Muscle-specific expression cassette

Abstract

This disclosure encompasses nucleic acid expression constructs that modulate the expression of heterologous proteins, particularly in muscle tissue (e.g., skeletal muscle and cardiac muscle). In specific embodiments of this disclosure, muscle-specific expression constructs encoding heterologous proteins are described. These heterologous proteins may be any proteins that are desirable to be expressed in muscle cells. In specific embodiments of this disclosure, proteins for the treatment of Duchenne muscular dystrophy (e.g., artificial nucleases such as artificial meganucleases) are described.

Description

[Technical Field]

[0001] This disclosure relates to the fields of molecular biology and recombinant nucleic acid technology. In certain embodiments, this disclosure relates to expression constructs that regulate the expression of heterologous proteins in muscle tissue (e.g., skeletal muscle and cardiac muscle). Such muscle-specific expression constructs are useful for the expression of heterologous proteins, such as artificial nucleases, for the treatment of muscle disorders (e.g., Duchenne muscular dystrophy).

[0002] References to sequence listings submitted as XML (ST.26) files via the United States Patent and Trademark Office (USPTO) Patent Center. This application includes a sequence listing filed in XML (ST.26) format via the United States Patent and Trademark Office (USPTO) Patent Center, which is incorporated herein by reference in its entirety. The XML (ST.26) copy, created on 11 April 2024, is named "PBIO-074PCT Seq List.xml" and has a size of 186,779 bytes. [Background technology]

[0003] Gene therapy is a potentially curative approach to muscle disorders caused by known gene mutations. However, to date, gene therapy approaches have not been successful due to the difficulty in delivering and expressing therapeutic proteins in muscle tissue in vivo. One such muscle disorder, Duchenne muscular dystrophy (DMD), is a rare X-linked muscle degeneration disease affecting approximately 1 in 3,500 boys worldwide. This disease is caused by mutations in the dystrophin gene, the largest known gene. The dystrophin gene occupies 2.2 Mb of the X chromosome and primarily encodes a 14 kb transcript derived from 79 exons. The full-length dystrophin protein, expressed in skeletal muscle, smooth muscle, and cardiac muscle, consists of 3,685 amino acids and has a molecular weight of 427 kD. Severe Duchenne phenotypes are generally associated with the loss of full-length dystrophin protein from skeletal muscle and cardiac muscle, which leads to debilitating muscle degeneration and ultimately heart failure. Numerous different dystrophin gene mutations have been described, many of which result in either severe DMD or mild Becker muscular dystrophy.

[0004] Several gene therapy strategies are being pursued for the treatment of DMD, which require the specific expression of heterologous proteins in muscle tissue. One strategy involves "gene replacement" (Non-Patent Literature 1; Non-Patent Literature 2; Non-Patent Literature 3; Non-Patent Literature 4). This approach requires the delivery of a functional copy of the dystrophin gene, such as "microdystrophin," to the patient using a viral delivery vector, typically adeno-associated virus (AAV).

[0005] Another strategy involves "gene editing" to correct gene expression at the genomic DNA level or to cause exon skipping so that harmful mutations in the DMD gene (e.g., out-of-frame exons) are not present in the mRNA. These approaches utilize artificial nucleases to excise portions of the mutant dystrophin gene or to edit individual parts of the gene. Such artificial nucleases include clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 enzymes, transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs), artificial meganucleases, CRISPR Cas9 base editors, and CRISPR Cas9 prime editors (Non-Patent Document 5). Another approach is to utilize multiple meganucleases to excise specific exons from the dystrophin coding sequence. This strategy is described in Patent Document 1.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0008] Each of these gene therapy strategies requires the expression of a heterologous protein in a patient. In the case of DMD, the therapeutic heterologous protein must be expressed in muscle tissue in order to have any therapeutic effect. In fact, in the case of gene editing, the expression of the artificial nuclease protein must be carefully controlled with respect to the muscle tissue in which gene editing is desired. Such muscle tissue affinity can be influenced by utilizing muscle tissue-affinity AAV. A secondary level of expression control can result from the use of tissue-specific promoters. However, prior to the present disclosure, the precise control of heterologous gene expression in muscle tissue had not been fully achieved to provide therapeutically relevant muscle protein expression levels. [Means for Solving the Problems]

[0009] This disclosure provides muscle-specific expression cassettes for specifically regulating gene expression in muscle tissue, including skeletal and cardiac muscle, for treating muscle disorders. In certain embodiments, the disclosure describes muscle-specific cassettes encoding artificial nucleases (e.g., artificial meganucleases) that bind to and cleave recognition sequences in the dystrophin gene (e.g., the human dystrophin gene), as well as compositions comprising such artificial nucleases and methods of use thereof. In some embodiments, the disclosure herein discloses muscle-specific expression cassettes encoding pairs of artificial nucleases (e.g., artificial meganucleases) used to remove multiple exons from the dystrophin gene by generating a first cleavage site in an intron upstream of a first exon and a second cleavage site in an intron downstream of a second exon. In certain examples described herein, the first cleavage site is generated in intron 5' upstream of exon 45 of the dystrophin gene, while the second cleavage site is generated in intron 3' downstream of exon 55. This process allows for the excision and removal of exons 45–55 from the dystrophin gene after annealing of the two cleavage sites and repair of the genome. The recognition sequences targeted by the disclosed artificial meganuclease are selected to have an identical 4-base pair central sequence such that the first and second cleavage sites have complementary 4-base pair 3' overhangs that can fully ligate each other (i.e., each base pair in one overhang pairs with its complement on the other overhang). By removing exons 45–55 from mutant dystrophin genes lacking one or more of these exons, 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 shortened, modified dystrophin protein in which the central spectrin repeat domain is absent, but the amino(N) and carboxy(C) terminal domains are intact. This often reduces the severity of the disease. In some cases, this results in a milder Becker phenotype.While the specific embodiments and examples disclosed herein relate to the strategies described above for the treatment of DMD, these muscle-specific expression cassettes are envisioned to be useful for the treatment of any muscle disorder in which the expression of xenotransgenes in muscle tissue is desired. Specific embodiments of this disclosure are provided and summarized below.

[0010] Accordingly, one aspect of the disclosure described herein is a muscle-specific expression cassette comprising a nucleic acid sequence encoding a heterologous protein operably linked to a muscle-specific promoter. In some embodiments described herein, the muscle-specific promoter is a muscle creatine kinase (MCK) promoter.

[0011] In some embodiments, the muscle-specific expression cassette described herein includes a muscle-specific enhancer. In some embodiments, the muscle-specific expression cassette described herein includes a Kozak sequence. In some embodiments, the muscle-specific expression cassette described herein includes a post-transcriptional regulatory element. In some embodiments, the post-transcriptional regulatory element includes a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0012] In some embodiments of the muscle-specific expression cassettes described herein, the muscle-specific enhancer comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 1.

[0013] In some embodiments of the muscle-specific expression cassettes described herein, the MCK promoter is a cleaved MCK (tMCK) promoter. In some embodiments, the tMCK promoter comprises a base promoter and one or more MCK enhancer elements. In some embodiments, the base promoter comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 3. In some embodiments, the base promoter comprises a nucleic acid sequence with the sequence shown in Sequence ID No. 3.

[0014] In some embodiments of the muscle-specific expression cassettes described herein, the MCK enhancer element includes an MCK-R control element. In some embodiments, the MCK enhancer element includes two MCK-R control elements. In some embodiments, the MCK-R control element includes a nucleic acid sequence represented by any one of sequence numbers 4 to 17.

[0015] In some embodiments of the muscle-specific expression cassettes described herein, the MCK enhancer element comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 21.

[0016] In some embodiments of the muscle-specific expression cassettes described herein, the MCK promoter is a tMCK promoter comprising three MCK enhancer elements.

[0017] In some embodiments of the muscle-specific expression cassettes described herein, the MCK promoter is a tMCK promoter comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 22.

[0018] In some embodiments of the muscle-specific expression cassettes described herein, the heterologous protein is a nucleoprotein. In some embodiments, the heterologous protein includes a nuclear localization sequence (NLS). In some embodiments, the NLS is located at the N-terminus of the heterologous protein. In some embodiments, the NLS is located at the C-terminus of the heterologous protein. In some embodiments, the heterologous protein includes a first NLS at the N-terminus and a second NLS at the C-terminus. In some embodiments, the first and second NLS are identical. In some embodiments, the first and second NLS are not identical. In some embodiments, the NLS includes SV40 NLS, c-myc NLS, or NLS5 NLS. In some embodiments, the NLS comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs.

[0019] In some embodiments, the SV40 NLS sequence includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 106. In some embodiments, the SV40 NLS sequence includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 107.

[0020] In some embodiments, the c-myc NLS sequence includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 108. In some embodiments, the c-myc NLS sequence includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 109.

[0021] In some embodiments, the SV40 NLS sequence includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 110. In some embodiments, the SV40 NLS sequence includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 111.

[0022] In some embodiments, the c-myc NLS sequence includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 112. In some embodiments, the c-myc NLS sequence includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 113.

[0023] In some embodiments of the muscle-specific expression cassette described herein, the expression cassette comprises a first nucleic acid sequence encoding a first heterologous protein and a second nucleic acid sequence encoding a second heterologous protein. In some embodiments, the first and second nucleic acid sequences are separated by an IRES or 2A sequence. In some embodiments, the 2A sequence further comprises a Fuhrin cleavage motif and a GSG linker sequence. In some embodiments, the 2A sequence is a T2A, P2A, E2A, or F2A sequence. In some embodiments, the 2A sequence further comprises a Fuhrin cleavage motif and a GSG linker sequence and comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 103. In some embodiments, the 2A sequence, comprising the Fuhrin cleavage motif and the GSG linker sequence, comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 104. In some embodiments, the heterologous protein is an artificial nuclease. In some embodiments, the first heterologous protein and the second heterologous protein are artificial nucleases.In some embodiments, the artificial nuclease is an artificial meganuclease, TALEN, zinc finger nuclease, CRISPR system nuclease, compact TALEN, megaTAL, base editor, or prime editor. In some embodiments, the artificial nuclease is an artificial meganuclease. In some embodiments, the artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs: 43-59. In some embodiments, the artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 50. In some embodiments, the artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 58. In some embodiments, the artificial meganuclease comprises an amino acid sequence represented by one of the sequences shown in SEQ ID NOs: 43 to 59. In some embodiments, the artificial meganuclease comprises an amino acid sequence represented by the sequence shown in SEQ ID NO: 50. In some embodiments, the artificial meganuclease comprises an amino acid sequence represented by the sequence shown in SEQ ID NO: 58.In some embodiments, the first heterologous protein is an artificial meganuclease comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 99 or 100. In some embodiments, the second heterologous protein is an artificial meganuclease comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 99. In some embodiments, the first heterologous protein is an artificial meganuclease comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 99. In some embodiments, the first heterologous protein is an artificial meganuclease comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 100.In some embodiments, the second heterologous protein is an artificial meganuclease comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 99. In some embodiments, the second heterologous protein is an artificial meganuclease comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 100.

[0024] In some embodiments, the first heterologous protein is an artificial meganuclease comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 101 or 102. In some embodiments, the second heterologous protein is an artificial meganuclease comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 101. In some embodiments, the first heterologous protein is an artificial meganuclease comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 101. In some embodiments, the first heterologous protein is an artificial meganuclease comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 102.In some embodiments, the second heterologous protein is an artificial meganuclease comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 101. In some embodiments, the second heterologous protein is an artificial meganuclease comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 102.

[0025] In some embodiments of the muscle-specific expression cassettes described herein, the expression cassette includes an SV40 polyadenylated signal sequence comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 114.

[0026] In some embodiments of the muscle-specific expression cassette described herein, the expression cassette comprises, from 5' to 3', a) a tMCK promoter with the sequence shown in Sequence ID No. 22, b) a Kozak sequence, c) a first nucleic acid sequence encoding a first heterologous protein, d) a 2A sequence, e) a second nucleic acid sequence encoding a second heterologous protein, and f) a WPRE sequence.

[0027] In some embodiments, the first heterologous protein is an artificial nuclease. In some embodiments, the second heterologous protein is an artificial nuclease. In some embodiments, the artificial nuclease is an artificial meganuclease, TALEN, zinc finger nuclease, CRISPR system nuclease, compact TALEN, megaTAL, base editor, or prime editor.

[0028] In some embodiments, the first heterologous protein is an artificial meganuclease. In some embodiments, the second heterologous protein is an artificial meganuclease.

[0029] In some embodiments, the first heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with one of SEQ ID NOs. In some embodiments, the second heterologous protein is an artificial meganuclease containing an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with one of SEQ ID NOs. In some embodiments, the first heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 50 or 58.In some embodiments, the second heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 50. In some embodiments, the first heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 50. In some embodiments, the first heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 58. In some embodiments, the second heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 50.In some embodiments, the second heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 58.

[0030] In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are modified to reduce the CpG content. In some embodiments, the first nucleic acid sequence or the second nucleic acid sequence is codon-modified to reduce the percentage sequence identity between the first and second nucleic acid sequences, and the codon modification does not alter the amino acid sequence of the first or second heterologous protein. In some embodiments, the first nucleic acid sequence has sequence identity of about 30% or less to about 90% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity of about 40% or less to about 60% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, about 75% or less, about 80% or less, about 85% or less, or about 90% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 40% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 50% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 60% or less.

[0031] In some embodiments, the expression cassette includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs: 78-87.

[0032] In another aspect described herein, a muscle-specific expression cassette comprising a nucleic acid sequence encoding a heterologous protein operably linked to a muscle-specific promoter, wherein the expression cassette comprises, from 5' to 3', a) a muscle-specific enhancer element according to SEQ ID NO: 1, b) a tMCK promoter according to SEQ ID NO: 22, c) a Kozak sequence, d) a first nucleic acid sequence encoding a first heterologous protein, e) a 2A sequence comprising a Fulin cleavage motif and a GSG linker, f) a second nucleic acid sequence encoding a second heterologous protein, and g) a WPRE sequence.

[0033] In some embodiments, the first heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs. In some embodiments, the second heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs. In some embodiments, the first heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 50 or 58.In some embodiments, the second heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 50. In some embodiments, the first heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 50. In some embodiments, the first heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 58. In some embodiments, the second heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 50.In some embodiments, the second heterologous protein is an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 58.

[0034] In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are modified to reduce the CpG content. In some embodiments, the first nucleic acid sequence or the second nucleic acid sequence is codon-modified to reduce the percentage sequence identity between the first and second nucleic acid sequences, and the codon modification does not alter the amino acid sequence of the first or second heterologous protein. In some embodiments, the first nucleic acid sequence has sequence identity of about 30% or less to about 90% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity of about 40% or less to about 60% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, about 75% or less, about 80% or less, about 85% or less, or about 90% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 40% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 50% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 60% or less. In some embodiments, the expression cassette includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs: 78-87.

[0035] In another aspect described herein, a muscle-specific expression cassette comprising a nucleic acid sequence encoding a heterologous protein operably linked to a muscle-specific promoter, wherein the expression cassette comprises, from 5' to 3', a) a muscle-specific enhancer element according to SEQ ID NO: 1, b) a tMCK promoter according to SEQ ID NO: 22, c) a Kozak sequence, d) a first nucleic acid sequence encoding a first artificial meganuclease comprising an amino acid sequence according to one of SEQ ID NOs: 43-51 or SEQ ID NOs: 52-59, e) a 2A sequence comprising a Fulin cleavage motif and a GSG linker, f) a second nucleic acid sequence encoding a second artificial meganuclease comprising an amino acid sequence according to one of SEQ ID NOs: 43-51 or SEQ ID NOs: 52-59, and g) a WPRE sequence.

[0036] In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are modified to reduce the CpG content. In some embodiments, the first nucleic acid sequence or the second nucleic acid sequence is codon-modified to reduce the percentage sequence identity between the first and second nucleic acid sequences, and the codon modification does not alter the amino acid sequence of the first or second heterologous protein. In some embodiments, the first nucleic acid sequence has sequence identity of about 30% or less to about 90% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity of about 40% or less to about 60% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, about 75% or less, about 80% or less, about 85% or less, or about 90% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 40% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 50% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 60% or less.

[0037] In some embodiments, the first artificial meganuclease comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the second artificial meganuclease comprises the amino acid sequence of SEQ ID NO: 58.

[0038] In some embodiments, the expression cassette includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs. In another aspect described herein, (a) an artificial meganuclease comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs: 43-51, wherein the artificial meganuclease binds to and cleaves a recognition sequence containing SEQ ID NO: 37 in the dystrophin gene, or (b) an artificial meganuclease comprising an amino acid sequence having at least 80% sequence identity with the sequence shown in any one of SEQ ID NOs: 52-59 An artificial meganuclease comprising an amino acid sequence having sequence identity of %, at least 81%, at least 82%, at least 83%, at least 84%, 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%, comprising a muscle-specific expression cassette comprising a nucleic acid sequence encoding the artificial meganuclease which binds to and cleaves a recognition sequence comprising SEQ ID NO: 39 in the dystrophin gene, wherein the nucleic acid sequence encoding the artificial meganuclease is operably linked to an MCK promoter.

[0039] In some embodiments, the muscle-specific expression cassette described herein includes a muscle-specific enhancer. In some embodiments, the muscle-specific expression cassette described herein includes a Kozak sequence. In some embodiments, the muscle-specific expression cassette described herein includes a post-transcriptional regulatory element. In some embodiments, the post-transcriptional regulatory element includes a WPRE.

[0040] In some embodiments of the muscle-specific expression cassettes described herein, the muscle-specific enhancer comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 1.

[0041] In some embodiments of the muscle-specific expression cassettes described herein, the MCK promoter is a tMCK promoter. In some embodiments, the tMCK promoter comprises a base promoter and one or more MCK enhancer elements. In some embodiments, the base promoter comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 3. In some embodiments, the base promoter comprises a nucleic acid sequence with the sequence shown in Sequence ID No. 3.

[0042] In some embodiments of the muscle-specific expression cassettes described herein, the MCK enhancer element includes an MCK-R control element. In some embodiments, the MCK enhancer element includes two MCK-R control elements. In some embodiments, the MCK-R control element includes a nucleic acid sequence represented by any one of sequence numbers 4 to 17.

[0043] In some embodiments of the muscle-specific expression cassettes described herein, the MCK enhancer element comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 21.

[0044] In some embodiments of the muscle-specific expression cassettes described herein, the MCK promoter is a tMCK promoter comprising three MCK enhancer elements.

[0045] In some embodiments of the muscle-specific expression cassettes described herein, the MCK promoter is a tMCK promoter comprising a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 22.

[0046] In some embodiments, the artificial meganuclease includes a nuclear localization sequence (NLS). In some embodiments, the NLS is located at the N-terminus of the artificial meganuclease. In some embodiments, the NLS is located at the C-terminus of the artificial meganuclease. In some embodiments, the artificial meganuclease includes a first NLS at the N-terminus and a second NLS at the C-terminus. In some embodiments, the first and second NLS are identical. In some embodiments, the first and second NLS are not identical. In some embodiments, the NLS includes an SV40 NLS, a c-myc NLS, or an NLS5 NLS. In some embodiments, the NLS comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs.

[0047] In some embodiments of the muscle-specific expression cassette described herein, the expression cassette comprises a first nucleic acid sequence encoding a first artificial meganuclease and a second nucleic acid sequence encoding a second artificial meganuclease. In some embodiments, the first and second nucleic acid sequences are separated by an IRES or 2A sequence. In some embodiments, the 2A sequence further comprises a furin cleavage motif and a GSG linker sequence. In some embodiments, the 2A sequence is a T2A, P2A, E2A, or F2A sequence.

[0048] In some embodiments, the first artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs: 43-51. In some embodiments, the second artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs: 43-51. In some embodiments, the first artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs. In some embodiments, the second artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs.

[0049] In some embodiments, the first artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 50. In some embodiments, the second artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 50. In some embodiments, the first artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 58. In some embodiments, the second artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 58.

[0050] In some embodiments, the first artificial meganuclease comprises the amino acid sequence shown in any one of SEQ ID NOs: 43-51. In some embodiments, the first artificial meganuclease comprises the amino acid sequence shown in any one of SEQ ID NOs: 52-59. In some embodiments, the second artificial meganuclease comprises the amino acid sequence shown in any one of SEQ ID NOs: 43-51. In some embodiments, the second artificial meganuclease comprises the amino acid sequence shown in any one of SEQ ID NOs: 52-59.

[0051] In some embodiments, the first artificial meganuclease includes the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the first artificial meganuclease includes the amino acid sequence shown in SEQ ID NO: 58. In some embodiments, the second artificial meganuclease includes the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the second artificial meganuclease includes the amino acid sequence shown in SEQ ID NO: 58.

[0052] In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are modified to reduce the CpG content. In some embodiments, the first nucleic acid sequence or the second nucleic acid sequence is codon-modified to reduce the percentage sequence identity between the first and second nucleic acid sequences, and the codon modification does not alter the amino acid sequence of the first or second heterologous protein. In some embodiments, the first nucleic acid sequence has sequence identity of about 30% or less to about 90% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity of about 40% or less to about 60% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, about 75% or less, about 80% or less, about 85% or less, or about 90% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 40% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 50% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 60% or less.

[0053] In another embodiment described herein, 5' to 3' have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with a) the tMCK promoter represented by the sequence shown in SEQ ID NO: 22, b) the Kozak sequence, or c) (i) the sequence shown in any one of SEQ ID NOs: 43 to 51. (ii) A first artificial meganuclease comprising an amino acid sequence which binds to and cleaves a recognition sequence containing SEQ ID NO: 37 in the dystrophin gene, or (ii) a sequence represented by any one of SEQ ID NOs: 52-59 which binds to at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 A first artificial meganuclease comprising an amino acid sequence having 99% or 100% sequence identity, comprising a first nucleic acid sequence encoding the first artificial meganuclease that binds to and cleaves a recognition sequence containing SEQ ID NO: 39 in the dystrophin gene, d) 2A sequence, e) (i) any one of the sequences shown in SEQ ID NOs: 43-51, and at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, and at least A second artificial meganuclease comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, wherein the second artificial meganuclease binds to and cleaves a recognition sequence containing SEQ ID NO: 37 in the dystrophin gene, or (ii) a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, or at least 92% sequence identity with any one of SEQ ID NOs: 52-59,A second artificial meganuclease comprising an amino acid sequence having 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, wherein the second artificial meganuclease binds to and cleaves a recognition sequence containing SEQ ID NO: 39 in the dystrophin gene, and a muscle-specific expression cassette comprising f) a WPRE sequence. In some embodiments, the first artificial meganuclease and the second artificial meganuclease are not identical.

[0054] In some embodiments, the first artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 50. In some embodiments, the first artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 58. In some embodiments, the second artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 50. In some embodiments, the second artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 58.

[0055] In some embodiments, the first artificial meganuclease comprises an amino acid sequence represented by either SEQ ID NOs: 43-51 or SEQ ID NOs: 52-59. In some embodiments, the second artificial meganuclease comprises an amino acid sequence represented by either SEQ ID NOs: 43-51 or SEQ ID NOs: 52-59.

[0056] In some embodiments, the first artificial meganuclease includes an amino acid sequence according to the sequence shown in SEQ ID NO: 50. In some embodiments, the first artificial meganuclease includes an amino acid sequence that is sequence-identical to the sequence shown in SEQ ID NO: 58. In some embodiments, the second artificial meganuclease includes an amino acid sequence according to the sequence shown in SEQ ID NO: 50. In some embodiments, the second artificial meganuclease includes an amino acid sequence according to the sequence shown in SEQ ID NO: 58.

[0057] In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are modified to reduce the CpG content. In some embodiments, the first nucleic acid sequence or the second nucleic acid sequence is codon-modified to reduce the percentage sequence identity between the first and second nucleic acid sequences, and the codon modification does not alter the amino acid sequence of the first or second heterologous protein. In some embodiments, the first nucleic acid sequence has sequence identity of about 30% or less to about 90% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity of about 40% or less to about 60% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, about 75% or less, about 80% or less, about 85% or less, or about 90% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 40% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 50% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 60% or less.

[0058] In some embodiments, the expression cassette includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs.

[0059] In another aspect described herein, a muscle-specific expression cassette comprising a nucleic acid sequence encoding an artificial meganuclease operably linked to a muscle-specific promoter, wherein the expression cassette comprises, from 5' to 3', a) a muscle-specific enhancer element according to SEQ ID NO: 1, b) a tMCK promoter according to SEQ ID NO: 22, c) a Kozak sequence, and d) (i) a sequence represented by any one of SEQ ID NOs: 43-51, with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, and at least 91% (ii) A first artificial meganuclease comprising an amino acid sequence having sequence identity of %, 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%, wherein the first artificial meganuclease binds to and cleaves a recognition sequence containing SEQ ID NO: 37 in the dystrophin gene, or (ii) a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, less than 100% of the sequence shown in any one of SEQ ID NOs: 52-59 A first artificial meganuclease comprising an amino acid sequence having sequence identity of 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, wherein the first artificial meganuclease comprises a first nucleic acid sequence encoding the first artificial meganuclease that binds to and cleaves a recognition sequence containing SEQ ID NO: 39 in the dystrophin gene, e) 2A sequence, f) (i) a sequence shown in any one of SEQ ID NOs: 43-51 (ii) a second artificial meganuclease containing an amino acid sequence having 80% sequence identity, which binds to and cleaves a recognition sequence containing SEQ ID NO: 37 in the dystrophin gene, or (ii) a second artificial meganuclease containing an amino acid sequence having at least 80% sequence identity with the sequence shown in any one of SEQ ID NOs: 52-59, which binds to and cleaves a recognition sequence containing SEQ ID NO: 39 in the dystrophin gene, or (g) a muscle-specific expression cassette containing a WPRE sequence.In some embodiments, the first artificial meganuclease and the second artificial meganuclease are not identical.

