEQ ID NO: 32 or 34. The method of claim 165 or claim 166.

168. The dystrophin gene is a mutant of the wild-type dystrophin gene, The modified dystrophin polypeptide encoding the dystrophin polypeptide of claim 1 is characterized in that it lacks the amino acids encoded by 168. The method according to any one of claims 165 to 167.

169. Any of claims 165 to 168, wherein the subject is converted to the Becker muscular dystrophy phenotype. The method according to any one of claims 1 to 5.

170. The method further comprises: and a second polynucleotide encoding the second engineered meganuclease. and delivering to said target cell a second polynucleotide comprising a second nucleic acid sequence that The method according to any one of claims 165 to 169.

171. 171. The method of claim 170, wherein the first polynucleotide is a first mRNA.

172. 170 or 171, wherein the second polynucleotide is a second mRNA. The method according to

173. The first mRNA and / or the second mRNA is the mRNA of claim 77. The method of claim 171 or claim 172,

174. 171. The method of claim 170, wherein the first polynucleotide is a first recombinant DNA construct. How to.

175. 170. The method of claim 170, wherein the second polynucleotide is a second recombinant DNA construct. The method of claim 174.

176. The first recombinant DNA construct and / or the second recombinant DNA construct are selected from the group consisting of: 174 or claim 175, which is the recombinant DNA construct according to any one of claims 8 to 85.

176. The method according to claim 175.

177. The first polynucleotide and the second polynucleotide are linked to one or more lipids. The method according to any one of claims 170 to 176, which is delivered to the target cells by nanoparticles. Method of posting.

178. The first polynucleotide is delivered to the target cell by a first lipid nanoparticle. The method of any one of claims 170 to 177,

179. The second polynucleotide is delivered to the target cell by a second lipid nanoparticle. The method of any one of claims 170 to 178,

180. The first polynucleotide is delivered to the target cell by a first recombinant virus. The method according to any one of claims 170 to 173,

181. The second polynucleotide is delivered to the target cell by a second recombinant virus. The method according to any one of claims 170 to 173 and 180,

182. The first recombinant virus and / or the second recombinant virus is a recombinant virus of any one of claims 86 to 82.

182. The recombinant virus of claim 180 or claim 181. Method of posting.

183. The method further comprises: providing a first nucleic acid encoding the first engineered meganuclease; and a second nucleic acid sequence encoding the second engineered meganuclease.

166. The method of claim 165, comprising delivering a nucleotide to the target cell.

184. 184. The method of claim 183, wherein the polynucleotide is mRNA.

185. The mRNA of claim 184 is the mRNA of claim 100 or 101. The method described.

186. 184. The method of claim 183, wherein the polynucleotide is a recombinant DNA construct.

187. The recombinant DNA construct according to any one of claims 102 to 113. The method of claim 186, which is a DNA construct.

188. 13. The method of claim 1, wherein the polynucleotide is delivered to the target cell by a lipid nanoparticle.

83. The method according to claim 83.

189. The polynucleotide is delivered to the target cell by a recombinant virus.

183. The method according to claim 183.

190. The recombinant virus according to any one of claims 115 to 125. The method of claim 189, wherein the

191. The method of any one of claims 165 to 190, wherein the subject is a mammal.

192. 192. The method of any one of claims 165 to 191, wherein the target cell is a muscle cell.

193. 193. The method of claim 192, wherein the muscle cell is a muscle progenitor cell, a skeletal muscle cell, or a cardiac muscle cell. How to.

194. The method of any one of claims 165 to 193, wherein the subject is a human.

195. A method of treating Duchenne muscular dystrophy (DMD) in a subject in need of such treatment. Thus, the DMD is characterized by a decreased expression of the dystrophin gene compared to a full-length wild-type dystrophin gene. It is characterized by a mutation in the dystrophin gene that changes the reading frame of the gene. , the method comprising: A first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease. an effective amount of one or more nucleic acids comprising a second nucleic acid sequence encoding an engineered meganuclease; administering to the subject a polynucleotide of The first engineered meganuclease is according to any one of claims 2 to 26. and the second engineered meganuclease. The enzyme is an engineered meganuclease according to any one of claims 27 to 49. or said second engineered meganuclease is any of claims 50 to 75.