[0060] In some embodiments, the first artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 50. In some embodiments, the first artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 58. In some embodiments, the second artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 50. In some embodiments, the second artificial meganuclease comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in Sequence ID No. 58.

[0061] In some embodiments, the first artificial meganuclease comprises an amino acid sequence represented by either SEQ ID NOs: 43-51 or SEQ ID NOs: 52-59. In some embodiments, the second artificial meganuclease comprises an amino acid sequence represented by either SEQ ID NOs: 43-51 or SEQ ID NOs: 52-59.

[0062] In some embodiments, the first artificial meganuclease includes an amino acid sequence according to the sequence shown in SEQ ID NO: 50. In some embodiments, the first artificial meganuclease includes an amino acid sequence that is sequence-identical to the sequence shown in SEQ ID NO: 58. In some embodiments, the second artificial meganuclease includes an amino acid sequence according to the sequence shown in SEQ ID NO: 50. In some embodiments, the second artificial meganuclease includes an amino acid sequence according to the sequence shown in SEQ ID NO: 58.

[0063] In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are modified to reduce the CpG content. In some embodiments, the first nucleic acid sequence or the second nucleic acid sequence is codon-modified to reduce the percentage sequence identity between the first and second nucleic acid sequences, and the codon modification does not alter the amino acid sequence of the first or second heterologous protein. In some embodiments, the first nucleic acid sequence has sequence identity of about 30% or less to about 90% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity of about 40% or less to about 60% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, about 75% or less, about 80% or less, about 85% or less, or about 90% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 40% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 50% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 60% or less. In some embodiments, the expression cassette includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs.

[0064] Another embodiment described herein is a muscle-specific expression cassette comprising a nucleic acid sequence encoding an artificial meganuclease operably linked to a muscle-specific promoter, wherein the expression cassette comprises, from 5' to 3', a) a muscle-specific enhancer element by the sequence shown in SEQ ID NO: 1, b) a tMCK promoter by the sequence shown in SEQ ID NO: 22, c) a Kozak sequence, and d) (i) a first artificial meganuclease comprising an amino acid sequence by any one of SEQ ID NOs: 43-51, which binds to and cleaves a recognition sequence in the dystrophin gene including SEQ ID NO: 37, or (ii) a first artificial meganuclease comprising an amino acid sequence by any one of SEQ ID NOs: 52-59, which binds to and cleaves a recognition sequence in the dystrophin gene. A muscle-specific expression cassette comprising: a first nucleic acid sequence encoding a first artificial meganuclease that binds to and cleaves a recognition sequence containing number 39; e) a 2A sequence; f) a second artificial meganuclease comprising an amino acid sequence represented by any one of sequence numbers 43-51, which binds to and cleaves a recognition sequence containing sequence number 37 in the dystrophin gene; or (ii) a second artificial meganuclease comprising an amino acid sequence represented by any one of sequence numbers 52-59, which binds to and cleaves a recognition sequence containing sequence number 39 in the dystrophin gene; and g) a WPRE sequence, wherein the first artificial meganuclease and the second artificial meganuclease are not identical.

[0065] In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are modified to reduce the CpG content. In some embodiments, the first nucleic acid sequence or the second nucleic acid sequence is codon-modified to reduce the percentage sequence identity between the first and second nucleic acid sequences, and the codon modification does not alter the amino acid sequence of the first or second heterologous protein. In some embodiments, the first nucleic acid sequence has sequence identity of about 30% or less to about 90% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity of about 40% or less to about 60% or less with the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, about 75% or less, about 80% or less, about 85% or less, or about 90% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 40% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 50% or less. In some embodiments, the first nucleic acid sequence has sequence identity with the second nucleic acid sequence of about 60% or less. In some embodiments, the expression cassette includes a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in any one of SEQ ID NOs.

[0066] In another aspect described herein, the polynucleotide comprises any muscle-specific expression cassette described herein.

[0067] In another embodiment described herein, the recombinant DNA construct comprises a polynucleotide including any muscle-specific expression cassette described herein. In some embodiments, the recombinant DNA construct described herein encodes any recombinant virus described herein. In some embodiments of the recombinant DNA construct described herein, the recombinant virus is a recombinant adenovirus, recombinant lentivirus, recombinant retrovirus, or recombinant adeno-associated virus (AAV). In some embodiments of the recombinant DNA construct described herein, the recombinant virus is recombinant AAV. In some embodiments of the recombinant DNA construct described herein, the recombinant AAV has an rh74 capsid or an AAV9 capsid. In some embodiments of the recombinant DNA construct described herein, the recombinant AAV has an AAV9 capsid. In some embodiments of the recombinant DNA construct described herein, the recombinant AAV includes a 5'ITR containing a first D sequence and a 3'ITR containing a second D sequence.

[0068] In another embodiment described herein, the recombinant virus is a polynucleotide comprising any muscle-specific expression cassette described herein. In some embodiments, the recombinant virus described herein is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant AAV. In some embodiments, the recombinant virus described herein is a recombinant AAV. In some embodiments of the recombinant virus described herein, the recombinant AAV has an rh74 capsid or an AAV9 capsid. In some embodiments of the recombinant virus described herein, the recombinant AAV has an AAV9 capsid. In some embodiments of the recombinant virus described herein, the recombinant AAV comprises a 5'ITR containing a first D sequence and a 3'ITR containing a second D sequence.

[0069] Another embodiment described herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and any polynucleotide described herein.

[0070] Another embodiment described herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and any recombinant DNA construct described herein.

[0071] Another embodiment described herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and any recombinant virus as described herein.

[0072] Another embodiment described herein is a method for expressing a heterologous protein in mammalian muscle cells, comprising introducing any muscle-specific expression cassette described herein into a mammalian cell composition containing mammalian muscle cells, wherein the heterologous protein is expressed in the mammalian muscle cells.

[0073] In some embodiments of the method, muscle cells include muscle progenitor cells, skeletal muscle cells, or cardiomyocytes. In some embodiments, mammalian muscle progenitor cells include muscle satellite cells expressing the Pax7 protein. In some embodiments, mammalian muscle cells are part of skeletal muscle tissue or cardiomyocytes. In some embodiments, mammalian cells are human cells. In some embodiments, heterologous proteins are expressed at higher levels in mammalian muscle cells compared to non-muscle cells. In some embodiments, non-muscle cells include non-muscle cells from the liver, non-muscle cells from the brain, germline cells, or non-muscle cells from the lungs. In some embodiments, heterologous proteins are expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells. In some embodiments, heterologous proteins are expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells from the liver. In some embodiments, heterologous proteins are expressed at least 15 to about 25 times more in skeletal muscle cells compared to cardiomyocytes.

[0074] In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any polynucleotide described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any recombinant DNA construct described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any recombinant virus described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any pharmaceutical composition described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition in vitro. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition in vivo. In some embodiments, the mammalian cell composition is a human cell composition.

[0075] Another embodiment described herein is a method for enhancing the expression of a heterologous protein in mammalian skeletal muscle cells compared to cardiomyocytes, comprising introducing any muscle-specific expression cassette described herein into a mammalian cell composition comprising mammalian skeletal muscle cells and cardiomyocytes, wherein the heterologous protein is expressed at a higher level in mammalian skeletal muscle cells compared to cardiomyocytes.

[0076] In some embodiments of the method, mammalian muscle cells are part of skeletal muscle tissue or cardiomyocytes. In some embodiments, heterologous proteins are expressed at higher levels in mammalian muscle cells compared to non-muscle cells. In some embodiments, non-muscle cells include liver non-muscle cells, brain non-muscle cells, germline cells, or lung non-muscle cells. In some embodiments, heterologous proteins are expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells. In some embodiments, heterologous proteins are expressed at least 15 to about 60 times more in muscle cells compared to liver non-muscle cells. In some embodiments, heterologous proteins are expressed at least 15 to about 25 times more in skeletal muscle cells compared to cardiomyocytes.

[0077] In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any polynucleotide described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any recombinant DNA construct described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any recombinant virus described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any pharmaceutical composition described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition in vitro. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition in vivo. In some embodiments, the mammalian cell composition is a human cell composition.

[0078] Another embodiment described herein is a method for expressing a heterologous protein in mammalian muscle progenitor cells, comprising introducing any muscle-specific expression cassette described herein into a mammalian cell composition containing mammalian muscle progenitor cells, wherein the heterologous protein is expressed in the muscle progenitor cells.

[0079] In some embodiments of the method, the muscle-specific expression cassette is any muscle-specific expression cassette described herein. In some embodiments, the mammalian muscle progenitor cells are a portion of skeletal muscle tissue or cardiomyocyte tissue. In some embodiments, the mammalian muscle progenitor cells include muscle satellite cells expressing the Pax7 protein.

[0080] In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any polynucleotide described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any recombinant DNA construct described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any recombinant virus described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any pharmaceutical composition described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition in vitro. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition in vivo. In some embodiments, the mammalian cell composition is a human cell composition.

[0081] Another aspect described herein is a method for selectively modifying the dystrophin gene in a target muscle cell, wherein the dystrophin gene is characterized by a mutation that alters the reading frame of the dystrophin gene from the wild type, the method comprising delivering any muscle-specific expression cassette described herein to the muscle cell, the heterologous protein being able to modify the dystrophin gene, the heterologous protein being expressed in the target muscle cell, and thereby modifying the dystrophin gene in the target muscle cell.

[0082] In some embodiments of the method, the heterologous protein is an artificial nuclease. In some embodiments, the expression cassette includes a first nucleic acid sequence encoding a first heterologous protein and a second nucleic acid sequence encoding a second heterologous protein. In some embodiments, the first heterologous protein is a first artificial nuclease, and the second heterologous protein is a second artificial nuclease.

[0083] In some embodiments of the method, a first artificial nuclease generates a first cleavage site within the dystrophin gene at a first recognition sequence located within the dystrophin gene, and a second artificial nuclease generates a second cleavage site within the dystrophin gene at a second recognition sequence located within the dystrophin gene, the first and second cleavage sites having complementary 3' overhangs, the intervening genomic DNA between the first and second cleavage sites being excised from the dystrophin gene, the dystrophin gene being annealed, and the normal reading frame of the dystrophin gene being restored compared to the full-length wild-type dystrophin gene. In some embodiments, the complementary 3' overhangs of the first and second cleavage sites are directly religated from each other. In some embodiments, the dystrophin gene comprises the nucleic acid sequence shown in SEQ ID NO: 41. 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 subject is transformed into a Becker muscular dystrophy phenotype.

[0084] In some embodiments of the method, muscle cells include muscle progenitor cells, skeletal muscle cells, or cardiomyocytes. In some embodiments, mammalian muscle progenitor cells include muscle satellite cells expressing the Pax7 protein. In some embodiments, muscle cells are part of skeletal muscle tissue or cardiomyocytes. In some embodiments, heterologous proteins are expressed at higher levels in muscle cells compared to non-muscle cells. In some embodiments, non-muscle cells include non-muscle cells from the liver, non-muscle cells from the brain, germline cells, or non-muscle cells from the lungs. In some embodiments, the first and second artificial meganucleases are expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells. In some embodiments, the first and second artificial meganucleases are expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells from the liver. In some embodiments, the first and second artificial meganucleases are expressed at least 15 to about 25 times more in skeletal muscle cells compared to cardiomyocytes. In some embodiments, the muscle-specific expression cassette is delivered to muscle cells by any polynucleotide described herein. In some embodiments, the muscle-specific expression cassette is delivered to muscle cells by any recombinant DNA construct described herein. In some embodiments, the muscle-specific expression cassette is delivered to muscle cells by any recombinant virus described herein. In some embodiments, the muscle-specific expression cassette is delivered to muscle cells by any pharmaceutical composition described herein. In some embodiments, the subjects are humans.

[0085] Another aspect described herein is a method for treating myopathy in a subject having myopathy, comprising administering any muscle-specific expression cassette described herein to the subject, wherein the heterologous protein is a therapeutic protein for treating myopathy, and the therapeutic protein is expressed in the muscle cells of the subject, thereby treating the myopathy in the subject.

[0086] In some embodiments of the method, the muscle disorder includes muscular dystrophy. In some embodiments, muscular dystrophy includes DMD. In some embodiments, the therapeutic protein includes a myotransgene, a DNA-binding regulatory protein, or an artificial nuclease. In some embodiments, the myotransgene includes a sarcoglycan or a dystrophin protein. In some embodiments, the dystrophin protein is a microdystrophin protein. In some embodiments, the therapeutic protein is an artificial nuclease.

[0087] In some embodiments of the method, the muscle disorder is a DMD characterized by a mutation in the dystrophin gene that alters the reading frame of the dystrophin gene from the wild type. In some embodiments, the muscle-specific expression cassette includes a first nucleic acid sequence encoding a first therapeutic protein and a second nucleic acid sequence encoding a second therapeutic protein. In some embodiments, the first therapeutic protein is a first artificial nuclease, and the second therapeutic protein is a second artificial nuclease.

[0088] In some embodiments of the method, a first artificial nuclease generates a first cleavage site within the dystrophin gene at a first recognition sequence located within the dystrophin gene, and a second artificial nuclease generates a second cleavage site within the dystrophin gene at a second recognition sequence located within the dystrophin gene, the first and second cleavage sites having complementary 3' overhangs, the intervening genomic DNA between the first and second cleavage sites being excised from the dystrophin gene, the dystrophin gene being annealed, and the normal reading frame of the dystrophin gene being restored compared to the full-length wild-type dystrophin gene. In some embodiments, the complementary 3' overhangs of the first and second cleavage sites are directly religated from each other. In some embodiments, the dystrophin gene comprises the nucleic acid sequence shown in SEQ ID NO: 41. 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 subject is transformed into a Becker muscular dystrophy phenotype.

[0089] In some embodiments of the method, muscle cells include muscle progenitor cells, skeletal muscle cells, or cardiomyocytes. In some embodiments, muscle progenitor cells include muscle satellite cells expressing the Pax7 protein. In some embodiments, muscle cells are part of skeletal muscle tissue or cardiomyocytes. In some embodiments, heterologous proteins are expressed at higher levels in muscle cells compared to non-muscle cells. In some embodiments, non-muscle cells include non-muscle cells of the liver, non-muscle cells of the brain, germline cells, or non-muscle cells of the lungs. In some embodiments, heterologous proteins are expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells. In some embodiments, heterologous proteins are expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells of the liver. In some embodiments, heterologous proteins are expressed at least 15 to about 25 times more in skeletal muscle cells compared to cardiomyocytes. In some embodiments, the muscle-specific expression cassette is administered to the subject by administering any polynucleotide described herein. In some embodiments, the muscle-specific expression cassette is administered to a subject by administering any recombinant DNA construct described herein. In some embodiments, the muscle-specific expression cassette is administered to a subject by administering any recombinant virus described herein. In some embodiments, the muscle-specific expression cassette is administered to a subject by administering any pharmaceutical composition described herein. In some embodiments, the subject is human.

[0090] Another embodiment described herein is a method for expressing an artificial meganuclease in mammalian muscle cells, comprising introducing any muscle-specific expression cassette described herein into a mammalian cell composition containing mammalian muscle cells, wherein the artificial meganuclease is expressed in the mammalian muscle cells.

[0091] In some embodiments of the method, muscle cells include muscle progenitor cells, skeletal muscle cells, or cardiomyocytes. In some embodiments, mammalian muscle progenitor cells include muscle satellite cells expressing the Pax7 protein. In some embodiments, mammalian muscle cells are part of skeletal muscle tissue or cardiomyocytes. In some embodiments, mammalian cells are human cells. In some embodiments, the artificial meganuclease is expressed at a higher level in mammalian muscle cells compared to non-muscle cells. In some embodiments, non-muscle cells include non-muscle cells from the liver, non-muscle cells from the brain, germline cells, or non-muscle cells from the lungs. In some embodiments, the artificial meganuclease is expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells. In some embodiments, the artificial meganuclease is expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells from the liver. In some embodiments, the artificial meganuclease is expressed at least 15 to about 25 times more in skeletal muscle cells compared to cardiomyocytes.

[0092] In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any polynucleotide described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any recombinant DNA construct described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any recombinant virus described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any pharmaceutical composition described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition in vitro. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition in vivo. In some embodiments, the mammalian cell composition is a human cell composition.

[0093] Another embodiment described herein is a method for enhancing the expression of an artificial meganuclease in mammalian skeletal muscle cells compared to cardiomyocytes, comprising introducing any muscle-specific expression cassette described herein into a mammalian cell composition comprising mammalian skeletal muscle cells and cardiomyocytes, wherein the artificial meganuclease is expressed at a higher level in mammalian skeletal muscle cells compared to cardiomyocytes.

[0094] In some embodiments of the method, mammalian muscle cells are part of skeletal muscle tissue or cardiomyocytes. In some embodiments, the artificial meganuclease is expressed at a higher level in mammalian muscle cells compared to non-muscle cells. In some embodiments, non-muscle cells include liver non-muscle cells, brain non-muscle cells, germline cells, or lung non-muscle cells. In some embodiments, the artificial meganuclease is expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells. In some embodiments, the artificial meganuclease is expressed at least 15 to about 60 times more in muscle cells compared to liver non-muscle cells. In some embodiments, the artificial meganuclease is expressed at least 15 to about 25 times more in skeletal muscle cells compared to cardiomyocytes.

[0095] In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any polynucleotide described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any recombinant DNA construct described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any recombinant virus described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any pharmaceutical composition described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition in vitro. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition in vivo. In some embodiments, the mammalian cell composition is a human cell composition.

[0096] Another embodiment described herein is a method for expressing an artificial meganuclease in mammalian muscle progenitor cells, comprising introducing any muscle-specific expression cassette described herein into a mammalian cell composition containing mammalian muscle progenitor cells, wherein the artificial meganuclease is expressed in the muscle progenitor cells.

[0097] In some embodiments of the method, the muscle-specific expression cassette is any muscle-specific expression cassette described herein. In some embodiments, the mammalian muscle progenitor cells are a portion of skeletal muscle tissue or cardiomyocyte tissue. In some embodiments, the mammalian muscle progenitor cells include muscle satellite cells expressing the Pax7 protein.

[0098] In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any polynucleotide described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any recombinant DNA construct described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any recombinant virus described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition by any pharmaceutical composition described herein. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition in vitro. In some embodiments, the muscle-specific expression cassette is introduced into the mammalian cell composition in vivo. In some embodiments, the mammalian cell composition is a human cell composition.

[0099] Another aspect described herein is a method for selectively modifying the dystrophin gene in a target muscle cell, wherein the dystrophin gene is characterized by a mutation that alters the reading frame of the dystrophin gene from the wild type, the method comprising delivering any muscle-specific expression cassette described herein to the muscle cell, the artificial meganuclease being able to modify the dystrophin gene, the artificial meganuclease being expressed in the target muscle cell, thereby modifying the dystrophin gene in the target muscle cell.

[0100] In some embodiments, the expression cassette includes a first nucleic acid sequence encoding a first artificial meganuclease and a second nucleic acid sequence encoding a second artificial meganuclease.

[0101] In some embodiments of the method, a first artificial meganuclease generates a first cleavage site within the dystrophin gene at a first recognition sequence located within the dystrophin gene, and a second artificial meganuclease generates a second cleavage site within the dystrophin gene at a second recognition sequence located within the dystrophin gene, the first and second cleavage sites having complementary 3' overhangs, the intervening genomic DNA between the first and second cleavage sites being excised from the dystrophin gene, the dystrophin gene being annealed, and the normal read frame of the dystrophin gene being restored compared to the full-length wild-type dystrophin gene. In some embodiments, the complementary 3' overhangs of the first and second cleavage sites are directly religated from each other. In some embodiments, the dystrophin gene comprises the nucleic acid sequence shown in SEQ ID NO: 41. 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 subject is transformed into a Becker muscular dystrophy phenotype.

[0102] In some embodiments of the method, muscle cells include muscle progenitor cells, skeletal muscle cells, or cardiomyocytes. In some embodiments, mammalian muscle progenitor cells include muscle satellite cells expressing the Pax7 protein. In some embodiments, muscle cells are part of skeletal muscle tissue or cardiomyocytes. In some embodiments, the artificial meganuclease is expressed at a higher level in muscle cells compared to non-muscle cells. In some embodiments, non-muscle cells include non-muscle cells of the liver, non-muscle cells of the brain, germline cells, or non-muscle cells of the lungs. In some embodiments, the first and second artificial meganucleases are expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells. In some embodiments, the first and second artificial meganucleases are expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells of the liver. In some embodiments, the first and second artificial meganucleases are expressed at least 15 to about 25 times more in skeletal muscle cells compared to cardiomyocytes. In some embodiments, the muscle-specific expression cassette is delivered to muscle cells by any polynucleotide described herein. In some embodiments, the muscle-specific expression cassette is delivered to muscle cells by any recombinant DNA construct described herein. In some embodiments, the muscle-specific expression cassette is delivered to muscle cells by any recombinant virus described herein. In some embodiments, the muscle-specific expression cassette is delivered to muscle cells by any pharmaceutical composition described herein. In some embodiments, the subjects are humans.

[0103] Another embodiment described herein is a method for treating DMD in a subject having DMD, comprising administering any muscle-specific expression cassette described herein to the subject, wherein the artificial meganuclease is a therapeutic protein for treating DMD, and the artificial meganuclease is expressed in the muscle cells of the subject, thereby treating DMD in the subject.

[0104] In some embodiments of the method, the DMD is characterized by a mutation in the dystrophin gene that alters the reading frame of the dystrophin gene from the wild type. In some embodiments, the muscle-specific expression cassette includes a first nucleic acid sequence encoding a first artificial meganuclease and a second nucleic acid sequence encoding a second artificial meganuclease.

[0105] In some embodiments of the method, a first artificial meganuclease generates a first cleavage site within the dystrophin gene at a first recognition sequence located within the dystrophin gene, and a second artificial meganuclease generates a second cleavage site within the dystrophin gene at a second recognition sequence located within the dystrophin gene, the first and second cleavage sites having complementary 3' overhangs, the intervening genomic DNA between the first and second cleavage sites being excised from the dystrophin gene, the dystrophin gene being annealed, and the normal read frame of the dystrophin gene being restored compared to the full-length wild-type dystrophin gene. In some embodiments, the complementary 3' overhangs of the first and second cleavage sites are directly religated from each other. In some embodiments, the dystrophin gene comprises the nucleic acid sequence shown in SEQ ID NO: 41. 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 subject is transformed into a Becker muscular dystrophy phenotype.