2. The engineered meganuclease according to claim 1 , the one or more polynucleotides are delivered to a target cell of the subject; The first engineered meganuclease and the second engineered meganuclease a nuclease is expressed in the target cell; The first engineered meganuclease has a recognition sequence comprising SEQ ID NO:6 A first cleavage site is generated in the dystrophin gene, and the second genetically engineered The ganucleases recognize the dystrophy at a recognition sequence comprising SEQ ID NO: 10 or SEQ ID NO:

12. creating a second cleavage site in the fin gene; the first cleavage site and the second cleavage site have complementary 3' overhangs; The intervening genomic DNA between the first and second cleavage sites is Cut out from the fin gene, the dystrophin gene is annealed; The normal reading frame of the dystrophin gene is a full-length, wild-type dystrophin gene. The method is as follows:

196. the complementary 3' overhangs of the first cleavage site and the second cleavage site are The method of claim 195, wherein the nucleic acid is directly religated to the nucleic acid.

197. The dystrophin gene comprises the nucleic acid sequence set forth in SEQ ID NO: 32 or 34. The method of claim 195 or claim 196.

198. The dystrophin gene is a mutant of the wild-type dystrophin gene, The modified dystrophin polypeptide encoding the dystrophin polypeptide of claim 1 is characterized in that it lacks the amino acids encoded by 198. The method of any one of claims 195 to 197.

199. Any of claims 195 to 198, wherein the subject is converted to a Becker muscular dystrophy phenotype. The method according to any one of claims 1 to 5.

200. The method further comprises: and a second polynucleotide encoding the second engineered meganuclease. administering to the subject a second polynucleotide comprising a second nucleic acid sequence that binds to the polypeptide. The method of any one of claims 195 to 199.

201. 201. The method of claim 200, wherein the first polynucleotide is a first mRNA.

202. 200 or 201, wherein the second polynucleotide is a second mRNA. The method described above.

203. The first mRNA and / or the second mRNA is the mRNA of claim 77. The method of claim 201 or claim 202,

204. 201. The method of claim 200, wherein the first polynucleotide is a first recombinant DNA construct. How to.

205. The method of claim 200, wherein the second polynucleotide is a second recombinant DNA construct. The method of claim 204.

206. The first recombinant DNA construct and / or the second recombinant DNA construct are selected from the group consisting of: 204 or claim 205, which is the recombinant DNA construct according to any one of claims 8 to 85. The method according to paragraph 205.

207. The first polynucleotide and the second polynucleotide are delivered by lipid nanoparticles. The method of any one of claims 200 to 206, wherein the method is administered to the subject via a

208. The first polynucleotide is administered to the subject via a first lipid nanoparticle.

208. The method of any one of claims 200 to 207.

209. The second polynucleotide is administered to the subject via a second lipid nanoparticle.

209. The method of any one of claims 200 to 208.

210. the first polynucleotide is administered to the subject via a first recombinant virus; 204. The method of any one of claims 200 to 203.

211. the second polynucleotide is administered to the subject via a second recombinant virus; 211. The method of any one of claims 200 to 203 and 210.

212. The first recombinant virus and / or the second recombinant virus is a recombinant virus according to any one of claims 85 to 91.

212. The recombinant virus of claim 210 or 211. Method of posting.

213. The method further comprises: providing a first nucleic acid encoding the first engineered meganuclease; and a second nucleic acid sequence encoding the second engineered meganuclease.

196. The method of claim 195, comprising administering a nucleotide to the subject.

214. The method of claim 213, wherein the polynucleotide is mRNA.

215. 2. The mRNA of claim 101 or claim 102.

15. The method according to claim 14.

216. 214. The method of claim 213, wherein the polynucleotide is a recombinant DNA construct.

217. The recombinant DNA construct according to any one of claims 103 to 114. The method of claim 216, which is a DNA construct.

218. 213. The method of claim 213, wherein the polynucleotide is administered to the subject by a lipid nanoparticle. The method according to

219. 22. The method of claim 21, wherein the polynucleotide is administered to the subject by a recombinant virus.

3. The method according to claim 3.

220. The recombinant virus according to any one of claims 115 to 125. The method of claim 219,

221. The method of any one of claims 195 to 220, wherein the subject is a mammal.

222. 222. The method of any one of claims 195 to 221, wherein the target cell is a muscle cell.

223. 223. The method of claim 222, wherein the muscle cell is a muscle progenitor cell, a skeletal muscle cell, or a cardiac muscle cell. How to.

224. The method of any one of claims 195 to 223, wherein the subject is a human.

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