[0106] In some embodiments of the method, muscle cells include muscle progenitor cells, skeletal muscle cells, or cardiomyocytes. In some embodiments, muscle progenitor cells include muscle satellite cells expressing the Pax7 protein. In some embodiments, muscle cells are part of skeletal muscle tissue or cardiomyocytes. In some embodiments, the artificial meganuclease is expressed at a higher level in muscle cells compared to non-muscle cells. In some embodiments, non-muscle cells include non-muscle cells of the liver, non-muscle cells of the brain, germline cells, or non-muscle cells of the lungs. In some embodiments, the artificial meganuclease is expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells. In some embodiments, the artificial meganuclease is expressed at least 15 to about 60 times more in muscle cells compared to non-muscle cells of the liver. In some embodiments, the artificial meganuclease is expressed at least 15 to about 25 times more in skeletal muscle cells compared to cardiomyocytes. In some embodiments, the muscle-specific expression cassette is administered to the subject by administering any polynucleotide described herein. In some embodiments, the muscle-specific expression cassette is administered to a subject by administering any recombinant DNA construct described herein. In some embodiments, the muscle-specific expression cassette is administered to a subject by administering any recombinant virus described herein. In some embodiments, the muscle-specific expression cassette is administered to a subject by administering any pharmaceutical composition described herein. In some embodiments, the subject is human. [Brief explanation of the drawing]

[0107] [Figure 1A]This provides charts of meganuclease expression in meganuclease (ng) / total cell protein (mg) from lysates derived from both quadriceps femoris and cardiac tissues of hDMDdel52 / mdx (hDMD) mice. For each tissue in Figure 1A, the dark dots on the left represent expression constructs utilizing the MHCK7 promoter, and the light dots in the center represent expression constructs utilizing the tMCK promoter operably linked to DMD 19-20L.329 and DMD 35-36L.349 pairs of meganuclease delivered to mice via the AAV9 capsid. The dots on the right represent mice treated with PBS alone. [Figure 1B] This provides charts of meganuclease expression in meganuclease (ng) / total cell protein (mg) from lysates derived from both quadriceps femoris and cardiac tissues of hDMDdel52 / mdx (hDMD) mice. For each tissue in Figure 1B, the dark dots on the left represent expression constructs utilizing the MHCK7 promoter, and the light dots in the center represent expression constructs utilizing the tMCK promoter operably ligated to DMD 19-20L.329 and DMD 35-36L.349 pairs of meganuclease delivered to mice via the AAVrh74 capsid. The dots on the right represent mice treated with PBS alone. [Figure 2A] This document provides bar graphs showing truncated modified dystrophin protein levels by WES analysis of DMD 19–20L.329 and DMD 35–36L.349 pairs of meganucleases from both quadriceps femoris and cardiac tissues of hDMDdel52 / mdx (hDMD) mice, normalized against vinculin loading controls. For each tissue in Figure 2A, the dark dots on the left represent expression constructs utilizing the MHCK7 promoter, and the light dots in the center represent expression constructs utilizing the tMCK promoter operably ligated to DMD 19–20L.329 and DMD 35–36L.349 pairs of meganucleases delivered to mice via AAV9 capsids. The dots on the right represent mice treated with PBS alone. [Figure 2B]This document provides bar graphs showing truncated modified dystrophin protein levels by WES analysis normalized against vinculin loading controls for DMD 19–20L.329 and DMD 35–36L.349 pairs of meganucleases from both quadriceps femoris and cardiac tissues of hDMDdel52 / mdx (hDMD) mice. For each tissue in Figure 2B, the dark dots on the left represent expression constructs utilizing the MHCK7 promoter, and the light dots in the center represent expression constructs utilizing the tMCK promoter operably ligated to DMD 19–20L.329 and DMD 35–36L.349 pairs of meganucleases delivered to mice via the AAVrh74 capsid. The dots on the right represent mice treated with PBS alone. [Figure 3A] Figure 3A provides bar graphs showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of DMD19-20 and DMD35-36 recognition sequences by meganuclease DMD 19-20L.329 and DMD35-36L.349 pairs, evaluated by ddPCR assay from both quadriceps femoris and cardiac tissues of hDMDdel52 / mdx (hDMD) mice. For each tissue in Figure 3A, the dark dots on the left represent expression constructs utilizing the MHCK7 promoter, and the light dots in the center represent expression constructs utilizing the tMCK promoter operably ligated to meganuclease DMD 19-20L.329 and DMD35-36L.349 pairs delivered to mice via AAV9 capsid. The dots on the right represent mice treated with PBS alone. [Figure 3B]Figure 3B provides bar graphs showing the percentage of total ligation (%) of genomic DNA adjacent to exons 45-55 after cleavage of DMD19-20 and DMD35-36 recognition sequences by meganuclease pairs DMD 19-20L.329 and DMD35-36L.349 from both quadriceps femoris and cardiac tissues of hDMDdel52 / mdx (hDMD) mice, as evaluated by ddPCR assay. For each tissue in Figure 3B, the dark dots on the left represent expression constructs utilizing the MHCK7 promoter, and the light dots in the center represent expression constructs utilizing the tMCK promoter operably ligated to meganuclease pairs DMD 19-20L.329 and DMD35-36L.349 delivered to mice via the AAVrh74 capsid. The dots on the right represent mice treated with PBS alone. [Figure 4] This document provides bar graphs showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD 19-20 recognition sequences and DMD 35-36 recognition sequences by meganuclease pairs DMD 19-20L.329 and DMD 35-36L.349 derived from liver tissue of hDMDdel52 / mdx (hDMD) mice, evaluated by ddPCR assay for each of the expression constructs shown. [Figure 5A]This provides fluorescence immunohistochemical imaging of mouse quadriceps femoris tissue after treatment with PBS or with DMD 19–20L.329 and DMD 35–36L.349 pairs of meganucleases expressed from expression constructs utilizing a tMCK promoter delivered to hDMDdel52 / mdx (hDMD) mice using either AAVrH74 capsid or AAV9 capsid. In the figure, the upper left panel is a control image showing meganuclease expression detected using a meganuclease-specific antibody. The upper right panel shows cells expressing Pax7, indicated by white arrows. The lower left panel shows the expression of both Pax7 (white arrows) and meganucleases within the same cells. The lower right panel shows Pax7, meganuclease expression, and nuclear staining (DAPI). Figure 5A provides imaging of meganuclease and Pax7 expression in quadriceps femoris tissue of mice treated with meganuclease using an expression construct that utilizes the tMCK promoter in AAV9 capsid at a dose of 1e14VG / kg. [Figure 5B] This provides fluorescence immunohistochemical imaging of mouse quadriceps femoris tissue after treatment with PBS or with DMD 19–20L.329 and DMD 35–36L.349 pairs of meganucleases expressed from expression constructs utilizing a tMCK promoter delivered to hDMDdel52 / mdx (hDMD) mice using either AAVrH74 capsid or AAV9 capsid. In the figure, the upper left panel is a control image showing meganuclease expression detected using a meganuclease-specific antibody. The upper right panel shows cells expressing Pax7, indicated by white arrows. The lower left panel shows the expression of both Pax7 (white arrows) and meganucleases within the same cells. The lower right panel shows Pax7, meganuclease expression, and nuclear staining (DAPI). Figure 5B provides imaging of meganuclease and Pax7 expression in quadriceps femoris tissue of mice treated with meganuclease using an expression construct that utilizes the tMCK promoter in rh74 AAV capsid at a dose of 1e14VG / kg. [Figure 5C]This provides fluorescence immunohistochemical imaging of mouse quadriceps femoris tissue after treatment with PBS or with DMD 19–20L.329 and DMD 35–36L.349 pairs of meganucleases expressed from expression constructs utilizing a tMCK promoter delivered to hDMDdel52 / mdx (hDMD) mice using either AAVrH74 capsid or AAV9 capsid. In the figure, the upper left panel is a control image showing meganuclease expression detected using a meganuclease-specific antibody. The upper right panel shows cells expressing Pax7, indicated by white arrows. The lower left panel shows the expression of both Pax7 (white arrows) and meganucleases within the same cells. The lower right panel shows Pax7, meganuclease expression, and nuclear staining (DAPI). Figure 5C provides imaging of meganuclease and Pax7 expression in quadriceps femoris tissue from mice treated with meganuclease using PBS alone. The asterisks in the upper left panel indicate nonspecific background detection from meganuclease-specific antibodies. [Figure 6A] Figure 6A provides bar graphs showing truncated modified dystrophin protein levels obtained by capillary Western immunoassay (WES) analysis normalized against vinculin loading controls for meganucleases DMD 19–20 L.329 and DMD 35–36 L.349 pairs from both quadriceps femoris and cardiac tissue, or PBS alone. In Figure 6A, mice were treated with an expression construct utilizing the tMCK promoter (with WPRE) in the indicated doses of AAV9 capsid. [Figure 6B] Figure 6B provides bar graphs showing truncated modified dystrophin protein levels obtained by capillary Western immunoassay (WES) analysis normalized against vinculin loading controls for meganucleases DMD 19–20 L.329 and DMD 35–36 L.349 pairs from both quadriceps femoris and cardiac tissue, or PBS alone. In Figure 6B, mice were treated with an expression construct utilizing the tMCK promoter (without WPRE) in the indicated doses of AAV9 capsid. [Figure 7] A table is provided showing a list of cardiac lesions from each group of hDMDdel52 / mdx (hDMD) mice treated with expression constructs encoding pairs of DMD 19-20L.329 meganuclease and DMD 35-36L.349 meganuclease. Group 1 shows mice treated with an expression construct utilizing the MHCK7 promoter in AAV9 capsid at a dose of 1e14VG / kg. Groups 2-4 show mice treated with an expression construct utilizing the MHCK7 promoter in AAVrh74 capsid at doses of 1e14VG / kg, 3e13VG / kg, and 1e13VG / kg, respectively. Groups 5-7 show mice treated with an expression construct utilizing the tMCK promoter in AAV9 capsid at doses of 1e14VG / kg, 3e13VG / kg, and 1e13VG / kg, respectively. Groups 8-10 represent mice treated with expression constructs utilizing the tMCK promoter in rh74 capsid at doses of 1e14VG / kg, 3e13VG / kg, and 1e13VG / kg, respectively. [Figure 8A] This document provides charts showing the percentage of truncated modified dystrophin protein from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or with DMD 19-20L.329 and DMD 35-36L.349 pairs of meganucleases expressed from expression constructs utilizing the tMCK promoter in either AAV9 capsid or AAVrH74 capsid at indicated doses, using WES analysis normalized to vinculin loading controls. Figure 8A shows the percentage of truncated modified dystrophin recovery from quadriceps femoris tissue. [Figure 8B]This document provides charts showing the percentage of truncated modified dystrophin protein from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or with DMD 19-20L.329 and DMD 35-36L.349 pairs of meganucleases expressed from expression constructs utilizing the tMCK promoter in either AAV9 capsid or AAVrH74 capsid at indicated doses, using WES analysis normalized to vinculin loading controls. Figure 8B shows the percentage of truncated modified dystrophin recovery from anterior tibial muscle tissue. [Figure 8C] We provide charts showing the percentage of truncated modified dystrophin protein by WES analysis normalized to vinculin loading controls from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or with DMD 19-20L.329 and DMD 35-36L.349 pairs of meganucleases expressed from expression constructs utilizing the tMCK promoter in either AAV9 capsid or AAVrH74 capsid at indicated doses. Figure 8C shows the percentage of truncated modified dystrophin recovery from cardiac tissue. [Figure 8D] We provide charts showing the percentage of truncated modified dystrophin protein by WES analysis normalized to vinculin loading controls from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or DMD 19-20L.329 and DMD 35-36L.349 pairs of meganucleases expressed from expression constructs utilizing the tMCK promoter in either AAV9 capsid or AAVrH74 capsid at indicated doses. Figure 8D shows the percentage of truncated modified dystrophin recovery from diaphragm tissue. [Figure 8E]This document provides charts showing the percentage of truncated modified dystrophin protein from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or DMD 19-20L.329 and DMD 35-36L.349 pairs of meganucleases expressed from expression constructs utilizing the tMCK promoter in either AAV9 capsid or AAVrH74 capsid at indicated doses, using WES analysis normalized to vinculin loading controls. Figure 8E shows the percentage of truncated modified dystrophin recovery from gastrocnemius muscle tissue. [Figure 9A] This document provides a chart showing the percentage of total ligation of genomic DNA adjacent to exons 45–55 after cleavage of the DMD19–20 and DMD35–36 recognition sequences by meganuclease pairs DMD19–20L.329 and DMD35–36L.349 in hDMDdel52 / mdx (hDMD) mice treated with either PBS or meganuclease. The meganuclease was expressed from an expression construct utilizing the tMCK promoter in either AAV9 capsid or AAVrh74 capsid at the indicated doses. Figure 9A shows the percentage of total ligation from quadriceps femoris tissue. [Figure 9B] This document provides a chart showing the percentage of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences by meganuclease pairs DMD19-20L.329 and DMD35-36L.349 in hDMDdel52 / mdx (hDMD) mice treated with either PBS or meganuclease. Meganuclease was expressed from an expression construct utilizing the tMCK promoter in either AAV9 capsid or AAVrh74 capsid at the indicated doses. Figure 9B shows the percentage of total ligation from anterior tibial muscle tissue. [Figure 9C]This document provides a chart showing the percentage of total ligation of genomic DNA adjacent to exons 45–55 after cleavage of the DMD19–20 and DMD35–36 recognition sequences by meganuclease pairs DMD19–20L.329 and DMD35–36L.349 in hDMDdel52 / mdx (hDMD) mice treated with either PBS or meganuclease. Meganuclease was expressed from an expression construct utilizing the tMCK promoter in either AAV9 capsid or AAVrh74 capsid at the indicated doses. Figure 9C shows the percentage of total ligation from cardiac tissue. [Figure 9D] This document provides a chart showing the percentage of total ligation of genomic DNA adjacent to exons 45–55 after cleavage of the DMD19–20 and DMD35–36 recognition sequences by meganuclease pairs DMD19–20L.329 and DMD35–36L.349 in hDMDdel52 / mdx (hDMD) mice treated with either PBS or meganuclease. The meganuclease was expressed from an expression construct utilizing the tMCK promoter in either AAV9 capsid or AAVrh74 capsid at the indicated doses. Figure 9D shows the percentage of total ligation from diaphragmatic tissue. [Figure 9E] This document provides a chart showing the percentage of total ligation of genomic DNA adjacent to exons 45–55 after cleavage of the DMD19–20 and DMD35–36 recognition sequences by meganuclease pairs DMD19–20L.329 and DMD35–36L.349 in hDMDdel52 / mdx (hDMD) mice treated with either PBS or meganuclease. Meganuclease was expressed from an expression construct utilizing the tMCK promoter in either AAV9 capsid or AAVrh74 capsid at the indicated doses. Figure 9E shows the percentage of total ligation from gastrocnemius muscle tissue. [Figure 10A]We provide a chart of meganuclease expression in meganuclease (ng) / total cell protein (mg) from lysates derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or with the indicated doses of 19-20 L.329 and 35-36 L.349 pairs of meganuclease. Meganuclease was expressed from an expression construct utilizing the tMCK promoter in the AAV9 capsid. Figure 10A shows meganuclease expression from quadriceps femoris tissue. [Figure 10B] We provide a chart of meganuclease expression in meganuclease (ng) / total cell protein (mg) from lysates derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or with the indicated doses of 19-20 L.329 and 35-36 L.349 pairs of meganuclease. Meganuclease was expressed from an expression construct utilizing the tMCK promoter in the AAV9 capsid. Figure 10B shows meganuclease expression from cardiac tissue. [Figure 10C] We provide a chart of meganuclease expression in meganuclease (ng) / total cell protein (mg) from lysates derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or with the indicated doses of 19-20 L.329 and 35-36 L.349 pairs of meganuclease. Meganuclease was expressed from an expression construct utilizing the tMCK promoter in the AAV9 capsid. Figure 10C shows meganuclease expression from gastrocnemius muscle tissue. [Figure 10D] We provide a chart of meganuclease expression in meganuclease (ng) / total cell protein (mg) from lysates derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or with the indicated doses of 19-20 L.329 and 35-36 L.349 pairs of meganuclease. Meganuclease was expressed from an expression construct utilizing the tMCK promoter in the AAV9 capsid. Figure 10D shows meganuclease expression from diaphragmatic tissue. [Figure 10E]We provide a chart of meganuclease expression in meganuclease (ng) / total cell protein (mg) from lysates derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or with the indicated doses of 19-20 L.329 and 35-36 L.349 pairs of meganuclease. Meganuclease was expressed from an expression construct utilizing the tMCK promoter in the AAV9 capsid. Figure 10E shows meganuclease expression from anterior tibial muscle tissue. [Figure 10F] We provide a chart of meganuclease expression in meganuclease (ng) / total cell protein (mg) from lysates derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or with the indicated doses of 19-20 L.329 and 35-36 L.349 pairs of meganuclease. Meganuclease was expressed from an expression construct utilizing the tMCK promoter in the AAV9 capsid. Figure 10F shows meganuclease expression from liver tissue. [Figure 11A] A bar graph is provided showing the percentage of Pax7-positive muscle satellite cells derived from quadriceps femoris tissue of hDMDdel52 / mdx (hDMD) mice treated with either PBS or expression constructs encoding DMD 19-20L.329 and DMD 35-36L.349 pairs of meganucleases in AAV9 capsid. [Figure 11B] This provides the percentage of Pax7-positive cells in which exons 45-55 of the dystrophin gene have been excised. [Figure 12A]This document provides a chart showing the percentage of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of DMD19-20 recognition sequences and DMD35-36 recognition sequences by meganuclease pairs DMD19-20L.329 and DMD35-36L.349 in hDMDdel52 / mdx (hDMD) mice treated with either PBS or meganuclease. Meganuclease was expressed from an expression construct utilizing a tMCK promoter in AAV9 capsid at a dose of 1e14VG / kg. Figure 12A shows the percentage of total ligation from quadriceps femoris tissue. [Figure 12B] This document provides a chart showing the percentage of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences by meganuclease pairs DMD19-20L.329 and DMD35-36L.349 in hDMDdel52 / mdx (hDMD) mice treated with either PBS or meganuclease. Meganuclease was expressed from an expression construct utilizing a tMCK promoter in an AAV9 capsid at a dose of 1e14VG / kg. Figure 12B shows the percentage of total ligation from gastrocnemius muscle tissue. [Figure 12C] This document provides a chart showing the percentage of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences by meganuclease pairs DMD19-20L.329 and DMD35-36L.349 in hDMDdel52 / mdx (hDMD) mice treated with either PBS or meganuclease. Meganuclease was expressed from an expression construct utilizing a tMCK promoter in AAV9 capsid at a dose of 1e14VG / kg. Figure 12C shows the percentage of total ligation from cardiac tissue. [Figure 12D]This document provides a chart showing the percentage of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences by meganuclease pairs DMD19-20L.329 and DMD35-36L.349 in hDMDdel52 / mdx (hDMD) mice treated with either PBS or meganuclease. Meganuclease was expressed from an expression construct utilizing a tMCK promoter in an AAV9 capsid at a dose of 1e14VG / kg. Figure 12D shows the percentage of total ligation from anterior tibial muscle tissue. [Figure 12E] This document provides a chart showing the percentage of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences by meganuclease pairs DMD19-20L.329 and DMD35-36L.349 in hDMDdel52 / mdx (hDMD) mice treated with either PBS or meganuclease. Meganuclease was expressed from an expression construct utilizing a tMCK promoter in an AAV9 capsid at a dose of 1e14VG / kg. Figure 12E shows the percentage of total ligation from diaphragmatic tissue. [Figure 12F] This document provides a chart showing the percentage of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences by meganuclease pairs DMD19-20L.329 and DMD35-36L.349 in hDMDdel52 / mdx (hDMD) mice treated with either PBS or meganuclease. Meganuclease was expressed from an expression construct utilizing a tMCK promoter in AAV9 capsid at a dose of 1e14VG / kg. Figure 12F shows the percentage of total ligation from liver tissue. [Figure 13A]This document provides charts showing the percentage of truncated modified dystrophin protein by WES analysis normalized to vinculin loading controls from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with DMD 19-20 L.329 and DMD 35-36 L.349 pairs of meganucleases expressed from expression constructs utilizing the tMCK promoter in PBS-treated AAV9 capsids at a dose of 1 e14 VG / kg. Figure 13A shows the percentage of truncated modified dystrophin recovery from quadriceps femoris tissue. [Figure 13B] This document provides charts showing the percentage of truncated modified dystrophin protein by WES analysis normalized to vinculin loading controls from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with DMD 19-20 L.329 and DMD 35-36 L.349 pairs of meganucleases expressed from expression constructs utilizing the tMCK promoter in PBS-treated or AAV9 capsids at a dose of 1 e14 VG / kg. Figure 13B shows the percentage of truncated modified dystrophin recovery from gastrocnemius tissue. [Figure 13C] This document provides charts showing the percentage of truncated modified dystrophin protein by WES analysis normalized to vinculin loading controls from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with DMD 19-20 L.329 and DMD 35-36 L.349 pairs of meganucleases expressed from expression constructs utilizing the tMCK promoter in AAV9 capsids treated with PBS or PBS. Figure 13C shows the percentage of truncated modified dystrophin recovery from cardiac tissue. [Figure 13D]This document provides charts showing the percentage of truncated modified dystrophin protein by WES analysis normalized to vinculin loading controls from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with DMD 19-20 L.329 and DMD 35-36 L.349 pairs of meganucleases expressed from expression constructs utilizing the tMCK promoter in PBS-treated AAV9 capsids at a dose of 1 e14 VG / kg. Figure 13D shows the percentage of truncated modified dystrophin recovery from anterior tibial muscle tissue. [Figure 13E] This document provides charts showing the percentage of truncated modified dystrophin protein by WES analysis normalized to vinculin loading controls from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with DMD 19-20 L.329 and DMD 35-36 L.349 pairs of meganucleases expressed from expression constructs utilizing the tMCK promoter in PBS-treated AAV9 capsids at a dose of 1 e14 VG / kg. Figure 13E shows the percentage of truncated modified dystrophin recovery from diaphragm tissue. [Figure 14] This document provides line graphs showing maximal muscle strength development in hDMDdel52 / mdx (hDMD) mice compared to non-disease mice. Mice were treated with DMD 19-20 L.329 and DMD 35-36 L.349 pairs of meganucleases expressed from an expression construct utilizing the tMCK promoter in AAV9 capsid at a dose of 1 e14 VG / kg. [Figure 15A] A chart is provided showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in hDMDdel52 / mdx (hDMD) mice treated with PBS or one of the indicated pair of meganucleases. The meganucleases were expressed from an expression construct utilizing a tMCK promoter in AAV9 capsid at a dose of 3e13VG / kg. Figure 15A shows the percentage of total ligation from quadriceps femoris tissue. [Figure 15B] A chart is provided showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in hDMDdel52 / mdx (hDMD) mice treated with PBS or one of the indicated pair of meganucleases. The meganucleases were expressed from an expression construct utilizing a tMCK promoter in AAV9 capsid at a dose of 3e13VG / kg. Figure 15B shows the percentage of total ligation from gastrocnemius muscle tissue. [Figure 15C] A chart is provided showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in hDMDdel52 / mdx (hDMD) mice treated with PBS or one of the indicated pair of meganucleases. The meganucleases were expressed from an expression construct utilizing a tMCK promoter in AAV9 capsid at a dose of 3e13VG / kg. Figure 15C shows the percentage of total ligation from cardiac tissue. [Figure 15D] A chart is provided showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in hDMDdel52 / mdx (hDMD) mice treated with PBS or one of the indicated pair of meganucleases. Meganucleases were expressed from an expression construct utilizing a tMCK promoter in AAV9 capsid at a dose of 3e13VG / kg. Figure 15D shows the percentage of total ligation from anterior tibial muscle tissue. [Figure 15E]A chart is provided showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45–55 after cleavage of the DMD19–20 and DMD35–36 recognition sequences in hDMDdel52 / mdx (hDMD) mice treated with PBS or one of the indicated pair of meganucleases. Meganucleases were expressed from an expression construct utilizing a tMCK promoter in AAV9 capsid at a dose of 3e13VG / kg. Figure 15E shows the percentage of total ligation from diaphragmatic tissue. [Figure 16A] This document provides charts showing the percentage of truncated modified dystrophin protein by WES analysis normalized to vinculin loading controls from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with the indicated pair of meganucleases, either treated with PBS or expressed from an expression construct utilizing the tMCK promoter in AAV9 capsid at a dose of 3e13VG / kg. Figure 16A shows the percentage of truncated modified dystrophin recovery from quadriceps femoris tissue. [Figure 16B] This document provides charts showing the percentage of truncated modified dystrophin protein from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with the indicated pair of meganucleases, either treated with PBS or expressed from an expression construct utilizing the tMCK promoter in AAV9 capsid at a dose of 3e13VG / kg, as measured by WES analysis normalized to vinculin loading controls. Figure 16B shows the percentage of truncated modified dystrophin recovery from gastrocnemius tissue. [Figure 16C]This document provides charts showing the percentage of truncated modified dystrophin protein from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with the indicated pair of meganucleases, either treated with PBS or expressed from an expression construct utilizing the tMCK promoter in AAV9 capsid at a dose of 3e13VG / kg, as measured by WES analysis normalized to vinculin loading controls. Figure 16C shows the percentage of truncated modified dystrophin recovery from cardiac tissue. [Figure 16D] This document provides charts showing the percentage of truncated modified dystrophin protein by WES analysis normalized to vinculin loading controls from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with the indicated pair of meganucleases, either treated with PBS or expressed from an expression construct utilizing the tMCK promoter in AAV9 capsid at a dose of 3e13VG / kg. Figure 16D shows the percentage of truncated modified dystrophin recovery from anterior tibial muscle tissue. [Figure 16E] This document provides charts showing the percentage of truncated modified dystrophin protein from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with the indicated pair of meganucleases, either treated with PBS or expressed from an expression construct utilizing the tMCK promoter in AAV9 capsid at a dose of 3e13VG / kg, as measured by WES analysis normalized to vinculin loading controls. Figure 16E shows the percentage of truncated modified dystrophin recovery from diaphragmatic tissue. [Figure 17] This provides a graph showing the percentage of dystrophin-positive fibers in hDMDdel52 / mdx (hDMD) mice treated with DMD19-20L.329 and DMD35-36L.349 pairs of meganuclease expressed from an expression construct containing the B-301 enhancer element and a tMCK promoter in AAV9 capsid at a dose of 1e14VG / kg. [Figure 18A]A chart is provided showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in hDMDdel52 / mdx (hDMD) mice treated with either PBS or the indicated expression construct. Meganucleases were expressed from an expression construct having either a B-301 enhancer element or an enhancer G element together with a tMCK promoter in an AAV9 capsid at a dose of either 3e13VG / kg or 1e14VG / kg. Figure 18A shows the percentage of total ligation from quadriceps femoris tissue. [Figure 18B] A chart is provided showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in hDMDdel52 / mdx (hDMD) mice treated with either PBS or the indicated expression construct. Meganucleases were expressed from an expression construct containing either the B-301 enhancer element or the enhancer G element along with the tMCK promoter in an AAV9 capsid at a dose of either 3e13VG / kg or 1e14VG / kg. Figure 18B shows the percentage of total ligation from cardiac tissue. [Figure 18C] A chart is provided showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in hDMDdel52 / mdx (hDMD) mice treated with either PBS or the indicated expression construct. Meganucleases were expressed from an expression construct having either a B-301 enhancer element or an enhancer G element along with a tMCK promoter in an AAV9 capsid at a dose of either 3e13VG / kg or 1e14VG / kg. Figure 18C shows the percentage of total ligation from diaphragmatic tissue. [Figure 18D]A chart is provided showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in hDMDdel52 / mdx (hDMD) mice treated with either PBS or the indicated expression construct. Meganucleases were expressed from an expression construct containing either the B-301 enhancer element or the enhancer G element along with the tMCK promoter in an AAV9 capsid at a dose of either 3e13VG / kg or 1e14VG / kg. Figure 18D shows the percentage of total ligation from gastrocnemius tissue. [Figure 19A] This chart provides the percentage of truncated modified dystrophin protein from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or the indicated pair of expression constructs, as measured by WES analysis normalized to vinculin loading controls. Meganuclease was expressed from expression constructs having either a B-301 enhancer element or an enhancer G element along with a tMCK promoter in an AAV9 capsid at either a dose of 3e13VG / kg or 1e14VG / kg. Figure 19A shows the percentage of truncated modified dystrophin protein from quadriceps femoris tissue. [Figure 19B] This chart provides the percentage of truncated modified dystrophin protein from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or the indicated pair of expression constructs, as measured by WES analysis normalized against vinculin loading controls. Meganuclease was expressed from expression constructs having either the B-301 enhancer element or the enhancer G element along with the tMCK promoter in AAV9 capsid at either 3e13VG / kg or 1e14VG / kg doses. Figure 19B shows the percentage of truncated modified dystrophin protein from cardiac tissue. [Figure 19C]This chart provides the percentage of truncated modified dystrophin protein from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or the indicated pair of expression constructs, as measured by WES analysis normalized to vinculin loading controls. Meganuclease was expressed from expression constructs containing either the B-301 enhancer element or the enhancer G element along with the tMCK promoter in AAV9 capsid at either 3e13VG / kg or 1e14VG / kg doses. Figure 19C shows the percentage of truncated modified dystrophin protein from diaphragm tissue. [Figure 19D] This chart provides the percentage of truncated modified dystrophin protein from various tissues derived from hDMDdel52 / mdx (hDMD) mice treated with PBS or the indicated pair of expression constructs, as measured by WES analysis normalized to vinculin loading controls. Meganuclease was expressed from expression constructs having either the B-301 enhancer element or the enhancer G element along with the tMCK promoter in AAV9 capsid at either 3e13VG / kg or 1e14VG / kg doses. Figure 19D shows the percentage of truncated modified dystrophin protein from gastrocnemius tissue. [Figure 20]This document provides a bar graph showing luciferin expression in AB1098, mouse muscle satellite cells, or HepG2 immortalized hepatocytes using an expression cassette with the indicated muscle enhancer, normalized to a muscle-specific expression cassette containing the tMCK promoter but without additional muscle-specific enhancers. Each bar for the treated cells (AB1098, satellite cells, or HepG2 cells) corresponds from left to right to a construct containing only the B-301 enhancer, MTF1 enhancer, SPDEF1 enhancer, enhancer F, enhancer G, enhancer I, and MHCK7 promoter. The first plasmid construct contained the B301 enhancer, muscle-specific promoter tMCK, firefly luciferase, WPRE element, and SV40 polyadenylation signal from 5' to 3'. The second plasmid construct contained an MTF1 enhancer, a muscle-specific promoter tMCK, firefly luciferase, a WPRE element, and an SV40 polyadenylation signal in the 5' to 3' region. The third plasmid construct contained an MTSPDEF1 enhancer, a muscle-specific promoter tMCK, firefly luciferase, a WPRE element, and an SV40 polyadenylation signal in the 5' to 3' region. The fourth plasmid construct contained enhancer F, a muscle-specific promoter tMCK, firefly luciferase, a WPRE element, and an SV40 polyadenylation signal in the 5' to 3' region. The fifth plasmid construct contained enhancer G, a muscle-specific promoter tMCK, firefly luciferase, a WPRE element, and an SV40 polyadenylation signal in the 5' to 3' region. The sixth plasmid construct contained enhancer I, muscle-specific promoter tMCK, firefly luciferase, WPRE element, and SV40 polyadenylation signal in 5' to 3'. The seventh plasmid construct contained muscle-specific promoter MHCK7, firefly luciferase, WPRE element, and SV40 polyadenylation signal in 5' to 3'. The eighth plasmid construct contained muscle-specific promoter tMCK, firefly luciferase, WPRE element, and SV40 polyadenylation signal in 5' to 3'. [Figure 21A] This chart provides the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in various muscle tissues from hDMDdel52 / mdx (hDMD) mice treated with 3e13VG / kg or 1e14VG / kg AAV encoding DMD19-20L.431 meganuclease and DMD35-36L.457 meganuclease, or with PBS as a control. The percentage of total ligation observed in the quadriceps femoris muscle is illustrated. [Figure 21B] This document provides charts showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in various muscle tissues from hDMDdel52 / mdx (hDMD) mice treated with 3e13VG / kg or 1e14VG / kg AAV encoding DMD19-20L.431 meganuclease and DMD35-36L.457 meganuclease, or with PBS as a control. The percentage of total ligation observed in the gastrocnemius muscle is illustrated. [Figure 21C] This chart provides the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in various muscle tissues from hDMDdel52 / mdx (hDMD) mice treated with 3e13VG / kg or 1e14VG / kg AAV encoding the DMD19-20 L.431 meganuclease and DMD35-36 L.457 meganuclease, or with PBS as a control. The percentage of total ligation observed in the tibialis anterior muscle (TA) is illustrated. [Figure 21D]This document provides charts showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in various muscle tissues from hDMDdel52 / mdx (hDMD) mice treated with 3e13VG / kg or 1e14VG / kg AAV encoding DMD19-20L.431 meganuclease and DMD35-36L.457 meganuclease, or with PBS as a control. The percentage of total ligation observed in the heart is illustrated. [Figure 21E] This document provides charts showing the percentage of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in various muscle tissues from hDMDdel52 / mdx (hDMD) mice treated with 3e13VG / kg or 1e14VG / kg AAV encoding the DMD19-20 L.431 meganuclease and DMD35-36 L.457 meganuclease, or with PBS as a control. The percentage of total ligation observed in the diaphragm is illustrated. [Figure 22A] This document provides charts showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in various tissues from hDMDdel52 / mdx (hDMD) mice treated with 3e13VG / kg or 1e14VG / kg AAV encoding the DMD19-20 L.431 meganuclease and DMD35-36 L.457 meganuclease, or with PBS as a control. The percentage of total ligation observed in the brain is illustrated. [Figure 22B]This document provides charts showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in various tissues from hDMDdel52 / mdx (hDMD) mice treated with 3e13VG / kg or 1e14VG / kg AAV encoding DMD19-20L.431 meganuclease and DMD35-36L.457 meganuclease, or with PBS as a control. The percentage of total ligation observed in the liver is illustrated. [Figure 22C] This document provides charts showing the percentage (%) of total ligation of genomic DNA adjacent to exons 45-55 after cleavage of the DMD19-20 and DMD35-36 recognition sequences in various tissues from hDMDdel52 / mdx (hDMD) mice treated with 3e13VG / kg or 1e14VG / kg AAV encoding DMD19-20L.431 meganuclease and DMD35-36L.457 meganuclease, or with PBS as a control. The percentage of total ligation observed in the lungs is illustrated.

[0108] A brief explanation of arrays Sequence ID 1 shows the nucleic acid sequence of the B-301 muscle-specific enhancer element. Sequence ID 2 shows the nucleic acid sequence of the MCK basic promoter region from position -358 to +7. Sequence ID 3 shows the nucleic acid sequence of the MCK basic promoter region starting from position -80+7. Sequence ID 4 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 5 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 6 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 7 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 8 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 9 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 10 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 11 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 12 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 13 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 14 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 15 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 16 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 17 shows the nucleic acid sequence of the MCK-R regulatory element. Sequence ID 18 shows the nucleic acid sequences of the mouse MCK promoter and enhancer. Sequence ID 19 shows the nucleic acid sequence of the human MCK promoter. Sequence ID 20 shows the nucleic acid sequence of the wild-type mouse MCK enhancer sequence. Sequence ID 21 shows the nucleic acid sequence of the modified mouse MCK enhancer sequence. Sequence ID 22 shows the nucleic acid sequence of the tMCK promoter sequence. Sequence ID 23 shows the nucleic acid sequence of the MHCK7 promoter sequence. Sequence ID 24 shows the nucleic acid sequence of the WPRE sequence. Sequence ID 25 shows the amino acid sequence of the SV40 nuclear localization sequence. Sequence ID 26 shows the amino acid sequence of NLS5. Sequence ID 27 shows the amino acid sequence of c-myc NLS. Sequence ID 28 shows the amino acid sequence of SV40H2 NLS. Sequence ID 29 shows the nucleic acid sequence of the SV40 polyA signal. Sequence ID 30 shows the amino acid sequence of the rh.74 capsid sequence. Sequence ID 31 shows the amino acid sequence of the AAV9 capsid sequence. Sequence ID 32 shows the nucleic acid sequence of the Kozak sequence. Sequence ID 33 shows the amino acid sequence of wild-type I-CreI meganuclease from Chlamydomonas reinhardtii. Sequence ID 34 shows the amino acid sequence of the LAGLIDADG motif. Sequence ID 35 shows the amino acid sequence of the wild-type dystrophin protein CCDS48091.1 (gene ID 1756). Sequence ID 36 shows the amino acid sequence of the wild-type dystrophin protein CCDS48091.1 (gene ID 1756), which lacks the amino acids encoded by exons 45-55. Sequence ID 37 shows the nucleic acid sequence of the sense strand of the DMD19-20 recognition sequence. Sequence ID 38 shows the nucleic acid sequence of the antisense strand of the DMD19-20 recognition sequence. Sequence ID 39 shows the nucleic acid sequence of the sense strand of the DMD35-36 recognition sequence. Sequence ID 40 shows the nucleic acid sequence of the antisense strand of the DMD35-36 recognition sequence. Sequence ID 41 shows the nucleic acid sequences of the ligated hybrid DMD19-20 / 35-36 sense strands. Sequence ID 42 shows the nucleic acid sequences of the ligated hybrid DMD19-20 / 35-36 antisense strands. Sequence ID 43 shows the amino acid sequence of the DMD 19-20x.13 artificial meganuclease. Sequence ID 44 shows the amino acid sequence of the DMD 19-20x.87 artificial meganuclease. Sequence ID 45 shows the amino acid sequence of the DMD 19-20L.249 artificial meganuclease. Sequence ID 46 shows the amino acid sequence of the DMD 19-20L.302 artificial meganuclease. Sequence ID 47 shows the amino acid sequence of the DMD 19-20L.329 artificial meganuclease. Sequence ID 48 shows the amino acid sequence of the DMD 19-20L.374 artificial meganuclease. Sequence ID 49 shows the amino acid sequence of the DMD 19-20L.375 artificial meganuclease. Sequence ID 50 shows the amino acid sequence of the DMD 19-20L.431 artificial meganuclease. Sequence ID 51 shows the amino acid sequence of the DMD 19-20L.458 artificial meganuclease. Sequence ID 52 shows the amino acid sequence of the DMD 35-36x.63 artificial meganuclease. Sequence ID 53 shows the amino acid sequence of the DMD 35-36x.81 artificial meganuclease. Sequence ID 54 shows the amino acid sequence of the DMD 35-36L.195 artificial meganuclease. Sequence ID 55 shows the amino acid sequence of the DMD 35-36L.282 artificial meganuclease. Sequence ID 56 shows the amino acid sequence of the DMD 35-36L.349 artificial meganuclease. Sequence ID 57 shows the amino acid sequence of the DMD 35-36L.376 artificial meganuclease. Sequence ID 58 shows the amino acid sequence of the DMD 35-36L.457 artificial meganuclease. Sequence ID 59 shows the amino acid sequence of the DMD 35-36L.469 artificial meganuclease. Sequence ID 60 shows the nucleic acid sequence of the DMD 19-20x.13 artificial meganuclease. Sequence ID 61 shows the nucleic acid sequence of the DMD 19-20x.87 artificial meganuclease. Sequence ID 62 shows the nucleic acid sequence of the DMD 19-20L.249 artificial meganuclease. Sequence ID 63 shows the nucleic acid sequence of the DMD 19-20L.302 artificial meganuclease. Sequence ID 64 shows the nucleic acid sequence of the DMD 19-20L.329 artificial meganuclease. Sequence ID 65 shows the nucleic acid sequence of the DMD 19-20L.374 artificial meganuclease. Sequence ID 66 shows the nucleic acid sequence of the DMD 19-20L.375 artificial meganuclease. Sequence ID 67 shows the nucleic acid sequence of the DMD 19-20L.431 artificial meganuclease. Sequence ID 68 shows the nucleic acid sequence of the DMD 19-20L.458 artificial meganuclease. Sequence ID 69 shows the nucleic acid sequence of the DMD 35-36x.63 artificial meganuclease. Sequence ID 70 shows the nucleic acid sequence of the DMD 35-36x.81 artificial meganuclease. Sequence ID 71 shows the nucleic acid sequence of the DMD 35-36L.195 artificial meganuclease. Sequence ID 72 shows the nucleic acid sequence of the DMD 35-36L.282 artificial meganuclease. Sequence ID 73 shows the nucleic acid sequence of the DMD 35-36L.349 artificial meganuclease. Sequence ID 74 shows the nucleic acid sequence of the DMD 35-36L.376 artificial meganuclease. Sequence ID 75 shows the nucleic acid sequence of the DMD 35-36L.457 artificial meganuclease. Sequence ID 76 shows the nucleic acid sequence of the DMD 35-36L.469 artificial meganuclease. Sequence ID 77 shows the amino acid sequence of the linker sequence. Sequence ID 78 shows the nucleic acid sequence of a muscle-specific expression cassette containing the muscle-specific promoter MHCK7, the coding sequences for DMD19-20L.329 meganucleases, the furin GSG P2A cleavage sequence, the coding sequences for DMD35-36L.349 meganucleases, the WPRE element, and the SV40 polyadenylation signal, from 5' to 3'. Sequence ID 79 shows the nucleic acid sequence of a muscle-specific expression cassette containing the muscle-specific promoter tMCK, coding sequences for DMD19-20L.329 nucleases, furin GSG P2A cleavage sequence, coding sequences for DMD35-36L.349 nucleases, WPRE element, and SV40 polyadenylation signal, from 5' to 3'. Sequence ID 80 shows the nucleic acid sequence of a muscle-specific expression cassette containing the muscle-specific promoter tMCK, coding sequences for DMD19-20L.329 meganucleases, furin GSG P2A cleavage sequence, coding sequences for DMD35-36L.349 meganucleases, WPRE elements, and the SV40 polyadenylation signal, from 5' to 3'. Sequence ID 81 shows the nucleic acid sequence of a muscle-specific expression cassette containing the muscle-specific promoter tMCK, coding sequences for DMD19-20L.431 meganucleases, furin GSG P2A cleavage sequence, coding sequences for DMD35-36L.469 meganucleases, WPRE elements, and the SV40 polyadenylation signal, from 5' to 3'. Sequence ID 82 shows the nucleic acid sequence of a muscle-specific expression cassette containing the muscle-specific promoter tMCK, the coding sequences for DMD19-20L.458 meganucleases, the furin GSG P2A cleavage sequence, the coding sequences for DMD35-36L.469 meganucleases, the WPRE element, and the SV40 polyadenylation signal, from 5' to 3'. Sequence ID 83 shows the nucleic acid sequence of a muscle-specific expression cassette containing the muscle-specific promoter tMCK, coding sequences for DMD19-20L.431 meganucleases, furin GSG P2A cleavage sequences, coding sequences for DMD35-36L.457 meganucleases, WPRE elements, and the SV40 polyadenylation signal, from 5' to 3'. Sequence ID 84 shows the nucleic acid sequence of a muscle-specific expression cassette containing the muscle-specific promoter tMCK, the coding sequences for DMD19-20L.458 meganucleases, the furin GSG P2A cleavage sequence, the coding sequences for DMD35-36L.457 meganucleases, the WPRE element, and the SV40 polyadenylation signal, from 5' to 3'. Sequence ID 85 shows the nucleic acid sequence of a muscle-specific expression cassette containing the B-301 enhancer, muscle-specific promoter tMCK, coding sequences for DMD19-20L.329 nucleases, furin GSG P2A cleavage sequence, coding sequences for DMD35-36L.349 nucleases, WPRE element, and SV40 polyadenylation signal, from 5' to 3'. Sequence ID 86 shows the nucleic acid sequence of a muscle-specific expression cassette containing the B-301 enhancer, muscle-specific promoter tMCK, coding sequences for DMD19-20L.431 meganuclease, furin GSG P2A cleavage sequence, coding sequences for DMD35-36L.457 meganuclease, WPRE element, and SV40 polyadenylation signal from 5' to 3', with the cassette elements having reduced CpG. Sequence ID 87 shows the nucleic acid sequence of a muscle-specific expression cassette containing the B-301 enhancer, muscle-specific promoter tMCK, coding sequences for DMD19-20L.431 meganuclease, furin GSG P2A cleavage sequence, coding sequences for DMD35-36L.457 meganuclease, WPRE element, and SV40 polyadenylation signal, from 5' to 3'. Sequence ID 88 shows the nucleic acid sequences of forward PCR primers used in ddPCR assays for ligated recognition sequences from DMD 19-20 to DMD 35-36. Sequence ID 89 shows the nucleic acid sequences of reverse PCR primers used in ddPCR assays for ligated recognition sequences from DMD 19-20 to DMD 35-36. Sequence ID 90 shows the nucleic acid sequence of the probe used in the ddPCR assay for ligated recognition sequences from DMD 19-20 to DMD 35-36. Sequence ID 91 shows the nucleic acid sequence of the primer used to generate the reference amplicon used in the ddPCR assay. Sequence ID 92 shows the nucleic acid sequence of the primer used to generate the reference amplicon used in the ddPCR assay. Sequence ID 93 shows the nucleic acid sequence of the primer used to generate the reference amplicon used in the ddPCR assay. Sequence ID 94 shows the nucleic acid sequence of the AAV9 5' ITR D sequence. Sequence ID 95 shows the nucleic acid sequence of AAV9 3' ITR D sequence. Sequence ID 96 shows the nucleic acid sequence of the DMD 35-36L.349 artificial meganuclease with no codon alteration. Sequence ID 97 shows the nucleic acid sequence of the DMD 35-36L.457 artificial meganuclease, which has not undergone codon modification. Sequence ID 98 shows the nucleic acid sequence of the DMD 35-36L.469 artificial meganuclease with no codon alteration. Sequence ID 99 shows the nucleic acid sequence of the DMD 19-20L.431 artificial meganuclease, which is composed of the construct shown in Sequence ID 87. Sequence ID 100 shows the nucleic acid sequence of the DMD 35-36L.457 artificial meganuclease, which is composed of the construct shown in Sequence ID 87. Sequence ID 101 shows a nucleic acid sequence with reduced CpG of the DMD 19-20L.431 artificial meganuclease, which is composed of the construct shown in Sequence ID 86. Sequence ID 102 shows a nucleic acid sequence with reduced CpG of the DMD 35-36L.457 artificial meganuclease, which is composed of the construct shown in Sequence ID 86. Sequence ID 103 shows the amino acid sequence of the furin GSG P2A cleavage sequence. Sequence ID 104 shows the nucleic acid sequence of the furin GSG P2A cleavage sequence. Sequence ID 105 shows a nucleic acid sequence in which the CpG of the furin GSG P2A cleavage sequence is reduced. Sequence ID 106 shows the nucleic acid sequence of the SV40 nuclear localization sequence. Sequence ID 107 shows a nucleic acid sequence in which the CpG of the SV40 nuclear localization sequence is reduced. Sequence ID 108 shows the nucleic acid sequence of the c-myc nuclear localization sequence. Sequence ID 109 shows a nucleic acid sequence in which the CpG of the c-myc nuclear localization sequence is reduced. Sequence ID 110 shows the nucleic acid sequence of the SV40 nuclear localization sequence. Sequence ID 111 shows a nucleic acid sequence in which the CpG of the SV40 nuclear localization sequence is reduced. Sequence ID 112 shows the nucleic acid sequence of the c-myc nuclear localization sequence. Sequence ID 113 shows a nucleic acid sequence in which the CpG of the c-myc nuclear localization sequence is reduced. Sequence ID 114 shows the nucleic acid sequence of the SV40 poly(A) signal. Detailed description of the invention

[0109] 1.1 References and Definitions The patents and scientific literature referenced herein establish knowledge available to those skilled in the art. References cited herein, including issued U.S. patents, granted patent applications, published foreign patent applications, and GenBank database arrays, are incorporated herein by reference to the same extent as each is specifically and individually indicated to be incorporated by reference.

[0110] This disclosure can be embodied in different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so as to make this disclosure thorough and complete and fully convey the scope of this disclosure to those skilled in the art. For example, features shown in one embodiment can be incorporated into other embodiments, and features shown in a particular embodiment can be omitted from that embodiment. Furthermore, numerous modifications and additions to the embodiments proposed herein are obvious to those skilled in the art in light of this disclosure and do not deviate from it.

[0111] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs. The terms used herein are intended solely to describe, and not to limit, specific embodiments.

[0112] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety.

[0113] As used herein, “a,” “an,” or “the” can mean one or more. For example, “a” cell can mean a single cell or a number of cells. Where used herein, unless otherwise specified, the word “or” is used in the comprehensive sense of “and / or” and not in the exclusive sense of “either / or.”

[0114] As used herein, all polynucleotide sequences written using the standard nucleic acid notation of the International Union of Pure and Applied Chemistry (IUPAC, Biochemistry (1970) Vol. 9: 4022-4027); adenine (A), thymine (T), guanine (G), and cytosine (C) are equivalent to their corresponding RNA polynucleotide sequences. Thus, "T" (thymine) in all sequences is equivalent to "U" (uracil). For example, the sequence AATAAA in the DNA coding strand also represents the corresponding mRNA sequence AAUAAA.

[0115] As used herein, the term “codon-modified” refers to the process by which a nucleotide sequence encoding a heterologous protein (e.g., an artificial nuclease such as an artificial meganuclease) is modified to utilize an alternative trinucleotide codon without altering the amino acid sequence originally encoded by the resulting nucleic acid coding sequence. Modification of nucleic acid sequence codons relies on the fact that in eukaryotic cells, there exist multiple trinucleotide codons encoding the same amino acid, where the third position in the codon can be selected from multiple nucleotides, i.e., “fluctuate.” As used herein, the terms “polynucleotide,” “DNA,” or “nucleic acid” are not intended to limit this disclosure to polynucleotides including DNA. Those skilled in the art will recognize that polynucleotides may include ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The polynucleotides described herein also encompass all forms of sequences, including but not limited to single-stranded, double-stranded, hairpin, and stem-and-loop structures.

[0116] As used herein, the term “Kozak sequence” refers to a nucleic acid motif that functions as a protein translation initiation site in most eukaryotic mRNA transcripts. Vertebrate Kozak sequences have a consensus sequence of “gcc A / G ccATGG”, where uppercase positions are conserved more than lowercase positions, and ATG is the start codon. Thus, the Kozak sequence spans the 5'UTR and the coding sequence, with the portion within the 5'UTR being the UTR Kozak sequence. For example, the UTR Kozak sequence is the portion of the Kozak sequence consisting of the first to sixth base pairs. In various embodiments, the first nucleotide of the Kozak sequence is A or G. In various embodiments, the second nucleotide of the Kozak sequence is C or T. In various embodiments, the third nucleotide of the Kozak sequence is A or C. In various embodiments, the fourth nucleotide of the Kozak sequence is A or G. In various embodiments, the fifth nucleotide of the Kozak sequence is A or C. In various embodiments, the sixth nucleotide of the Kozak sequence is A, C, or G. In certain embodiments, the Kozak sequence includes the sequence GCCACC, which is part of the 5'UTR. In various embodiments, the seventh to tenth nucleotides of the Kozak sequence are ATGG. In certain embodiments, the Kozak sequence may include part of a polynucleotide NLS. A suitable Kozak sequence is described in PCT International Patent Application No. PCT / US2023 / 060258. For example, the Kozak sequence may include the sequence ATGGC, which is part of the SV40 NLS. In various embodiments, the Kozak sequence includes SEQ ID NO: 32.

[0117] As used herein, the term “muscle-specific enhancer” refers to a nucleotide element operably ligated to a promoter (e.g., a muscle-specific promoter) that increases the expression of a transgene in a muscle cell. The muscle-specific enhancers described herein are less than 1 kb, less than 500 bp, less than 250 bp, and less than 150 bp. These muscle-specific enhancers include one or more transcription factor binding sites, including an E-box motif, an AP-1 binding motif, and a homing domain binding motif. In some embodiments, such muscle-specific enhancers drive the expression of a transgene in muscle progenitor cells (e.g., Pax7-positive muscle satellite cells). In some embodiments, the muscle-specific enhancer includes SEQ ID NO: 1.

[0118] As used herein, the term “MCK-R control element” refers to a control element typically found in an MCK promoter enhancer array having a consensus sequence AACAc / gc / gTGCa / t. In some embodiments, the MCK-R control element includes one of sequence numbers 4 to 17.

[0119] As used herein, the term “basic promoter” refers to a portion of promoters that interact with regulatory elements and transcription factors involved in the initiation of transcription of a gene or, in some embodiments described herein, an exogenous gene. Such basic promoters typically include TATA boxes and / or downstream promoter elements (DPEs). In some embodiments, the basic promoter is derived from an MCK promoter. In some embodiments, the basic promoter derived from an MCK promoter is about 50 bp to about 1,000 bp long. In some embodiments, the basic promoter derived from an MCK promoter is about 400 bp long. In some embodiments, the basic promoter derived from an MCK promoter is about 90 bp long. In some embodiments, the basic promoter includes a nucleic acid sequence represented by the sequence shown in Sequence ID No. 2. In some embodiments, the basic promoter includes a nucleic acid sequence represented by the sequence shown in Sequence ID No. 3.

[0120] As used herein, the term truncated MCK promoter (or tMCK) refers to an MCK promoter that is abbreviated compared to a wild-type MCK promoter. Useful tMCK promoters as described herein may include truncated mouse or human MCK promoters. In some embodiments, the tMCK promoter includes a truncated promoter that includes a portion of a WT mouse MCK promoter with the sequence shown in Sequence ID No. 18. In some embodiments, the tMCK promoter includes a truncated promoter that includes a portion of a WT human MCK promoter with the sequence shown in Sequence ID No. 19. A suitable tMCK promoter includes at least one MCK enhancer element. In some embodiments, the tMCK promoter described herein includes three MCK enhancer elements as described herein. In some embodiments, the tMCK promoter includes a nucleic acid sequence with the sequence shown in Sequence ID No. 22.

[0121] As used herein, the term MCK enhancer element refers to a portion of an MCK promoter that enhances or increases the expression of a gene operably linked to the MCK promoter. Such MCK enhancers include various transcription factor binding sites. In some embodiments, an MCK enhancer element refers to a sequence containing one or more transcription factor binding motifs, including CArg, AP2, Trex, E-Box, NcoI, or MEF2 motifs. In some embodiments, an MCK enhancer element includes SEQ ID NO: 20 or 21.

[0122] As used herein, the term post-transcriptional regulatory element (PTRE) refers to a nucleotide sequence that functions to increase mRNA stability and mRNA cytoplasmic transport. Stability of intron-less genes is typically enhanced by facilitating mRNA transport from the nucleus to the cytoplasm and increasing 3'-terminus processing and stability. Suitable PTREs include, but are not limited to, PTREs derived from viruses, including, hepatitis B virus (HPRE) and woodchuck hepatitis virus (WPRE). In some embodiments described herein, the PTRE is a WPRE. In some embodiments, the WPRE comprises a nucleotide sequence represented by the sequence shown in SEQ ID NO: 24.

[0123] As used herein, the term “nucleoprotein” refers to any recombinant or naturally occurring protein that exhibits any biological effect within the nucleus of a cell. In some embodiments described herein, nucleoproteins include DNA-binding regulatory proteins and artificial nucleases. In certain embodiments, nucleoproteins described herein include artificial meganucleases.

[0124] As used herein, the term “DNA-binding regulatory protein” refers to any protein that binds to DNA and regulates gene expression without directly editing the DNA. Such proteins typically function within the cell nucleus. Gene expression regulation includes influencing the transcription of mRNA from DNA. Mechanisms by which DNA-binding regulatory proteins influence mRNA transcription may include both epigenetic modifications (e.g., methylation, histone modification, and nucleosome positioning) and direct influences on transcription rate by functioning as transcription factors (e.g., artificial transcription factors having activator or repressor domains). Such artificial transcription factors are well known; see, for example, PCT International Patent Application Publication WO / 2003 / 066828.

[0125] As used herein, the terms “nuclease” and “endonuclease” are used interchangeably to refer to naturally occurring or artificial enzymes that cleave phosphodiester bonds within polynucleotide chains. Artificial nucleases include, but are not limited to, artificial meganucleases, zinc finger nucleases, TALENs, compact TALENs, CRISPR system nucleases, and megaTAL. Furthermore, any artificial nuclease capable of generating an overhang at its cleavage site is also conceivable.

[0126] As used herein, the term “compact TALEN” refers to an endonuclease containing a DNA-binding domain having one or more TAL domain repeats fused in any orientation to any portion of an I-TevI ​​homing endonuclease or any of the endonucleases listed in Table 2 of U.S. Patent Application Publication No. 2013 / 0117869, including but not limited to MmeI, EndA, End1, I-BasI, I-TevII, I-TevIII, I-TwoI, MspI, MvaI, NucA, and NucM. Compact TALENs do not require dimerization for DNA processing activity and reduce the need for a dual target site with an intervening DNA spacer. In some embodiments, compact TALENs contain 16 to 22 TAL domain repeats.

[0127] As used herein, the terms “CRISPR nuclease” or “CRISPR system nuclease” refer to a CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) endonucleases, such as Cas9 or Cas12a, or their variants, that associate with a guide RNA that directs nucleic acid cleavage by the associated endonucleases by hybridizing to a recognition site within a polynucleotide. In certain embodiments, the CRISPR nuclease is a class 2 CRISPR enzyme. In some of these embodiments, the CRISPR nuclease is a class 2 type II enzyme, e.g., Cas9. In other embodiments, the CRISPR nuclease is a class 2 type V enzyme, e.g., Cpf1. The guide RNA comprises a direct repeat and a guide sequence complementary to the target recognition site (often referred to as a spacer in relation to the endogenous CRISPR system). In certain embodiments, the CRISPR system further includes a tracrRNA (trans-activated CRISPR RNA) that is complementary (completely or partially) to a direct repeat sequence (sometimes called a tracrmate sequence) present on the guide RNA. In certain embodiments, the CRISPR nuclease can be mutated from the corresponding wild-type enzyme so that the enzyme functions as a nickase and lacks the ability to cleave single strands of target polynucleotides, cleaving only single strands of target DNA. Non-limiting examples of CRISPR enzymes that function as nickases include the Cas9 enzyme having a D10A mutation in the RuvC I catalytic domain, or having H840A, N854A, or N863A mutations.Given a given DNA locus, the recognition sequence can be identified using many programs known in the art (Labun et al. (2016). CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Research; doi:10.1093 / nar / gkw398; Montague et al. (2014). CHOPCHOP: a CRISPR / Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42. W401-W407). Further CRISPR system nucleases include CRISPR-based prime editor systems and base editor systems. CRISPR system nucleases can further include CRISPR-based epigenetic editing systems that modify the epigenome without directly altering gene sequences (Nakamura, M., Gao, Y., Dominguez, AA et al. CRISPR technologies for precise epigenome editing. Nat Cell Biol 23, 11-22 (2021)).

[0128] As used herein, a prime editor is an enzyme-based nuclease that mediates insertions, deletions, and base transpositions without producing double-strand breaks. An example of such a prime editor is the CRISPR / Cas9 prime editor described in Anzalone et al. (2019) Nature 576(7785)149-157. In this system, the pegRNA comprises a single guide RNA having a primer-binding site and a reverse transcriptase template. When editing a genome, the DNA is cleaved, the primer-binding site hybridizes the cleaved DNA strand with the pegRNA, and the template acts as a synthetic template for editing the gene. Compositions and methods of this disclosure utilizing a prime editing system may include a CRISPR nuclease (e.g., Cas9 H840A nickase) and pegRNA(or nucleic acid) encoding a CRISPR nuclease and / or pegRNA(or more).

[0129] As used herein, a base editor is based on an enzyme capable of catalyzing the conversion of cytosine / guanosine to thymidine / alanine. Such base editors include a CRISPR / Cas9 enzyme fused to a cytidine deaminase enzyme that does not induce double-strand breaks. The Cas9 enzyme is typically inactivated to allow for longer cleavage of DNA, but still functions to bind to DNA along with guide RNA. Mutations that inactivate the Cas9 enzyme include the Asp10Ala mutation and the His840Ala mutation. These base editors perform cytosine to thymidine or guanosine to adenosine substitutions, as described in Komor et al. (2016) Nature 533(7603)420-424. Another exemplary base editor includes a CRISPR-free system based on a transcription activator-like effector (TALE) protein fused to a double-stranded DNA-specific cytidine deaminase, as described by Mok et al., (2022) Nature Biotechnology 40 1378-1387.

[0130] As used herein, the term "megaTAL" refers to a single-stranded endonuclease containing a TALE DNA-binding domain having an artificial sequence-specific homing endonuclease.

[0131] As used herein, the term "TALEN" refers to an endonuclease containing a DNA-binding domain that includes a nuclease domain or active portion fused to a TAL domain repeat from an endonuclease or exonuclease, including but not limited to restriction endonucleases, homing endonucleases, S1 nucleases, manguinea nucleases, pancreatic DNAse I, micrococcus nucleases, and yeast HO endonucleases. See, for example, Christian et al. (2010) Genetics 186:757-761, which is incorporated in its entirety by reference. Nuclease domains useful for TALEN design include those derived from type IIs restriction endonucleases, including but not limited to FokI, FoM, StsI, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI. Further IIs-type restriction endonucleases are described in International Publication No. WO2007 / 014275, which is incorporated in its entirety by reference. In some embodiments, the nuclease domain of the TALEN is the FokI nuclease domain or its active portion. The TAL domain repeat may be derived from the TALE family of proteins used in the infection process by plant pathogens of the genus Xanthomonas. The TAL domain repeat is a 33-34 amino acid sequence having different 12th and 13th amino acids. These two positions are called repeat variable dipeptides (RVDs), which are highly variable and strongly correlate with specific nucleotide recognition. Each base pair in the DNA target sequence is brought into contact with a single TAL repeat with specificity arising from the RVD. In some embodiments, the TALEN contains 16-22 TAL domain repeats. DNA cleavage by the TALEN requires two DNA recognition regions (i.e., "half-sites") adjacent to a nonspecific central region (i.e., "spacer"). In relation to TALENs, the term "spacer" refers to a nucleic acid sequence that separates two nucleic acid sequences that are recognized and bound by each monomer that makes up the TALEN.TAL domain repeats may be naturally occurring sequences derived from naturally occurring TALE proteins, or they may be redesigned by reasonable or experimental means to produce proteins that bind to a given DNA sequence (see, for example, Boch et al. (2009) Science 326(5959):1509-1512 and Moscou and Bogdanove (2009) Science 326(5959):1501, which are incorporated in their entirety by reference). See also U.S. Patent Application Publication 2011 / 0145940 and PCT International Patent Application Publication WO / 2010 / 079430 for methods of manipulating TALENs to recognize and bind to specific sequences, as well as examples of RVDs and their corresponding target nucleotides. In some embodiments, each nuclease (e.g., FokI) monomer can fuse to a TAL effector sequence that recognizes and binds to a different DNA sequence, and inactive monomers combine to produce a functional enzyme only when the two recognition sites are in close proximity. It should be understood that the term "TALEN" can refer to a single TALEN protein or a pair of TALEN proteins (i.e., a left TALEN protein and a right TALEN protein) that bind to upstream and downstream half-sites adjacent to a TALEN spacer sequence and work together to generate cleavage sites within the spacer sequence. Given a given DNA locus or spacer sequence, the upstream and downstream half-sites can be identified using several programs known in the art (Kornel Labun; Tessa G. Montague; James A. Gagnon; Summer B. Thyme; Eivind Valen. (2016)).CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Research; doi:10.1093 / nar / gkw398; Tessa G. Montague; Jose M. Cruz; James A. Gagnon; George M. Church; Eivind Valen. (2014). CHOPCHOP: a CRISPR / Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42. W401-W407). It should also be understood that the TALEN recognition sequence can be defined as the DNA binding sequence of a single TALEN protein (i.e., the halfsite), or as a DNA sequence containing an upstream halfsite, a spacer sequence, and a downstream halfsite.

[0132] As used herein, the terms “zinc finger nuclease” or “ZFN” refer to a chimeric protein comprising a zinc finger DNA-binding domain fused to a nuclease domain from an endonuclease or exonuclease, including but not limited to restriction endonucleases, homing endonucleases, S1 nucleases, manguinea nucleases, pancreatic DNAse I, micrococcus nucleases, and yeast HO endonucleases. Nuclease domains useful for designing zinc finger nucleases include those derived from IIs-type restriction endonucleases, including but not limited to FokI, FoM, and StsI restriction enzymes. Further IIs-type restriction endonucleases are described in PCT International Patent Publication WO2007 / 014275, which is incorporated in whole by reference. The structure of the zinc finger domain is stabilized by the coordination of zinc ions. DNA-binding proteins comprising one or more zinc finger domains bind to DNA in a sequence-specific manner. Zinc finger domains can be naturally occurring sequences or redesigned by rational or experimental means to produce proteins that bind to a given DNA sequence of approximately 18 base pairs in length, containing a pair of 9 base pairs of half-sites separated by 2 to 10 base pairs. For example, see U.S. Patents 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,200,759, and PCT International Patent Application Publications WO95 / 19431, WO96 / 06166, WO98 / 53057, WO98 / 54311, WO00 / 27878, WO01 / 60970, WO01 / 88197, and WO02 / 099084 (each of these is incorporated in its entirety by reference). By fusing this artificial protein domain to a nuclease domain such as FokI nuclease, it is possible to target DNA cleavage with genome-level specificity.The selection of target sites, zinc finger proteins, and methods for designing and constructing zinc finger nucleases are known to those skilled in the art and are described in detail in U.S. Patent Applications Publications 2003 / 0232410, 2005 / 0208489, 2005 / 064474, 2005 / 0026157, 2006 / 0188987 and PCT International Patent Application Publication WO 07 / 014275, each of which is incorporated in whole by reference. In the case of zinc fingers, the DNA-binding domain typically recognizes an 18 bp recognition sequence containing a pair of 9 base pair "half-sites" separated by a 2-10 base pair "spacer sequence," and cleavage by the nuclease produces variable-length (often 4 base pairs) blunt ends or 5' overhangs. It should be understood that the term "zinc finger nuclease" can refer to a single zinc finger protein or a pair of zinc finger proteins (i.e., a left ZFN protein and a right ZFN protein) that bind to upstream and downstream half-sites adjacent to a zinc finger nuclease spacer sequence and work together to generate cleavage sites within the spacer sequence. Given a given DNA locus or spacer sequence, the upstream and downstream half-sites can be identified using several programs known in the art (Mandell and Barbas 3rd. Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res. 2006 Jul 1;34 (Web Server issue):W516-23). ​​It should also be understood that a zinc finger nuclease recognition sequence can be defined as the DNA-binding sequence (i.e., half-site) of a single zinc finger nuclease protein, or as a DNA sequence containing the upstream half-site, the spacer sequence, and the downstream half-site.

[0133] As used herein, the terms “cleave” or “cleave” refer to the hydrolysis of a phosphodiester bond within the backbone of a recognition sequence in the target sequence, which results in a double-strand break in the target sequence, as referred herein to as a “cleavage site.”

[0134] As used herein, the term “meganucleases” refers to endonucleases that bind to double-stranded DNA with a recognition sequence of more than 12 base pairs. In some embodiments, the recognition sequences of the meganucleases of this disclosure are 22 base pairs. Meganucleases may be endonucleases derived from I-CreI (SEQ ID NO: 33) and may refer to engineered variants of I-CreI modified from the native I-CreI, for example, with respect to DNA binding specificity, DNA cleavage activity, DNA binding affinity, or dimerization properties. Methods for producing such engineered variants of I-CreI are known in the art (e.g., PCT International Patent Application Publication WO / 2007 / 047859, which is incorporated in whole by reference). Meganucleases as used herein bind to double-stranded DNA as heterodimers. Meganucleases may 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 this disclosure are substantially nontoxic when expressed in target cells described herein, so as to be transfected and maintained at 37°C, without observing any adverse effects on cell viability or a significant decrease in meganuclease cleavage activity, as measured using the methods described herein.

[0135] As used herein, the term “single-stranded meganuclease” refers to a polypeptide containing a pair of nuclease subunits linked by a linker. A single-stranded meganuclease has an N-terminal subunit-linker-C-terminal subunit configuration. The two meganuclease subunits typically have non-identical amino acid sequences and bind to non-identical DNA sequences. Therefore, single-stranded meganucleases typically cleave pseudopalindromic or non-palindromic recognition sequences. Although single-stranded meganucleases are not actually dimers, they are sometimes referred to as “single-stranded heterodimers” or “single-stranded heterodimer meganucleases.” For clarity, unless otherwise specified, the term “meganuclease” can refer to a dimer or a single-stranded meganuclease.

[0136] As used herein, the term “linker” refers to an exogenous peptide sequence used to link two nuclease subunits to a single polypeptide. Linkers may have sequences found in native proteins or artificial sequences not found in any native proteins. Linkers may be flexible and lack secondary structures, or they may have a tendency to form specific three-dimensional structures under physiological conditions. Examples of linkers include, but are not limited to, those covered in U.S. Patents 8,445,251, 9,340,777, 9,434,931, and 10,041,053. In some embodiments, the linker may have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 with the sequence shown in SEQ ID NO: 77, which is shown at residues 154–195 of any one of SEQ ID NOs: 43–59.

[0137] As used herein, the terms “nuclear localization sequence” or “NLS” refer to generally short peptides that act as signaling fragments mediating protein transport from the cytoplasm to the nucleus. Classical NLSs encompass two categories: monopartite (MP) and bipartite NLSs. Monopartite NLSs have a single cluster consisting of 4 to 8 basic amino acids, which generally contain 4 or more positively charged residues, i.e., arginine (R) or lysine (K). The characteristic motif of MP NLSs is usually defined as K(K / R)X(K / R), where X can be any residue. For example, the NLS of the SV40 large T antigen is 126PKKKRKV132, which has 5 consecutive positively charged amino acids (KKKRK). Bipartite NLSs are characterized by two clusters of 2 to 3 positively charged amino acids separated by a 9 to 12 amino acid linker region containing several proline (P) residues. The consensus sequence can be represented as R / K(X)10~12KRXK. In particular, in bipartite NLS, the upstream and downstream amino acid clusters are interdependent and essential, jointly determining the intracellular localization of the protein. Non-classical nuclear localization sequences are not similar to standard signals and are not rich in arginine or lysine residues. Among non-classical nuclear localization sequences, the "proline-tyrosine" category has been studied in the most detail. PY-NLS are characterized by 20-30 amino acids with a disordered structure consisting of a hydrophobic or basic motif at the N-terminus and an R / K / H(X)2~5PY motif at the C-terminus (X2~5 is any sequence of 2-5 residues). Two subclasses, hPY-NLS and bPY-NLS, were defined by their N-terminal motifs. hPY-NLS contains a φG / A / Sφφ motif (where φ is a hydrophobic residue), while bPY-NLS is rich in basic residues. In summary, the PY-NLS consensus corresponds to [basic / hydrophobic]-Xn-[R / H / K]-(X)2~5-PY, where X can be any residue.Human heteronuclear ribonucleoprotein A1 (hnRNP A1) is known as hPY-NLS due to its sequence 263FGNYNNQSSNFGPMKGGNFGGRSSGPY289, which contains the hydrophobic region (273FGPM276) necessary for its nuclear localization.

[0138] In some embodiments, the NLS includes SV40 NLS (SEQ ID NO: 25), NLS5 (SEQ ID NO: 26), c-myc NLS (SEQ ID NO: 27), or SV40H2 NLS (SEQ ID NO: 28). In some embodiments, the NLS includes an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 more sequence identity with the sequence shown in any one of SEQ ID NOs: 25-28. In some embodiments, the NLS includes an amino acid sequence in any one of SEQ ID NOs: 25-28.

[0139] As used herein, the terms “recombinant” or “artificial” with respect to proteins mean having an amino acid sequence altered as a result of the application of genetic engineering techniques to the nucleic acid encoding the protein and to the cells or organisms expressing the protein. With respect to nucleic acids, the terms “recombinant” or “artificial” mean having a nucleic acid sequence altered as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques, transfection, transformation, and other gene transfer techniques, homologous recombination, site-directed mutagenesis, and gene fusion. According to this definition, a protein that has the same amino acid sequence as a naturally occurring protein but is produced by cloning and expression in a heterologous host is not considered recombinant or artificial.

[0140] As used herein, the term “wild-type” refers to the most common native allele (i.e., polynucleotide sequence) in the allele population of the same type of gene, the polypeptide encoded by the wild-type allele having its original function. The term “wild-type” also refers to the polypeptide encoded by the wild-type allele. Wild-type alleles (i.e., polynucleotides) and polypeptides are distinguishable from mutant or variant alleles and polypeptides containing one or more mutations and / or substitutions, compared to the wild-type sequence. Wild-type alleles or polypeptides can confer a normal phenotype in an organism, while mutant or variant alleles or polypeptides may, in some cases, confer an altered phenotype. Wild-type nucleases are distinguishable from recombinant nucleases or nucleases that do not exist naturally. The term “wild-type” may also refer to cells, organisms, and / or subjects having the wild-type allele of a particular gene, or cells, organisms, and / or subjects used for comparative purposes.

[0141] As used herein, the term “genetically modified” refers to a cell or organism whose genomic DNA sequence has been intentionally altered by recombinant DNA technology, or whose ancestors have had their genomic DNA sequence intentionally altered by recombinant DNA technology. As used herein, the term “genetically modified” encompasses the term “transgenic.”

[0142] As used herein, the term “modification” in relation to recombinant proteins means any insertion, deletion, or substitution of amino acid residues within a recombinant sequence relative to a reference sequence (e.g., a wild-type sequence or a native sequence).

[0143] As used herein, the terms “recognition sequence” or “recognition site” refer to the DNA sequence bound and cleaved by a nuclease. In the case of meganucleases, the recognition sequence contains a pair of inverted 9-base pair “half-sites” separated by 4 base pairs. In the case of single-stranded meganucleases, 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 the meganuclease results in a 4-base pair 3' overhang. An “overhang” or “attached 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 I-CreI-derived meganucleases and single-stranded meganucleases, the overhang contains bases 10–13 of the 22-base pair recognition sequence. In the case of compact TALENs, the recognition sequence includes a first CNNNGN sequence recognized by the I-TevI ​​domain, followed by a 4-16 base pair nonspecific spacer, and then a second sequence of 16-22 bp length recognized by the TAL-effector domain (this sequence typically has a 5' T base). Cleavage by compact TALENs results in a two-base pair 3' overhang. In the case of CRISPR nucleases, the recognition sequence is the sequence to which the guide RNA binds and directly cleaves, typically 16-24 base pairs. Perfect complementarity between the guide sequence and the recognition sequence is not necessarily required to affect cleavage. Cleavage by CRISPR nucleases can result in blunt ends (e.g., by class 2 type II CRISPR nucleases) or overhang ends (e.g., by class 2 type V CRISPR nucleases), depending on the CRISPR nuclease. In embodiments where CpfI CRISPR nucleases are utilized, cleavage by the CRISPR complex containing the CpfI CRISPR nuclease results in a 5' overhang, and in certain embodiments, a 5-nucleotide 5' overhang. Each CRISPR nuclease enzyme also requires recognition of a PAM (protospacer adjacent motif) sequence located near the recognition sequence complementary to the guide RNA.The exact sequence, PAM length requirements, and distance from the target sequence vary depending on the CRISPR nuclease enzyme, but the PAM is typically a 2-5 base pair sequence adjacent to the target / recognition sequence. PAM sequences for specific CRISPR nuclease enzymes are publicly known in the art (see, e.g., U.S. Patent No. 8,697,359 and U.S. Patent Application Publication No. 2016 / 0208243), and PAM sequences for novel or artificial CRISPR nuclease enzymes can be identified using methods known in the art, such as PAM depletion assays (see, e.g., Karvelis et al. (2017) Methods 121-122:3-8). In the case of zinc fingers, the DNA-binding domain typically recognizes an 18 bp recognition sequence containing a pair of 9 base pair “half-sites” separated by 2-10 base pairs, and cleavage by the nuclease produces variable-length (often 4 base pairs) blunt ends or 5' overhangs.

[0144] As used herein, the terms “target site” or “target sequence” refer to a region of cellular chromosomal DNA containing the nuclease recognition sequence.

[0145] As used herein, the terms “DNA binding affinity” or “binding affinity” mean the tendency of a nuclease to non-covalently bind to a reference DNA molecule (e.g., a recognition sequence or any sequence). Binding affinity is measured by the dissociation constant Kd. As used herein, a nuclease has “altered” binding affinity if its Kd for a reference recognition sequence increases or decreases by a statistically significant percentage change relative to the reference nuclease.

[0146] As used herein, the term “specificity” means the ability of a nuclease to bind to and cleave a double-stranded DNA molecule only at a specific sequence of base pairs called a recognition sequence, or only at a specific set of recognition sequences. A set of recognition sequences may share certain conserved positions or sequence motifs, but may be degenerate at one or more positions. A highly specific nuclease may cleave only one or a very small number of recognition sequences. Specificity can be determined by any method known in the art.

[0147] 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 native 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 Dp427m dystrophin variant. The amino acid sequence of the full-length wild-type Dp427m dystrophin polypeptide is shown in SEQ ID NO: 35. NCBI reference numbers NM_004006.3 and NP_003997.2 represent the dystrophin Dp427m mRNA and polypeptide, respectively. In some embodiments described herein, the dystrophin gene is edited with a pair of artificial meganucleases, resulting in the excision of exons 45–55 and subsequent complete ligation of the dystrophin gene. Removal of exons 45–55 from the wild-type dystrophin gene may result in a dystrophin polypeptide containing the amino acid sequence shown in SEQ ID NO: 36.

[0148] As used herein, the terms “complete ligation” or “to completely ligate” refer to the ligation (i.e., annealing) of all four bases of the 3' overhang of the first cleavage site and all four bases of the complementary 3' overhang of the second cleavage site within the dystrophin gene after cleavage by the pair of artificial meganucleases described herein. The recognition sequences targeted by the disclosed artificial meganucleases have an identical 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, resulting in ligation via a DNA ligase enzyme. An example of a sequence resulting from such complete ligation is shown in Sequence ID No. 41 (i.e., complete ligation of the DMD 19-20 and DMD 35-36 recognition sequences).

[0149] As used herein, the term “Becker muscular dystrophy phenotype” refers to a less severe form of muscular dystrophy compared to DMD. Individuals with Becker muscular dystrophy still have mutations in the dystrophin gene, but express more functional dystrophin protein in muscle cells (e.g., muscle progenitor cells, skeletal muscle cells, and cardiomyocytes) compared to individuals with DMD, which generally leads to a better clinical prognosis.

[0150] As used herein, the terms “homologous recombination” or “HR” refer to the innate cellular process in which double-strand DNA breaks are repaired using homologous DNA sequences as repair templates (see, for example, Cahill et al. (2006) Front. Biosci. 11:1958-76). The homologous DNA sequence may be an endogenous chromosome sequence or an exogenous nucleic acid delivered to the cell.

[0151] As used herein, the terms “non-homologous end joining” or “NHEJ” refer to the innate cellular process in which double-strand DNA breaks are repaired by the direct joining of two non-homologous DNA segments (see, e.g., Cahill et al. (2006)). DNA repair by non-homologous end joining is error-prone and frequently results in the addition or deletion of a template-less DNA sequence at the repair site. In some cases, breaks at target recognition sequences result in an NHEJ at the target recognition site. Nuclease-induced breaks at target sites in the coding sequence of a gene, followed by DNA repair by an NHEJ, can introduce mutations into the coding sequence, such as frameshift mutations that disrupt gene function. Therefore, artificial nucleases can be used to effectively knock out genes in a cell population.

[0152] As used herein, the terms “homology arm” or “sequence homologous to a sequence adjacent to a nuclease cleavage site” refer to sequences adjacent to the 5' and 3' ends of a nucleic acid molecule that facilitate the insertion of the nucleic acid molecule into a cleavage site produced by a nuclease. Generally, homology arms can be at least 50 base pairs, preferably at least 100 base pairs, and up to 2000 base pairs or more in length, and can have at least 90%, preferably at least 95% or more sequence homology with their corresponding sequences in the genome. In some embodiments, homology arms are about 500 base pairs.

[0153] As used herein, terms relating to both amino acid sequences and nucleic acid sequences, such as "percent identity," "sequence identity," "percentage similarity," and "sequence similarity," refer to a measure of the degree of similarity between two sequences based on sequence alignment, which 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, maximizing the similarity between aligned amino acid residues or nucleotides. Various algorithms and computer programs are available to determine sequence similarity using standard parameters. Where 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 through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ) and are described, for example, Altschul et al. (1990) J.Mol.Biol.215:403-10, Gish & States (1993) Nature Genet.3:266-72, Madden et al. (1996) Meth.Enzymol.266:131-41, Altschul et al. (1997) Nucleic Acids Res.25:3389-3402, and Zhang et al. (2000) J.Comput.Biol.7:203-14. As used herein, the percentage similarity of two amino acid sequences is a score based on the following parameters for the BLASTp algorithm: word size = 3, gap opening penalty = -11, gap extension penalty = -1, score matrix = BLOSUM62. As used herein, the percentage similarity of two nucleic acid sequences is a score based on the following parameters for the BLASTn algorithm: word size = 11, gap opening penalty = -5, gap extension penalty = -2, match reward = 1, and mismatch penalty = -3.

[0154] As used herein, the term “corresponds” with respect to modifications of two proteins or amino acid sequences is used to indicate that a particular modification of the first protein is the same amino acid residue substitution as that in the modification of the second protein, and that when the two proteins are subjected to standard sequence alignment (e.g., using the BLASTp program), the amino acid position of the modification of the first protein corresponds to or matches the amino acid position of the modification of the second protein. Thus, a modification of residue “X” in the first protein to amino acid “A” corresponds to a modification of residue “Y” in the second protein to amino acid “A,” despite the fact that residues X and Y can be different numbers, provided that residues X and Y correspond to each other in sequence alignment.

[0155] As used herein, the terms “recognition halfsite,” “recognition sequence halfsite,” or simply “halfsite” mean 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.

[0156] As used herein, the term “hypervariable region” refers to a localization sequence within a meganuclease monomer or subunit containing amino acids with relatively high variability. A hypervariable region may include about 50–60 consecutive residues, about 53–57 consecutive residues, or preferably about 56 residues. In some embodiments, the residues of the hypervariable region may correspond to positions 24–79 or 215–270 of any one of SEQ ID NOs. 43–59. A hypervariable region may include one or more residues that contact DNA bases in the recognition sequence and may be modified to alter the base selectivity of the monomer or subunit. A hypervariable region may also include one or more residues that bind to the DNA backbone when the meganuclease associates with a double-stranded DNA recognition sequence. Such residues may be modified to alter the binding affinity of the meganuclease to the DNA backbone and the target recognition sequence. In different embodiments, a hypervariable region may include 1–20 residues that exhibit variability and may be modified to affect base selectivity and / or DNA binding affinity. In certain embodiments, the hypervariable region contains about 15–20 residues that can be modified to exhibit variability and 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: 43–59. In certain embodiments, the variable residues within the hypervariable region may further correspond to residues 48, 50, and 71–73 of any one of SEQ ID NOs: 43–59. 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 268 of any one of SEQ ID NOs.43-59. In certain embodiments, the variable residues within the hypervariable region may further correspond to residues 239, 241, and 263-265 of any one of SEQ ID NOs.43-59.

[0157] In relation to dystrophin protein or mRNA levels, the term “increase” refers to any increase in the level of dystrophin protein or mRNA expression compared to a reference level, including an increase of at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more compared to a reference level or control. In some embodiments, an increase in dystrophin protein or mRNA levels refers to an increase in a shortened dystrophin polypeptide or mRNA transcript (e.g., a polypeptide lacking a portion encoded by at least one exon (e.g., the portion encoded by exons 45–55), or a portion of the mRNA corresponding to exons 45–55) compared to a wild-type dystrophin polypeptide or gene.

[0158] As used herein, the term “reference level” in relation to dystrophin protein or mRNA levels means, for example, the level of dystrophin protein or mRNA measured at a previous point in time in the treated control cells, control cell population, or control subject (e.g., a pre-administration baseline level obtained from the control cells, control cell population, or control subject), or a predefined threshold level of dystrophin protein or mRNA (e.g., a threshold level identified through previous experiments).

[0159] 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. Control cells may include, for example, (a) wild-type cells, i.e., wild-type cells with the same genotype as the starting material of the genetic mutation that resulted in the genetically modified cell; (b) cells with the same genotype as the genetically modified cell but transformed with a null construct (i.e., a construct that does not have a known effect on the trait of interest); or (c) cells that are genetically identical to the genetically modified cell but have not been exposed to conditions or stimuli that induce the expression of the altered genotype or phenotype, or to further genetic modification. Control subjects may include, for example, wild-type subjects, i.e., wild-type subjects with the same genotype as the starting material of the genetic mutation that resulted in the genetically modified subject (e.g., a subject with the same mutation in the dystrophin gene), and which have not been exposed to conditions or stimuli that induce the expression of the altered genotype or phenotype in the subject, or to further genetic modification.

[0160] As used herein, the terms “recombinant DNA construct,” “recombinant construct,” “expression cassette,” “expression construct,” “chimeric construct,” “construct,” and “recombinant DNA fragment” are used interchangeably herein and refer to single-stranded or double-stranded polynucleotides. Recombinant constructs include artificial combinations of nucleic acid fragments that include, but are not limited to, regulatory and coding sequences not found together in nature. For example, a recombinant DNA construct may include regulatory and coding sequences from different sources, or regulatory and coding sequences from the same source arranged in a manner different from that found in nature. Such constructs may be used on their own or in conjunction with a vector.

[0161] As used herein, the terms “vector” or “recombinant DNA vector” may refer to a construct comprising a replication system and sequence capable of transcribing and translating a polypeptide-encoding sequence in a given host cell. Where a vector is used, the choice of vector depends, as is well known to those skilled in the art, on the method used to transform the host cell. Examples of vectors include, but are not limited to, plasmid vectors and recombinant AAV vectors, or any other vectors known in the art suitable for delivering genes to target cells. Those skilled in the art will have a good understanding of the genetic elements that must be present on a vector in order to successfully transform, select, and grow host cells containing any of the isolated nucleotide or nucleic acid sequences described herein. In some embodiments, “vector” also refers to viral vectors. Examples of viral vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, and AAVs.

[0162] As used herein, the term “operably linked” is intended to mean a functional linkage between two or more elements. For example, the operable linkage between a nucleic acid sequence encoding a nuclease and a regulatory sequence (e.g., a promoter) disclosed herein is a functional linkage that enables the expression of the nucleic acid sequence encoding the nuclease. The operable linked elements may be contiguous or discontinuous. When used to refer to the linkage of two protein-coding regions, operable linkage means that the coding regions are within the same reading frame.

[0163] As used herein, the terms “treatment” or “treat a subject” mean administering to a subject having DMD an artificial meganuclease described herein, or a polynucleotide encoding an artificial meganuclease described herein, or a pair of such artificial meganucleases or polynucleotides, for the purpose of increasing the level of the subject’s dystrophin protein. In some embodiments, the expression of a truncated version of the dystrophin protein (e.g., lacking amino acids encoded by several exons) is increased. In some embodiments, the expression of a version of the dystrophin protein lacking the amino acids encoded by exons 45–55 is increased. Such treatment, in some embodiments, causes the DMD phenotype to transition to a Becker dystrophy phenotype.

[0164] As used herein, the terms “gc / kg” or “gene copies / kilogram” refer to the number of copies of the nucleic acid sequence encoding the artificial meganuclease described herein per kilogram of body weight of the subject to whom the polynucleotide containing the nucleic acid sequence is administered.

[0165] Where used herein, the terms “effective dose” or “therapeutic effective dose” refer to an amount sufficient to produce a beneficial or desirable biological and / or clinical outcome. The therapeutic effective dose varies depending on the formulation or composition used, the disease and its severity, and the age, weight, physical condition and responsiveness of the person being treated. In certain embodiments, an effective dose of the synthetic meganuclease or synthetic meganuclease pair described herein, or the polynucleotide or polynucleotide pair encoding it, or the pharmaceutical composition disclosed herein, increases the expression level of dystrophin protein (e.g., truncated dystrophin protein lacking the amino acid encoded by exons 45-55) and improves at least one symptom associated with DMD.

[0166] Where used herein, the enumeration of numerical ranges for a variable is intended to convey that the disclosure may be carried out with a variable equal to any value within that range. Thus, for a variable that is inherently discrete, the variable may be equal to any integer value within the numerical range that includes the endpoints of the range. Similarly, for a variable that is inherently continuous, the variable may be equal to any real number within the numerical range that includes the endpoints of the range. As an example, a variable that is stated to have values ​​between 0 and 2, but is not limited thereto, can take the values ​​of 0, 1, or 2 if the variable is inherently discrete, and can take the values ​​of 0.0, 0.1, 0.01, 0.001, or any other real number between 0 and 2 if the variable is inherently continuous.

[0167] 2.1 Principles of this Disclosure This disclosure is based on the discovery of muscle-specific expression cassettes that function to better express heterologous proteins, particularly in muscle tissue. Specifically, these expression cassettes utilize muscle-specific promoters as part of nucleic acid expression cassettes that, when delivered in vivo to mammals using AAVs (e.g., AAV9 or AAVrh74), result in the specific expression of heterologous proteins in skeletal and cardiomyocyte tissues. The expression cassettes described herein further demonstrate the ability to express heterologous proteins in muscle satellite cells (e.g., Pax7-positive muscle satellite cells).

[0168] Furthermore, it was unexpectedly discovered that the muscle-specific expression cassettes described herein yield superior expression in skeletal muscle tissue compared to cardiomyocyte tissue. These favorable expression profiles allow for the modulation of heterologous protein expression in cardiac and muscle tissue to maximize the efficacy of the delivered heterologous protein. As described herein, such delivery of artificial nucleases (e.g., artificial meganucleases) increases the potency in gene editing. The muscle-specific expression cassettes described herein are useful for heterologous protein expression in muscle cells and tissues in vitro and in vivo. These muscle-specific expression cassettes are useful for the treatment of muscle disorders in which heterologous protein expression is desired in affected or diseased muscle cells. In certain embodiments disclosed herein, the muscle-specific expression cassettes described herein are useful for the treatment of DMD utilizing site-specific nucleases.

[0169] 2.2 Muscle-Specific Expression Cassettes for Heterogeneous Protein Expression in Cells Heterogeneous proteins (e.g., artificial nucleases) can be specifically expressed in muscle cells by muscle-specific expression cassettes described herein. These muscle-specific expression cassettes comprise a nucleic acid sequence containing the heterogeneous protein. These muscle-specific expression cassettes are contained within a larger delivery vehicle (e.g., as part of a viral vector). According to this disclosure, the coding sequence of the heterogeneous protein described herein is delivered to muscle cells in a muscle-specific expression cassette, which is in DNA form. As part of the muscle-specific expression cassette, the heterogeneous protein should be operably ligated to a promoter (i.e., a muscle-specific promoter) to facilitate the transcription of the heterogeneous protein transgene.

[0170] Therefore, the nucleic acid sequences encoding heterologous proteins described herein are operably linked to muscle-specific promoters. In some specific embodiments, the promoter can express the artificial meganucleases described herein in muscle progenitor cells (e.g., satellite cells or stem cells). Exemplary and non-limiting muscle promoters include C5-12 (Liu et al. (2004) Hum Gene Ther. 15:783-92), muscle-specific creatine kinase (MCK) promoters (Yuasa et al. (2002) Gene Ther. 9:1576-88), or smooth muscle 22 (SM22) promoters (Haase et al. (2013) BMC Biotechnol. 13:49-54).

[0171] In some embodiments, the muscle-specific promoter in the muscle-specific expression cassette described herein is based on or includes a portion of the MCK promoter. These promoters include an MCK enhancer element region and an MCK base promoter region. In some embodiments, the MCK promoter is the tMCK promoter described herein. In some embodiments, the base MCK promoter includes a sequence from a WT human or mouse MCK promoter sequence. In some embodiments, the base MCK promoter is about 50 bp to about 1,000 bp in length. In some embodiments, the basic MCK promoter is approximately 50bp, 55bp, 60bp, 65bp, 70bp, 75bp, 80bp, 85bp, 90bp, 95bp, 100bp, 150bp, 200bp, 250bp, 300bp, 350bp, 400bp, 450bp, 500bp, 600bp, 700bp, 800bp, 900bp, or 1000bp in length. In some embodiments, the basic MCK promoter is approximately 350bp in length. In some embodiments, the basic MCK promoter is approximately 90bp in length.

[0172] In some embodiments, the basic MCK promoter includes a nucleic acid sequence that is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the basic MCK promoter includes a nucleic acid sequence that is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3. In some embodiments, the basic MCK promoter includes a nucleic acid sequence represented by the sequence shown in SEQ ID NO: 2. In some embodiments, the basic MCK promoter includes a nucleic acid sequence represented by the sequence shown in SEQ ID NO: 3. In some embodiments, the MCK enhancer element includes one or more MCK-R control elements. In some embodiments, the MCK-R control element includes a sequence represented by any one of SEQ ID NOs: 4 to 17. In some embodiments, the MCK promoter includes a sequence that is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the mouse MCK promoter including SEQ ID NO: 18. In some embodiments, the MCK promoter includes an array that is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the human MCK promoter including array 19.

[0173] In some embodiments, the MCK promoter includes an MCK enhancer element array that is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the MCK enhancer element array including sequence number 20. In some embodiments, the MCK promoter includes a modified MCK enhancer array that is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the modified MCK enhancer including sequence number 21.

[0174] In some embodiments, the MCK promoter is a tMCK promoter containing a nucleic acid sequence that is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22.

[0175] In some embodiments, the muscle-specific expression cassette further comprises a post-transcriptional regulatory element described herein. In certain embodiments, the post-transcriptional regulatory element is a WPRE described herein. In some embodiments, the WPRE comprises a nucleic acid sequence that is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24. In some embodiments, the WPRE comprises a nucleic acid sequence represented by the sequence shown in SEQ ID NO: 24.

[0176] In some embodiments, the muscle-specific expression cassette further includes a polyA termination sequence. In certain embodiments, the polyA termination sequence is SV40 polyA. In some embodiments, the polyA termination sequence includes a nucleic acid sequence that is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29. In some embodiments, the polyA termination sequence includes a nucleic acid sequence represented by the sequence shown in SEQ ID NO: 29.

[0177] In some embodiments, the muscle-specific promoter includes a consensus Kozak sequence described herein. Such Kozak sequences are known in the art and are generally suitable for the embodiments of the present disclosure described herein. In some embodiments, the Kozak sequence includes a sequence represented by sequence number 32.

[0178] In some embodiments, the muscle-specific expression cassette includes a muscle-specific enhancer located either 5' upstream or 3' downstream of the muscle-specific promoter. According to this disclosure, the inclusion of a muscle-specific enhancer increases the expression of the transgene in muscle tissue and muscle cell types without significantly increasing the expression of the transgene in other tissues or cell types. In particular, the B-301 enhancer has been found to increase the expression of heterologous proteins in Pax7-positive muscle satellite cells and differentiated muscle cells. According to some embodiments, the muscle-specific enhancer is located 5' upstream of the muscle-specific promoter described herein. In some embodiments, the muscle-specific enhancer includes a B-301 enhancer sequence. In some embodiments, the B-301 enhancer sequence includes a nucleic acid sequence that is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the B-301 enhancer sequence includes a nucleic acid sequence represented by the sequence shown in SEQ ID NO: 1.

[0179] In some embodiments, a heteroprotein transgene, comprising a single polynucleotide, each comprising two distinct nucleic acid sequences encoding heteroproteins (e.g., two distinct nucleic acid sequences encoding the artificial nucleases described herein), is operably linked to two or more muscle-specific promoters described herein. These two or more muscle-specific promoters may be the same or different. In alternative embodiments, two heteroprotein transgenes are operably linked to a single promoter, which in some embodiments may be separated by an intraribosome entry site (IRES) or a 2A peptide sequence (Szymczak & Vignali (2005) Expert Opin Biol Ther. 5:627-38). Such a 2A peptide sequence may include, for example, a T2A, P2A, E2A, or F2A sequence. In some further embodiments, the 2A element comprises a Fulin cleavage motif. In some further embodiments, the 2A element comprises a Fulin cleavage motif and a GSG linker.

[0180] In embodiments of the methods and compositions described herein, the muscle-specific expression cassette increases the expression of heterologous proteins in muscle cells compared to non-muscle cells. Non-muscle cells can be any mammalian cells that are not of muscle cell origin, such as cells that have not differentiated from Pax7+ muscle stem cells. In some embodiments, non-muscle cells are non-muscle cells from the liver, brain, kidney, pancreas, spleen, germ cells, or lung.

[0181] In some embodiments, the heterologous proteins described herein, expressed from the muscle-specific expression cassette described herein, show an increase in expression of the heterologous proteins described herein in muscle cells of about 1.5 to about 2 times, or about 2 to about 5 times, or about 5 to about 10 times, or about 10 to about 15 times, or about 15 to about 20 times, or about 25 to about 30 times, or about 30 to about 40 times, or about 40 to about 50 times, or about 50 to about 60 times, or about 60 to about 70 times, or about 70 to about 80 times, or about 80 to about 90 times, or about 90 to about 100 times, compared to non-muscle cells described herein. In some embodiments, the heterologous proteins described herein, expressed from the muscle-specific expression cassette described herein, show an increase in expression of the heterologous proteins described herein in muscle cells of about 2 to about 80 times, compared to non-muscle cells described herein. In some other embodiments, the heterologous proteins described herein, expressed from the muscle-specific expression cassette described herein, show an increase in expression of the heterologous proteins described herein in muscle cells of about 5 to about 60 times compared with non-muscle cells described herein. In some additional embodiments, the heterologous proteins described herein, expressed from the muscle-specific expression cassette described herein, show an increase in expression of the heterologous proteins described herein in muscle cells of about 15 to about 60 times compared with non-muscle cells described herein.

[0182] In embodiments of the methods and compositions described herein, the muscle-specific expression cassette increases the expression of heterologous proteins in muscle cells compared to non-muscle cells. In such embodiments, the heterologous proteins are still expressed in cardiomyocytes compared to skeletal muscle cells, but to a lower degree.

[0183] In some embodiments, the heterologous protein described herein expressed from the muscle-specific expression cassette described herein shows an increase in the expression of the heterologous protein described herein in muscle cells compared to cardiomyocytes of about 1.5-fold to about 2-fold, or about 2-fold to about 5-fold, or about 5-fold to about 10-fold, or about 10-fold to about 15-fold, or about 15-fold to about 20-fold, or about 25-fold to about 30-fold, or about 30-fold to about 40-fold, or about 40-fold to about 50-fold, or about 50-fold to about 60-fold, or about 60-fold to about 70-fold, or about 70-fold to about 80-fold, or about 80-fold to about 90-fold, or about 90-fold to about 100-fold. In some embodiments, the heterologous protein described herein expressed from the muscle-specific expression cassette described herein shows an increase in the expression of the heterologous protein described herein in muscle cells compared to cardiomyocytes of about 2-fold to about 80-fold. In some other embodiments, the heterologous protein described herein expressed from the muscle-specific expression cassette described herein shows an increase in the expression of the heterologous protein described herein in muscle cells compared to cardiomyocytes of about 5-fold to about 60-fold. In some additional embodiments, the heterologous protein described herein expressed from the muscle-specific expression cassette described herein shows an increase in the expression of the heterologous protein described herein in muscle cells compared to cardiomyocytes of about 15-fold to about 60-fold.

[0184] 2.3 Heterologous Proteins Encoded by Muscle-Specific Expression Cassettes The muscle-specific expression cassettes described herein can encode any heterologous protein that is desired to be expressed in muscle cells. The heterologous protein can be a protein for which it is desirable to study muscle cell function, such as a fluorescently labeled (e.g., green fluorescent protein) version of a naturally occurring muscle protein. Alternatively, the heterologous protein can be a therapeutic protein that is a muscle protein for treating a muscle disorder (e.g., DMD) described herein. Exemplary and non-limiting therapeutic agents include dystrophin proteins such as microdystrophin 1 (MD1), follistatin (FST), sarcoglycan proteins, α-glucosidase, and myotubularin-1 (MTM-1).

[0185] In some embodiments, the heterologous protein is a nuclear protein as described herein. Such nuclear proteins can include proteins that perform their primary function in the cell nucleus and regulate gene expression at the DNA level without editing the DNA. These proteins are referred to herein as DNA-binding regulatory proteins. Such proteins include, but are not limited to, naturally occurring transcription factors, nuclease-inactivated versions of artificial nucleases, and artificial transcription factors. Examples of nuclease-inactivated versions of DNA-binding regulatory proteins include CRISPR-Cas proteins in which the CAS enzyme is inactivated and linked to a gene activator domain or a gene repressor domain. In addition, in combination with gene repressor domains and activator domains, TALE, artificial ZFN, and artificial meganucleases can be utilized to regulate gene expression. Alternative nuclear proteins described herein directly edit genomic DNA and include artificial nucleases.

[0186] Thus, in some embodiments, the heterologous protein is an artificial nuclease. As described herein, muscle-specific expression cassettes are suitable for expressing an artificial nuclease for binding to and cleaving a recognition sequence within the genome of mammalian muscle cells. In some embodiments, the methods described herein are useful for modifying the dystrophin gene using the artificial nucleases described herein. In some other embodiments, the methods described herein are useful for treating DMD in a subject having DMD. Non-limiting examples of artificial nucleases useful in the present disclosure include, inter alia, artificial meganucleases, CRISPR system nucleases, ZFN, TALEN, compact TALEN, megaTAL, base editors, and prime editors.

[0187] ZFNs can be manipulated to recognize and cleave specific sites in the genome. ZFNs are chimeric proteins containing a zinc finger DNA-binding domain fused to a nuclease domain from an endonuclease or exonuclease (e.g., a type IIs restriction endonuclease such as Fok I restriction enzyme). The zinc finger domain may be a naturally occurring sequence or can be redesigned by rational or experimental means to produce a protein that binds to a specific DNA sequence of approximately 18 base pairs in length. By fusing this artificial protein domain to the nuclease domain, it is possible to target DNA cleavage with genome-level specificity. ZFNs are widely used to target gene addition, deletion, and substitution in a wide range of eukaryotes (as outlined in S. Durai et al., Nucleic Acids Res 33, 5978 (2005)).

[0188] Similarly, TALENs can be generated to cleave specific sites in genomic DNA. Like ZFNs, TALENs contain an engineered site-specific DNA-binding domain fused to an endonuclease or exonuclease (e.g., an Iis-type restriction endonuclease such as Fok I restriction enzyme) (as outlined in Mak, et al. (2013) Curr Opin Struct Biol. 23:93-9). However, in this case, the DNA-binding domain contains a tandem array of TAL-effector domains, each specifically recognizing a single DNA base pair.

[0189] Compact TALENs are an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley, et al. (2013) Nat Commun. 4:1762). Compact TALENs contain an engineered site-specific TAL-effector DNA-binding domain fused to a nuclease domain from either an I-TevI ​​homing endonuclease or one of the endonucleases listed in Table 2 of U.S. Patent Application Publication No. 2013 / 0117869. Because compact TALENs do not require dimerization for DNA processing activity, compact TALENs function as monomers.

[0190] Artificial endonucleases based on the CRISPR / Cas system are also known in the art (Ran, et al. (2013) Nat Protoc. 8:2281-2308, Mali et al. (2013) Nat Methods. 10:957-63). In those embodiments in which the CRISPR system is used to insert a donor nucleic acid sequence into a heterologous polynucleotide or genomic locus, the CRISPR system comprises two components: (1) a CRISPR nuclease, and (2) a short “guide RNA” containing a target sequence of about 20 nucleotides that guides the nuclease to the desired site in the genome or on a polynucleotide. The CRISPR system may also include tracrRNA. By expressing multiple guide RNAs, each with a different target sequence, in the same cell, it is possible to simultaneously target multiple sites in the genome with DNA breaks. The compositions and methods of this disclosure utilizing the CRISPR system may include a CRISPR nuclease and a guide RNA(s) or nucleic acid encoding the CRISPR nuclease and / or guide RNA(s).

[0191] The nuclease known as megaTAL is a single-stranded endonuclease containing a transcription activator-like effector (TALE) DNA-binding domain with an engineered sequence-specific homing endonuclease.

[0192] An exemplary and non-limiting prime editor consists of a fusion protein of Cas9 H840A nickase fused to a reverse transcriptase and a prime editing guide RNA (pegRNA). The prime editor mediates insertions, deletions, and base transpositions without producing double-strand breaks (Anzalone et al. (2019) Nature 576(7785) 149-157). The pegRNA contains a single guide RNA having a primer-binding site and a reverse transcriptase template. When editing a genome, the DNA is cleaved, the primer-binding site hybridizes the cleaved DNA strand with the pegRNA, and the template acts as a synthetic template for editing the gene. Compositions and methods of the present disclosure utilizing a prime editing system may include a CRISPR nuclease (e.g., Cas9 H840A nickase) and pegRNA(s) or nucleic acids encoding a CRISPR nuclease and / or pegRNA(s).

[0193] Exemplary and non-limiting base editors are based on enzymes that can catalyze the conversion of cytosine / guanosine to thymidine / alanine. For example, base editors utilizing the CRISPR / CAS9 enzyme include a CRISPR / Cas9 enzyme fused to a cytidine deaminase enzyme that does not induce double-strand breaks. The Cas9 enzyme is typically inactivated so that it can no longer cleave DNA, but it still functions to bind to DNA along with the guide RNA. Mutations that inactivate the Cas9 enzyme include the Asp10Ala mutation and the His840Ala mutation. These base editors perform cytosine to thymidine or guanosine to adenosine substitution (Komor et al. (2016) Nature 533(7603) 420-424). Another exemplary and non-limiting base editor is a CRISPR-free system based on a transcription activator-like effector (TALE) protein fused to a double-stranded DNA-specific cytidine deaminase, as described by Mok et al., (2022) Nature Biotechnology 40 1378-1387.

[0194] The compositions and methods of this disclosure may utilize purified nuclease proteins or nucleic acids encoding nucleases. These may be delivered to cells to cleave genomic DNA or polynucleotides by various different mechanisms known in the art, including those further detailed elsewhere in this specification. In some embodiments utilizing CRISPR system nucleases, a ribonucleoprotein complex comprising a CRISPR nuclease and guide RNA(s) may be introduced into cells.

[0195] Artificial meganucleases may be, for example, endonucleases derived from I-CreI, and may refer to engineered variants of I-CreI that are modified from the natural I-CreI in terms of, for example, DNA binding specificity, DNA cleavage activity, DNA binding affinity, or dimerization properties. Methods for producing such modified variants of I-CreI are known in the art (e.g., WO / 2007 / 047859, which is incorporated in whole by reference). The meganucleases used herein bind to double-stranded DNA as heterodimers. Meganucleases may also be “single-stranded meganucleases” in which a pair of DNA-binding domains are linked to a single polypeptide using a peptide linker.

[0196] In certain embodiments, the meganuclease used to carry out embodiments of the present disclosure is a single-stranded meganuclease. The single-stranded meganuclease comprises an N-terminal subunit and a C-terminal subunit linked by a linker peptide. Each of the two subunits recognizes and binds to half of the recognition sequence (i.e., the recognition half-site), and the site of DNA cleavage is located in the middle of the recognition sequence near the interface of the two subunits. The DNA strand cleavage is offset by four base pairs such that the DNA cleavage by the meganuclease results in a 3' single-stranded overhang of a pair of four base pairs. The first subunit of the single-stranded meganuclease comprises a first hypervariable (HVR1) region, and the second subunit comprises a second hypervariable (HVR2) region. Furthermore, the first subunit binds to a first recognition half-site in the recognition sequence, and the second subunit binds to a second recognition half-site in the recognition sequence.

[0197] In embodiments where the artificial meganuclease is a single-stranded meganuclease, the first and second subunits may be oriented such that the first subunit, which includes an HVR1 region and binds to the first half-site, is positioned as the N-terminal subunit, and the second subunit, which includes an HVR2 region and binds to the second half-site, is positioned as the C-terminal subunit. In alternative embodiments, the first and second subunits may be oriented such that the first subunit, which includes an HVR1 region and binds to the first half-site, is positioned as the C-terminal subunit, and the second subunit, which includes an HVR2 region and binds to the second half-site, is positioned as the N-terminal subunit.

[0198] Recognition array It is known in the art that site-specific nucleases can be used to cleave DNA within the genome of living cells, and that such DNA cleavage can lead to permanent genomic alterations via mutagenic NHEJ repair or homologous recombination with transgenic DNA sequences. NHEJs can induce mutagenesis at the cleavage site, resulting in allelic inactivation. NHEJ-associated mutagenesis can inactivate alleles through the generation of early stop codons, frameshift mutations that produce abnormal nonfunctional proteins, or by activating mechanisms such as nonsense mutation-dependent mRNA degradation mechanisms. The use of nucleases that induce mutagenesis via NHEJs can be used to target specific mutations or sequences present in wild-type alleles. Furthermore, the use of nucleases to induce double-strand breaks at target loci is known to stimulate homologous recombination of transgenic DNA sequences adjacent to sequences homologous to genomic targets in particular. In this way, exogenous polynucleotides can be inserted into target loci. Such exogenous polynucleotides can encode any sequence or polypeptide of interest.

[0199] In certain embodiments, the artificial meganucleases described herein are designed to bind to and cleave the DMD 19-20 recognition sequence (SEQ ID NO: 37) or the DMD 35-36 recognition sequence (SEQ ID NO: 39). Exemplary meganucleases that bind to and cleave the DMD 19-20 recognition sequence are provided in SEQ ID NOs: 43-51. Exemplary meganucleases that bind to and cleave the DMD 35-36 recognition sequence are provided in SEQ ID NOs: 52-59. The sequences of each recognition sequence and the 4-base pair 3' overhang produced when cleaved by the artificial meganucleases described herein are provided in Table 1 below.

[0200] [Table 1]

[0201] In some embodiments for modifying the dystrophin gene, a pair of artificial meganucleases described herein are used together in the same cell. Such a pair of artificial meganucleases is described in PCT International Patent Application Publication No. WO / 2022 / 104062. These meganucleases are designed to generate a first cleavage site in the intron upstream of exon 45 and a second cleavage site in the intron downstream of exon 55, enabling the removal of a 500,000 bp intervening genomic sequence. In some embodiments, the meganuclease recognition sequence has a complementary 4-base pair 3' overhang after cleavage, which frequently promotes dystrophin gene repair by complete ligation of the 3' overhangs of the two cleavage sites. An example of a fully ligated recognition sequence after removal of exons 45–55 of the dystrophin gene is provided in Sequence ID No. 41.

[0202] In some embodiments, the recognition sequence is further selected to be located within an intron sequence that is normally excised by splicing during post-transcriptional modification cellular processes. This reduces the likelihood of mutations being introduced into the dystrophin gene and the polypeptide it encodes. Exemplary artificial meganucleases

[0203] The artificial meganuclease described herein comprises a first subunit containing an HVR1 region and a second subunit containing an HVR2 region. Furthermore, the first subunit binds to a first recognition half-site in the recognition sequence (e.g., the DMD19 half-site), and the second subunit binds to a second recognition half-site in the recognition sequence (e.g., the DMD20 half-site).

[0204] In certain embodiments, the meganuclease is a single-stranded meganuclease. The single-stranded meganuclease comprises an N-terminal subunit and a C-terminal subunit (i.e., the first subunit and the second subunit described above) linked by a linker peptide. Each of the two subunits recognizes and binds to a half-site of the recognition sequence, and the site of DNA cleavage is at the center of the recognition sequence near the interface of the two subunits. As discussed, DNA strand cleavage is offset by four base pairs such that DNA cleavage by the meganuclease results in a pair of 3´ single-stranded overhangs of four base pairs.

[0205] In embodiments where the engineered meganuclease is a single-stranded meganuclease, the first subunit and the second subunit can be oriented such that the first subunit, which contains the HVR1 region and binds to the first half-site, is positioned as the N-terminal subunit and the second subunit, which contains the HVR2 region and binds to the second half-site, is positioned as the C-terminal subunit. In an alternative embodiment, the first subunit and the second subunit can be oriented such that the first subunit, which contains the HVR1 region and binds to the first half-site, is positioned as the C-terminal subunit and the second subunit, which contains the HVR2 region and binds to the second half-site, is positioned as the N-terminal subunit.

[0206] Exemplary DMD meganucleases suitable for expression from the muscle-specific expression cassettes and methods described herein are provided in SEQ ID NOs: 43-59. As described herein, combinations of meganucleases can be used to excise exons 45-55 from the dystrophin gene. Thus, in some embodiments, the first artificial meganuclease is an artificial meganuclease described herein that binds to and cleaves a recognition sequence including SEQ ID NO: 37, and the second artificial meganuclease is an artificial meganuclease described herein that binds to and cleaves a recognition sequence including SEQ ID NO: 39. In some embodiments, the first and second artificial meganucleases encoded by the muscle-specific expression cassette are selected from combinations of meganucleases (and their variants described herein) provided in Table 2.

[0207] [Table 2] TIFF2026515786000004.tif248170

[0208] In different embodiments, the Disclosure provides an artificial meganuclease described herein that is useful for binding to and cleaving a recognition sequence within a cellular dystrophin gene (e.g., the human dystrophin gene). The Disclosure also provides various methods for modifying the intracellular dystrophin gene using the artificial meganuclease described herein, methods for creating genetically modified cells containing the modified dystrophin gene, and methods for modifying the dystrophin gene within a target cell of interest. In further embodiments, the Disclosure provides a method for treating a subject's DMD by administering the artificial meganuclease of the operation described herein, or a polynucleotide encoding it, to the subject, possibly as part of a pharmaceutical composition.

[0209] Detection and expression The expression of modified dystrophin (i.e., a gene lacking exons 45–55, or a protein lacking the amino acids encoded by exons 45–55) in genetically modified cells or subjects can be detected using standard methods in the art. For example, the level of such modified dystrophin can be assessed based on the level of any variable related to dystrophin gene expression, such as dystrophin mRNA levels or dystrophin protein levels. An increase in the level or expression of such modified dystrophin can be assessed by an increase in the absolute or relative level of one or more of these variables relative to a reference level. Such modified dystrophin levels can be measured in biological samples isolated from subjects, such as tissue biopsies or bodily fluids including blood, serum, plasma, cerebrospinal fluid, or urine. Optionally, such modified dystrophin levels are normalized to a standard protein or substance in the sample. Furthermore, such modified dystrophin levels can be assessed at any point before, during, or after treatment by the methods herein.

[0210] In various embodiments, the methods described herein can increase the protein level of modified dystrophin (i.e., lacking the amino acid encoded by exons 45-55) in genetically modified cells, target cells, or subjects (e.g., measured in cells, tissues, organs, or biological samples obtained from the subject) to at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than the reference level (i.e., the protein level of dystrophin in wild-type cells or subjects). In some embodiments, the methods herein are effective in increasing the level of such modified dystrophin protein to about 10% to about 100% (e.g., 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, or more) of the reference level of dystrophin (i.e., the protein level of dystrophin in wild-type cells or subjects).

[0211] 2.4 Introduction of muscle-specific expression cassettes into cells The heterologous proteins (e.g., artificial nucleases) disclosed herein can be delivered to cells by various different mechanisms known in the art, including those further detailed below herein. In certain embodiments, the muscle-specific expression cassette described herein is delivered onto a recombinant DNA construct. For example, the recombinant DNA construct includes the muscle-specific expression cassette described herein.

[0212] In another specific embodiment, the muscle-specific expression cassette described herein is introduced into cells using a single-stranded DNA template. The single-stranded DNA may further include 5' and / or 3' AAV inverted end sequences (ITRs) upstream and / or downstream of the sequence encoding the artificial nuclease. The single-stranded DNA may further include 5' and / or 3' homology arms upstream and / or downstream of the sequence encoding the artificial meganuclease.

[0213] In another specific embodiment, the muscle-specific expression cassette described herein may be introduced into cells using a linearized DNA template. Such a linearized DNA template can be prepared by methods known in the art. For example, plasmid DNA encoding a nuclease can be digested with one or more restriction enzymes so that the circular plasmid DNA is linearized before it is introduced into cells.

[0214] In some embodiments, the muscle-specific expression cassettes described herein are bound to cell-permeable peptides or target ligands to facilitate cell uptake. Examples of cell-permeable peptides known in the art include polyarginine (Jearawiriyapaisarn et al. (2008) Mol Ther. 16:1624-29), HIV virus-derived TAT peptide (Hudecz et al. (2005) Med. Res. Rev. 25:679-736), MPG (Simeoni et al. (2003) Nucleic Acids Res. 31:2717-24), Pep-1 (Deshayes et al. (2004) Biochemistry 43:7698-7706), and HSV-1 VP-22 (Deshayes et al. (2005) Cell Mol Life). Examples include Sci.62:1839-49). In alternative embodiments, the muscle-specific expression cassette described herein is covalently or non-covalently bound to an antibody that recognizes a specific cell surface receptor expressed on the target cell, so that the muscle-specific expression cassette binds to the target cell and is internalized by the target cell. Alternatively, the muscle-specific expression cassette described herein may be covalently or non-covalently bound to a native ligand (or a part of a native ligand) of such a cell surface receptor. (McCall et al.(2014)Tissue Barriers.2(4):e944449, Dinda et al.(2013)Curr.Pharm.Biotechnol.14:1264-74, Kang et al.(2014)Curr.Pharm.Biotechnol.15:220-30, and Qian et al.(2014)Expert Opin.Drug Metab Toxicol.10:1491-508).

[0215] In some embodiments, the muscle-specific expression cassette described herein is encapsulated in a biodegradable hydrogel for injection or implantation into a desired region of the liver (e.g., a region adjacent to hepatic sinusoidal endothelial cells or hematopoietic endothelial cells, or progenitor cells that differentiate into them). The hydrogel can provide sustained and controllable release of the therapeutic payload to a desired region of target tissue without requiring frequent injections, and stimulus-responsive materials (e.g., temperature and pH-responsive hydrogels) can be designed to release the payload in response to environmental cues or externally applied cues (Derwent et al. (2008) Trans Am. Ophthalmol. Soc. 106:206-14).

[0216] In some embodiments, the muscle-specific expression cassettes described herein are covalently or preferably non-covalently bonded to nanoparticles or encapsulated within such nanoparticles using methods known in the art (Sharma et al. (2014) Biomed.Res.Int. 2014:156010). The nanoparticles are nanoscale delivery systems with a length scale of less than 1 μm, preferably less than 100 nm. Such nanoparticles may be designed using a core composed of a metal, lipid, polymer, or biomacromolecule, and multiple copies of the muscle-specific expression cassettes described herein may be attached to or encapsulated within the nanoparticle core. This increases the number of DNA copies delivered to each cell and, therefore, increases the intracellular expression of each meganuclease, maximizing the likelihood that the target recognition sequence will be cleaved. The surface of such nanoparticles may be further modified with polymers or lipids (e.g., chitosan, cationic polymers, or cationic lipids) to form core-shell nanoparticles in which the surface confers additional functionality for enhancing cell delivery and payload uptake (Jian et al. (2012) Biomaterials. 33:7621-30). Nanoparticles may be further advantageously bound to target molecules in order to direct the nanoparticles to the appropriate cell type and / or to increase the likelihood of cell uptake. Examples of such target molecules include antibodies specific to cell surface receptors and native ligands (or parts of native ligands) of cell surface receptors.

[0217] In some embodiments, the muscle-specific expression cassettes described herein are encapsulated within liposomes or complexed using cationic lipids (see, for example, LIPOFECTAMINE®, Life Technologies Corp., Carlsbad, CA; Zuris et al. (2015) Nat. Biotechnol. 33:73-80, Mishra et al. (2011) J. Drug Deliv. 2011:863734). Liposomes and lipoplex formulations can protect the payload from degradation, enhance accumulation and retention at target sites, and promote cellular uptake and delivery efficiency through fusion with and / or disruption of the cell membrane of target cells.

[0218] In some embodiments, the muscle-specific expression cassettes described herein are encapsulated within a polymer scaffold (e.g., PLGA) or compounded using cationic polymers (e.g., PEI, PLL) (Tamboli et al. (2011) Ther Deliv. 2:523-36). The polymer carrier can be designed to provide a drug release rate tunable by controlling polymer erosion and drug diffusion, and high drug encapsulation efficiency can provide protection of the therapeutic payload until intracellular delivery to the desired target cell population.

[0219] In some embodiments, the muscle-specific expression cassette described herein is combined with amphiphilic molecules that self-assemble into micelles (Tong et al. (2007) J. Gene Med. 9:956-66). Examples of polymeric micelles include micelle shells formed from hydrophilic polymers (e.g., polyethylene glycol) that can prevent aggregation, block charge interactions, and reduce nonspecific interactions.

[0220] In some embodiments, the muscle-specific expression cassettes described herein are formulated into emulsions or nanoemulsions (i.e., having an average particle size of less than 1 nm) for administration and / or delivery to target cells. The term “emulsion” refers to any oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil dispersion or droplet, but is not limited to those containing lipid structures that can be formed as a result of hydrophobic forces that repel nonpolar residues (e.g., long hydrocarbon chains) away from water and direct polar head groups toward water when a water-immiscible phase is mixed with an aqueous phase. These other lipid structures include, but are not limited to, monolayer, sparselayer, and multilayer lipid vesicles, micelles, and lamellar phases. Emulsions consist of an aqueous phase and a lipophilic phase (typically containing oil and organic solvents). Emulsions also often contain one or more surfactants. Nanoemulsion formulations are well known and are described, for example, in U.S. Patents 6,015,832, 6,506,803, 6,635,676, 6,559,189, and 7,767,216.

[0221] In some embodiments, the muscle-specific expression cassettes described herein are covalently or acovalently bonded to polyfunctional polymer conjugates, DNA dendrimers, and polymer dendrimers (Mastorakos et al. (2015) Nanoscale. 7:3845-56, Cheng et al. (2008) J. Pharm Sci. 97:123-43). Dendrimer generation can control payload capacity and size, and can provide high payload capacity. Furthermore, by utilizing the display of multiple surface groups, stability can be improved, nonspecific interactions can be reduced, and cell-specific targeting and drug release can be enhanced.

[0222] In some embodiments, the muscle-specific expression cassettes described herein are introduced into cells using recombinant viruses (i.e., recombinant viral vectors). Such recombinant viruses are known in the art and include recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, and recombinant AAVs (as outlined in Vannucci et al. (2013) New Microbiol. 36:1-22). Recombinant AAVs useful herein may have any serotype that enables transduction of the virus into target cell types and expression of meganuclease genes in target cells. For example, in some embodiments, the recombinant AAV may have serotypes (i.e., capsids) of AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, or AAVrh.74. It is known in the art that different AAVs tend to localize to different tissues (Wang et al. (2014) Expert Opin Drug Deliv 11:345-34). The AAVrh.74 serotype, closely related to AAV8, is further described as targeting muscle tissue, including skeletal and cardiomyocyte tissue (Mendell et al. (2020) JAMA Neurol. 77:1122-31). Therefore, in some embodiments, the AAV serotype is AAV1. In some embodiments, the AAV serotype is AAV2. In some embodiments, the AAV serotype is AAV5. In some embodiments, the AAV serotype is AAV6. In some embodiments, the AAV serotype is AAV7. In some embodiments, the AAV serotype is AAV8. In some embodiments, the AAV serotype is AAV9. In some embodiments, the AAV serotype is AAV12. In some embodiments, the AAV serotype is AAVrh.74. AAVs can also be self-complementary so as not to require second-strand DNA synthesis in host cells (McCarty et al. (2001) Gene Ther. 8:1248-54). Polynucleotides delivered by recombinant AAV may contain left (5') and right (3') inverted terminal sequences as part of the viral genome.In some embodiments, the recombinant virus is injected directly into the target tissue. In alternative embodiments, the recombinant virus is delivered systemically via the circulatory system.

[0223] In one embodiment, the recombinant virus used to deliver the meganuclease gene is a self-restricting recombinant virus. Because the self-restricting virus has a recognition sequence for the artificial meganuclease within its viral genome, it can have a limited duration in a cell or organism. Therefore, the self-restricting recombinant virus can be manipulated to provide the promoter coding sequence, the artificial meganuclease described herein, and the meganuclease recognition site within the ITR. The self-restricting recombinant virus delivers the meganuclease gene to a cell, tissue, or organism so that the meganuclease is expressed and can cleave the cell's genome at the endogenous recognition sequence within the genome. The delivered meganuclease also finds its target site within the self-restricting recombinant viral genome and cleaves the recombinant viral genome at this target site. Upon cleavage, the 5' and 3' ends of the viral genome are exposed and degraded by the exonuclease, thus killing the virus and halting meganuclease production.

[0224] Such polynucleotides containing exogenous nucleic acids can be introduced into and / or delivered to target cells by any of the means described above. In certain embodiments, such polynucleotides containing exogenous nucleic acid molecules are introduced by recombinant viruses (i.e., viral vectors), such as recombinant lentiviruses, recombinant retroviruses, recombinant adenoviruses, or recombinant AAVs. Recombinant AAVs useful for introducing polynucleotides containing exogenous nucleic acid molecules can have any serotype (i.e., capsid) that allows for the transduction of the virus into cells and the insertion of the exogenous nucleic acid molecular sequence into the cellular genome. In some embodiments, the recombinant AAV has serotypes AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, or AAVrh.74. In some embodiments, the AAV serotype is AAV1. In some embodiments, the AAV serotype is AAV2. In some embodiments, the AAV serotype is AAV5. In some embodiments, the AAV serotype is AAV6. In some embodiments, the AAV serotype is AAV7. In some embodiments, the AAV serotype is AAV8. In some embodiments, the AAV serotype is AAV9. In some embodiments, the AAV serotype is AAV12. In some embodiments, the AAV serotype is AAVrh.74. Recombinant AAVs may also be self-complementary so as not to require second-strand DNA synthesis in host cells. Exogenous nucleic acid molecules introduced using recombinant AAVs can be flanked by 5' (left) and 3' (right) inverted terminal sequences in the viral genome.

[0225] In another specific embodiment, an exogenous nucleic acid molecule can be introduced into cells using a single-stranded DNA template. The single-stranded DNA may include an exogenous nucleic acid molecule and, in a specific embodiment, may include 5' and 3' homology arms to facilitate the insertion of the nucleic acid sequence into a nuclease cleavage site by homologous recombination. The single-stranded DNA may further include a 5' AAV ITR sequence 5' upstream of the 5' homology arm and a 3' AAV ITR sequence 3' downstream of the 3' homology arm.

[0226] In another specific embodiment, the genes encoding the nucleases and / or exogenous nucleic acid molecules described herein can be introduced into cells by transfection with a linearized DNA template. Plasmid DNA encoding artificial nucleases and / or exogenous nucleic acid molecules can be digested with one or more restriction enzymes, for example, circular plasmid DNA, so that it is linearized before transfection into cells.

[0227] Administration Target tissues(s) or target cells(s) include, but are not limited to, muscle cells such as skeletal muscle cells, cardiomyocytes, or diaphragmatic muscle cells. In some embodiments, target cells are muscle progenitor cells such as skeletal muscle progenitor cells or cardiomyocytes. Such muscle progenitor cells may be described in the art and present in the subject, or may originate from other stem cell populations such as induced pluripotent stem cells or embryonic stem cells (Tey et al. (2019) Front. Cell Dev. Biol. 7:284 and Amini et al. (2017) J. Cardiovasc. Thorac. Res. 9:127-32).

[0228] In some embodiments, the muscle-specific expression cassette described herein is delivered to cells in vitro. In some embodiments, the muscle-specific expression cassette described herein is delivered to target cells in vivo. In some embodiments, the muscle-specific expression cassette described herein is delivered to target cells (e.g., muscle cells or muscle progenitor cells) by direct injection into target tissue. Alternatively, the muscle-specific expression cassette described herein can be delivered systemically via the circulatory system.

[0229] In various embodiments of this method, the compositions described herein, such as the muscle-specific expression cassette, recombinant virus containing such polynucleotides, or lipid nanoparticles containing such polynucleotides, can be administered via any suitable route of administration known in the art. Such routes of administration include, for example, intravenous, intramuscular, intraperitoneal, subcutaneous, intrahepatic, transmucosal, transdermal, intra-arterial, and sublingual. In some embodiments, the muscle-specific expression cassette, recombinant virus containing such polynucleotides, or lipid nanoparticles containing such polynucleotides are delivered to target cells (e.g., muscle cells or muscle progenitor cells) via direct injection into target tissue (e.g., muscle tissue). Other suitable routes of administration can be readily determined by the physician administering the procedure, as needed.

[0230] In some embodiments, a therapeutically effective dose of the therapeutic protein described herein (e.g., the artificial nuclease described herein), encoded by the muscle-specific expression cassette described herein, is administered to a subject in need to treat a disease. If necessary, the dose or frequency of administration of the muscle-specific expression cassette described herein encoding the therapeutic protein described herein may be adjusted during treatment at the discretion of the administering physician. The appropriate dose depends, among other factors, on the details of any selected AAV (e.g., serotype), any selected lipid nanoparticles, the route of administration, the subject being treated (i.e., the subject's age, weight, sex, and general condition), and the mode of administration. Therefore, the appropriate dose may vary from patient to patient. The appropriate effective dose can be readily determined by a person skilled in the art or the treating physician. The drug treatment may be a single-dose schedule or a multi-dose schedule if multiple doses are required. Furthermore, the subject may be administered multiple times as needed. A person skilled in the art can readily determine the appropriate number of doses. The dose may need to be adjusted to take alternative routes of administration into consideration or to balance the therapeutic benefits with any side effects.

[0231] In some embodiments, the method further comprises administering a polynucleotide comprising a nucleic acid sequence that encodes a secretory-impairing hepatotoxin or tPA that stimulates hepatocyte regeneration without acting as a hepatotoxin.

[0232] In some embodiments, a subject is administered a pharmaceutical composition comprising an arbitrary muscle-specific expression cassette encoding a therapeutic protein (e.g., an artificial nuclease) as described herein, wherein the encoding muscle-specific expression cassette nucleic acid sequence is approximately 1 × 10⁻¹⁶ 10 gc / kg ~ approx. 1×10 14 gc / kg (for example, approximately 1 × 10⁻⁶) 10 gc / kg, approximately 1×10 11 gc / kg, approximately 1×10 12 gc / kg, approximately 1×10 13 gc / kg, or approximately 1 × 10⁻⁶ 14administered at a dose of (e.g., 10 gc / kg). In some embodiments, a subject is administered a pharmaceutical composition comprising any muscle-specific expression cassette encoding a therapeutic protein (e.g., an artificial nuclease) described herein, wherein the nucleic acid sequence of the encoding muscle-specific expression cassette is about 1×10 10 gc / kg, about 1×10 11 gc / kg, about 1×10 12 gc / kg, about 1×10 13 gc / kg, or about 1×10 14 gc / kg. In some embodiments, a subject is administered a pharmaceutical composition comprising any muscle-specific expression cassette encoding a therapeutic protein (e.g., an artificial nuclease) described herein, wherein the nucleic acid sequence of the encoding muscle-specific expression cassette is about 1×10 10 gc / kg to about 1×10 11 gc / kg, about 1×10 11 gc / kg to about 1×10 12 gc / kg, about 1×10 12 gc / kg to about 1×10 13 gc / kg, or about 1×10 13 gc / kg to about 1×10 14 gc / kg. In some embodiments, a subject is administered a pharmaceutical composition comprising any muscle-specific expression cassette encoding a therapeutic protein (e.g., an artificial nuclease) described herein, wherein the nucleic acid sequence of the encoding muscle-specific expression cassette is about 1×10 13 [[ID=2】]gc / kg, 2×10 13 gc / kg, 3×10 13 gc / kg, 4×10 13 gc / kg, 5×10 13 gc / kg, 6×10 13 gc / kg, 7×10 13 gc / kg, 8×10 13 gc / kg, 9×10 13 gc / kg, 1×10 14 gc / kg, 2×10 14 gc / kg, 3×10 14 gc / kg, 4×10 14 gc / kg, 5×10 14 gc / kg, 6×10 14gc / kg, 7 × 10 14 gc / kg, 8 × 10 14 gc / kg, 9x10 14 gc / kg, 1x10 15 gc / kg, 2x10 15 gc / kg, 3x10 15 gc / kg, 4x10 15 gc / kg, 5x10 15 gc / kg, 6x10 15 gc / kg, 7x10 15 gc / kg, 8x10 15 gc / kg, 9x10 15 gc / kg, 1x10 15 gc / kg, 2x10 15 gc / kg, 3x10 15 gc / kg, 4x10 15 gc / kg, 5x10 15 gc / kg, 6x10 15 gc / kg, 7x10 15 gc / kg, 8x10 15 gc / kg, or 9x10 15 It is administered at a dose of gc / kg.

[0233] These doses may relate to the administration of a single polynucleotide containing a single nucleic acid sequence encoding a single artificial meganuclease described herein, or to a single polynucleotide containing a first nucleic acid sequence encoding a first artificial meganuclease described herein and a second nucleic acid sequence encoding a second artificial meganuclease described herein (each of the two encoding nucleic acids)

[0234] 2.5 Pharmaceutical Compositions This disclosure also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an artificial meganuclease as described herein, or a polynucleotide as described herein comprising a pharmaceutically acceptable carrier and an arbitrary muscle-specific expression cassette encoding a heterologous protein (e.g., an artificial nuclease) as described herein. In some such examples, the polynucleotide comprising an arbitrary muscle-specific expression cassette as described herein in the pharmaceutical composition may consist of lipid nanoparticles or recombinant viruses (e.g., recombinant AAV). In other embodiments, this disclosure provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a genetically modified cell as described herein, which can be delivered to a target tissue expressing a heterologous protein (e.g., an artificial nuclease) as disclosed herein. Such pharmaceutical compositions are formulated, for example, for systemic administration or administration to a target tissue.

[0235] In various embodiments, the pharmaceutical composition may be useful for treating a muscle disorder of interest and / or alleviating symptoms associated with the muscle disorder. Exemplary and non-limiting muscle disorders include neuromuscular disorders and cardiac diseases, e.g., abetalipoproteinemia (Bassen-Kornzwieg), acetylcholine receptor deficiency (myasthenia gravis), Charlevoix-Saguenet syndrome / disease, benign congenital myopathy, Brody's disease, central nucleus myopathy (myotubular myopathy), chondrodystrophic myotonia (Schwarz-Jampel syndrome), Chudley syndrome, fingerprint myopathy, hereditary neuropathic muscular atrophy (Personage-Turner syndrome), inclusion body myopathy Chi (e.g., type 2 or 3), inclusion body myositis, Isaacs syndrome (neurogenic myotonia), Kennedy disease (bulbar spinal atrophy), macrophage fasciitis, McArdle disease (muscle phosphorylase deficiency / glycostored storage disease type V), polyneuropathy, myo-ophthalmopathy, nemaline myopathy, nonaka myopathy, wave-like muscle contraction, tibial muscular dystrophy (distal Wood myopathy), distal Welander's muscular dystrophy Myopathy, acid maltase deficiency (Pompe disease / glycostoredisease type II), Danon disease (glycostoredisease type IIb / vacuolar myopathy), debranching enzyme deficiency (glycostoredisease type III / Forbes disease), Anderson's disease / syndrome (glycostoredisease type IV / branching enzyme deficiency), Tauri disease (glycostoredisease type VII / phosphofructokinase deficiency), desmin accumulation myopathy (myofibrillar myopathy), myodenylate deaminase deficiency, adrenoleukodystrophy, congenital polyarthritis, ataxia with congenital glaucoma, ataxia due to isolated vitamin E deficiency, Barth syndrome, Bethlem myopathy, carnitine palmityltransferase deficiency, carnitine deficiency, central core myopathy, hereditary sensorimotor neuropathy [e.g., Charcot-Marie-Tooth disease (CMT)] CMT Type I, CMT Type II, CMT Type III (Dejerin-Sottas disease), CMT Type IV (Refsum disease), CMT[Type V, etc.], peroneal muscular atrophy, neuronal type peroneal muscular atrophy, hereditary sensory and autonomic neuropathy (e.g., Type I, Type III (familial autonomic dysregulation / Riley-Day syndrome), Type IV (congenital insensitivity to pain and anhidrosis), congenital fibrous disequilibrium myopathy, distal spinal muscular atrophy, familial amyloid neuropathy, familial dilated cardiomyopathy with muscular dystrophy, Friedreich's ataxia, hypercholesterolemia Muhememic periodic paralysis (Gumstorp disease), giant axonal neuropathy, Guillain-Barré syndrome (acute inflammatory demyelinating / polyneuropathy), hyperthermia (malignant hyperthermia), hypokalemic periodic paralysis, iatrogenic myopathy, Kearns-Sayer syndrome, Kugelberg-Welander disease (spinal muscular atrophy type III), distal Laing myopathy, Lambert-Eaton (myasthenia gravis) syndrome, Leigh syndrome Group, minicore myopathy / multicore myopathy, mitochondrial myopathy and / or neuropathy, mixed connective tissue duplication disease, Miyoshi myopathy, multifocal motor neuropathy with conduction block, myasthenia gravis, congenital myotonia (Thomsen disease), myotonic muscular dystrophy [e.g., type I (Steinert's disease), type II (proximal myotonic myopathy)], oculopharyngeal muscular dystrophy, Ori Pontocerebellar atrophy, congenital myotonia, paraneoplastic neuropathy, polymyositis, reductal body myopathy, scapulaebyneal muscle atrophy, tubular cluster myopathy, Walker-Warburg syndrome, Werdnig-Hoffmann disease (spinal muscular atrophy type 1), zebra body myopathy, nuclear membrane disease, muscular dystrophy, motor neuron disease (MND), e.g., Charcot-Marie-Tooth disease (CMT), e.g., CMT This includes Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), congenital muscular dystrophy, congenital myopathy, limb-girdle muscular dystrophy, metabolic myopathy, myoinflammatory disease, myasthenia gravis, mitochondrial myopathy, ion channel abnormalities, nuclear membrane diseases, cardiac myopathy, cardiac hypertrophy, heart failure, and distal myopathy, cardiovascular disease.

[0236] In various embodiments, the pharmaceutical composition may be useful for treating DMD, converting the DMD disease phenotype to the Becker muscular dystrophy phenotype, and / or alleviating symptoms associated with the target DMD.

[0237] Such pharmaceutical compositions can be prepared by known techniques. See, for example, Remington, The Science and Practice of Pharmacy (21st ed., Philadelphia, Lippincott, Williams & Wilkins, 2005). In the manufacture of the pharmaceutical formulations described herein, the artificial meganuclease, the polynucleotide encoding it, or the cells expressing it are typically mixed with a pharmaceutically acceptable carrier, and the resulting composition is administered to a subject. The carrier must be acceptable in the sense that it is compatible with any other components in the formulation and must not be harmful to the subject. The carrier may be solid, liquid, or both, and may be formulated together with the compound as a unit-dose formulation.

[0238] In some embodiments, the pharmaceutical compositions described herein may further include one or more additional agents or biological molecules useful for treating the disease of interest. Similarly, the additional agents and / or biological molecules may be administered concurrently as separate compositions.

[0239] The pharmaceutical compositions described herein may include any polynucleotide described herein, comprising any muscle-specific expression cassette encoding any heterologous protein (e.g., an artificial nuclease disclosed herein) or any artificial meganuclease described herein. For example, in some embodiments, the pharmaceutical composition may include a polynucleotide comprising any muscle-specific expression cassette described herein at any dose described herein (e.g., gc / kg of the encoding nucleic acid sequence or mg / kg of mRNA).

[0240] In certain embodiments, the pharmaceutical composition described herein may include one or more recombinant viruses (e.g., recombinant AAV) described herein, each comprising a polynucleotide comprising a muscle-specific expression cassette described herein, each comprising a polynucleotide comprising a different artificial meganuclease described herein, as described herein. For example, a first recombinant virus (e.g., recombinant AAV) may comprise a second recombinant virus (e.g., recombinant AAV) comprising a first polynucleotide comprising a first muscle-specific expression cassette described herein, which has specificity for DMD 19-20 recognition sequences, and a second polynucleotide comprising a second muscle-specific expression cassette described herein, which has specificity for a second artificial meganuclease described herein, as described herein. The expression of such a pair of artificial meganucleases in the same cell (e.g., muscle cells) allows for the excision of exons 45-55 from the dystrophin gene according to the specific embodiments described herein.

[0241] In other specific embodiments, the pharmaceutical compositions described herein may include a recombinant virus (e.g., recombinant AAV) comprising a polynucleotide (i.e., packaged within a viral genome) comprising a muscle-specific expression cassette described herein that encodes two distinct artificial meganucleases described herein. For example, the recombinant virus (e.g., recombinant AAV) may include a polynucleotide comprising a muscle-specific expression cassette described herein that comprises a first nucleic acid sequence encoding a first artificial meganuclease described herein having specificity for DMD 19-20 recognition sequences and a second nucleic acid sequence encoding a second artificial meganuclease described herein having specificity for DMD 35-36 recognition sequences. Expression of such a pair of artificial meganucleases allows for the excision of exons 45-55 from the dystrophin gene.

[0242] In some embodiments, the pharmaceutical compositions described herein may further comprise one or more additional agents useful for treating the target DMD.

[0243] This disclosure also provides a muscle-specific expression cassette encoding a heterologous protein (e.g., an artificial nuclease) as described herein, or cells comprising a muscle-specific expression cassette as described herein, wherein the heterologous protein is expressed from the muscle-specific expression cassette in muscle cells or muscle tissue for use as a pharmaceutical. This disclosure further provides the use of a muscle-specific expression cassette encoding a heterologous protein (e.g., an artificial nuclease) as described herein, expressing the heterologous protein (e.g., an artificial nuclease) as described herein, in the manufacture of a pharmaceutical for treating DMD, to increase the level of modified dystrophin protein (i.e., lacking the amino acids encoded by exons 45-55 of the dystrophin gene), or to alleviate symptoms associated with DMD.

[0244] 2.6 Method for producing recombinant viruses This disclosure also provides recombinant viruses, such as recombinant AAV, for use in the methods described herein. Recombinant AAV is typically produced in mammalian cell lines such as HEK-293. The viral cap and rep genes are removed from the recombinant virus to prevent its self-replication and make room for the delivery of therapeutic genes (or more) (e.g., meganuclease genes), and therefore need to be provided trans to the packaging cell line. Furthermore, it is necessary to provide “helper” (e.g., adenovirus) components necessary to support replication (Cots et al. (2013) Curr. Gene Ther. 13:370-81). Often, recombinant AAV is produced using triple transfection, which transfects the cell line with a first plasmid encoding “helper” components, a second plasmid containing the cap and rep genes, and a third plasmid containing a viral ITR containing an intervening DNA sequence to be packaged into the virus. The third plasmid may contain an ITR having one or more D sequences. Alternatively, the third plasmid may contain an ITR having one D sequence and a second ITR without a D sequence. Finally, the third plasmid may contain a first ITR having one D sequence and a second ITR having one D sequence. The AAV D sequences are typically 20 nucleotides long and do not contribute to the formation of the hairpin structure. Instead, they serve as viral packaging signals. In some embodiments, the D sequences include sequences shown in SEQ ID NOs. 94-95. The viral particles, containing the capsid-encapsulated genome (the ITR of interest and intervening genes), are then isolated from cells by freeze-thaw cycles, sonication, detergents, or other means known in the art. The particles are then purified using cesium chloride density gradient centrifugation or affinity chromatography and subsequently delivered to the gene(s), cells, tissues, or organisms such as human patients.

[0245] Since recombinant AAV particles are typically produced (manufactured) intracellularly, in certain embodiments disclosed herein, precautions must be taken to ensure that artificial nucleases are not expressed in the packaging cells. Since the recombinant viral genomes described herein may contain nuclease recognition sequences, any nuclease expressed in the packaging cell line may be able to cleave the viral genome before it can be packaged into viral particles. This results in reduced packaging efficiency and / or packaging of fragmented genomes. Several approaches can be used to prevent nuclease expression in the packaging cells.

[0246] As described herein, the nuclease is encoded by a muscle-specific expression cassette comprising a muscle-specific promoter (e.g., an MCK promoter) operably linked to a nucleic acid sequence encoding the artificial nuclease. This muscle-specific promoter is not active in packaging cells, and therefore, any expression in packaging cells is significantly reduced or completely eliminated.

[0247] Furthermore, recombinant viruses can be packaged into cells of different species where nucleases are less likely to be expressed. For example, viral particles can be produced in microbial, insect, or plant cells using mammalian promoters such as well-known cytomegaloviruses or SV40 virus early promoters that are not active in non-mammalian packaging cells. In certain embodiments, viral particles are produced in insect cells using a baculovirus system described in Gao et al. (2007) J. Biotechnol. 131:138-43. Nucleases under the control of mammalian promoters are less likely to be expressed in these cells (Airenne et al. (2013) Mol. Ther. 21:739-49). In addition, insect cells utilize different mRNA splicing motifs than mammalian cells. Therefore, it is possible to incorporate mammalian introns, such as human growth hormone (HGH) introns or SV40 large T antigen introns, into the coding sequence of the nuclease. Because these introns are not efficiently spliced ​​from premRNA transcripts in insect cells, insect cells do not express functional nucleases and package the full genome. In contrast, mammalian cells to which the resulting recombinant AAV particles are delivered properly splice the premRNA and express functional nuclease proteins. Chen reported using HGH and SV40 large T antigen introns to attenuate the expression of the toxic protein barnase and diphtheria toxin fragment A in insect packaging cells, enabling the production of recombinant AAV vectors carrying these toxin genes (Chen (2012) Mol. Ther. Nucleic Acids. 1:e57).

[0248] Furthermore, artificial nuclease genes can be operably linked to induction promoter elements such that small molecule inducers are required for nuclease expression. Examples of induction promoters include the Tet-On system (Clontech; Chen et al. (2015) BMC Biotechnol. 15:4) and the RheoSwitch system (Intrexon; Sowa i (2011) Spine 36:E623-8). Both systems, as well as similar systems known in the art, rely on ligand-inducible transcription factors (mutants of the Tet repressor and ecdysone receptor, respectively) that activate transcription in response to small molecule activators (doxycycline or ecdysone, respectively). Such methods using such ligand-inducible transcription activators include 1) placing an artificial nuclease gene under the control of a promoter that responds to the corresponding transcription factor, wherein the nuclease gene has (a) binding sites for the transcription factor, and 2) including a gene encoding the transcription factor in a packaged viral genome. The latter step is necessary because artificial nucleases will not be expressed in target cells or tissues after recombinant AAV delivery unless the transcriptional activator is also provided to the same cells. The transcriptional activator then induces meganuclease gene expression only in cells or tissues treated with the congeneral small molecule activator. This approach is advantageous because it allows for spatiotemporal regulation of nuclease gene expression by selecting when and to which tissues the small molecule inducer is delivered. However, the need to include the inducer in the viral genome, which severely limits its transport capacity, presents a drawback to this approach.

[0249] In another specific embodiment, recombinant AAV particles are produced in mammalian cell lines expressing transcriptional repressors that inhibit nuclease expression. Transcriptional repressors are known in the art and include Tet repressors, Lac repressors, Cro repressors, and lambda repressors. Many nuclear hormone receptors, such as ecdysone receptors, also act as transcriptional repressors in the absence of their congener hormone ligands. Thus, packaging cells are transfected / transduced with a vector encoding a transcriptional repressor, and the nuclease gene in the viral genome (packaging vector) is operably ligated to a muscle-specific promoter modified to include a binding site for the repressor, so that the repressor terminates the promoter's expression. The gene encoding the transcriptional repressor can be positioned in a variety of locations. It can be encoded on a separate vector, incorporated into an ITR-extra-sequence packaging vector, incorporated into a cap / rep vector or adenovirus helper vector, or stably incorporated into the genome of the packaging cell for constitutive expression. Methods for modifying common mammalian promoters to incorporate transcriptional repressor sites are known in the art. For example, Chang and Roninson modified potent constitutive CMV and RSV promoters to include a Lac repressor operator and showed that gene expression from the modified promoters was significantly attenuated in cells expressing the repressor (Chang & Roninson (1996) Gene 183:137-42). The use of non-human transcriptional repressors ensures that the transcription of meganuclease genes is repressed only in packaging cells expressing the repressor and not in target cells or tissues transduced with the resulting recombinant AAV. [Examples]

[0250] This disclosure is further supplemented by the following embodiments, which should not be construed as limiting. Those skilled in the art will be able to recognize or confirm numerous equivalents to the specific substances and procedures described herein without using experiments beyond routine experimentation. Such equivalents are intended to be included in the claims following the embodiments.

[0251] Example 1 Comparison of in vivo meganuclease expression and dystrophin gene editing in hDMD mouse studies.

[0252] 1. Method In vivo studies were conducted in hDMDdel52 / mdx (hDMD) mice to investigate the restoration of in vivo editing and truncated human dystrophin protein induced by delivery of meganuclease pairs DMD 19-20L.329 and DMD 35-36L.349 using different muscle-specific promoters and AAV capsid types. Four different constructs encapsulated in either AAV9 or AAVrh74 (1e14VG / kg) were injected into mice by post-orbital systemic injection. The first AAV9 inclusion product contained a viral genome including Sequence ID No. 78, from 5' to 3', which included the muscle-specific promoter MHCK7, the first SV40 nuclear localization sequence (NLS) coding sequence, the DMD 19-20L.329 meganuclease coding sequence, the first c-myc NLS coding sequence, the furin GSG P2A cleavage sequence, the second SV40 NLS coding sequence, the DMD 35-36L.349 meganuclease coding sequence, the second c-myc NLS coding sequence, a WPRE element, and the SV40 polyadenylation signal. The second AAV9 inclusion product contained a viral genome including Sequence ID No. 79, from 5' to 3', which included a muscle-specific promoter tMCK, a first SV40 NLS coding sequence, a DMD 19-20L.329 nuclease coding sequence, a first c-myc NLS coding sequence, a furin GSG P2A cleavage sequence, a second SV40 NLS coding sequence, a DMD 35-36L.349 nuclease coding sequence, a second c-myc NLS coding sequence, a WPRE element, and an SV40 polyadenylation signal. The first AAVrh74 inclusion product contained a viral genome including Sequence ID No. 78, from 5' to 3', which included the muscle-specific promoter MHCK7, the first SV40 NLS coding sequence, the DMD 19-20L.329 meganuclease coding sequence, the first c-myc NLS coding sequence, the furin GSG P2A cleavage sequence, the second SV40 NLS coding sequence, the DMD 35-36L.349 meganuclease coding sequence, the second c-myc NLS coding sequence, a WPRE element, and the SV40 polyadenylation signal.The second AAVrh74 inclusion product contained a viral genome including SEQ ID NO: 79, from 5' to 3', which included a muscle-specific promoter tMCK, the first SV40 NLS coding sequence, the coding sequence for DMD 19-20L.329 meganuclease, the first c-myc NLS coding sequence, a furin GSG P2A cleavage sequence, the second SV40 NLS coding sequence, the coding sequence for DMD 35-36L.349 meganuclease, the second c-myc NLS coding sequence, a WPRE element, and an SV40 polyadenylation signal. The nucleic acid coding sequence for the DMD 35-36L.349 artificial meganuclease was codon-modified to reduce the percentage identity between the nucleic acid sequences common to both meganucleases without altering the encoded amino acid sequence. The unmodified nucleic acid coding sequence for the DMD 35-36L.349 artificial meganuclease is provided herein as SEQ ID NO: 96. This artificial meganuclease was codon-modified so that approximately 40% of the nucleotides were altered in the DMD 35-36L.349 artificial meganuclease. Therefore, the codon-modified DMD 35-36L.349 meganuclease had less than 60% nucleotide sequence identity with the DMD 19-20L.329 artificial meganuclease. Twenty-eight days after injection, the mice were sacrificed, and tissue sections from skeletal muscle (quadriceps femoris), heart, and liver were collected for molecular, protein, and histological analysis.

[0253] Meganuclease protein expression on day 28 was measured using the Meso Scale Discovery (MSD) system, an electrochemiluminescence assay used for the quantification of meganuclease protein expression with high sensitivity. Briefly, standard 96-well plates were coated with anti-meganuclease R54 rabbit antibody (prepared in-house by Precision BioSciences) and left overnight at 4°C. Cell lysates were combined with MSD lysis buffer, incubated at room temperature for 15 minutes, centrifuged at 10,000 g for 30 minutes, and the supernatant was collected. Protein concentration was determined using BCA and diluted to 1 mg / ml with MSD Diluent 100. Plates were removed from 4°C, allowed to rise to room temperature for 30 minutes, blotted to remove excess R54 antibody, and MSD blocker was added for 1 hour. Meganuclease standards were prepared by serially diluting a 1 mg / ml stock to produce seven standards. Next, the plate was washed to remove excess fluid, the standard substance and sample were added to the plate, and then incubated at room temperature for 90 minutes. After incubation, the plate was washed four times with MSD buffer, MSD sulfo-labeled V34 antibody was added to each well, and incubated at room temperature for 1 hour. After this final incubation, the plate was washed four times in MSD buffer and tapped gently to remove excess fluid. MSD Gold Read buffer was added, the plate was sealed, and analyzed in an MSD instrument. When electricity was applied to the plate, the luminescence from the sulfo label was measured to quantify the meganuclease level.

[0254] To identify and quantify the dystrophin protein, samples were analyzed using the WES® system (Protein Simple). Briefly, lysates were prepared from tissue samples using RIPA protein lysis, diluted to a concentration of 500 ng / μl with sample buffer, and 4 μl of the diluted sample was transferred to a PCR tube. The lysates were mixed with a fluorescent master mix (Protein Simple) and denatured. Detection reagents containing primary antibodies (anti-dystrophin MANDYS106 [Sigma, MABT827], anti-vinculin [Abcam, ab129002]) and secondary antibodies (Wes 20X rabbit, Wes 1X mouse, and goat anti-rabbit [Abcam, AB6702]) along with the samples were transferred to 66–440 kilodalton (kDa) cassettes and run on the Wes system according to the standard manufacturer's protocol. After execution, peak analysis was performed by assigning peaks to dystrophin, which has sizes of 285 kDa and 125 kDa relative to vinculin. To determine dystrophin recovery, standard curves for vinculin and dystrophin were created from lysates of corresponding tissues derived from hDMD mice and included in each run (no purified dystrophin protein is currently available due to its large size and instability). The relative loading for each sample was then calculated based on the vinculin and dystrophin signals using a trend line equation. The signals were corrected for variations in the sample loading (using the relative loading from the vinculin calculation), and this loading was divided by the total loading of 500 ng to determine the dystrophin recovery rate.

[0255] Using droplet digital PCR, the ligation frequency (i.e., total ligation %) between the cleaved DMD 19-20 target sites and DMD 35-36 target sites was determined using primer pairs and probes extending to the junction between the 19-20 and corresponding 35-36 target sites. This ddPCR assay used a forward primer 5' (primer 143) for the 19-20 binding site, a reverse primer 3' (primer 145) for the 35-36 site, and a probe (primer 134) specific to the 51-base pair sequence of 5' for the ligated 19-20 / 35-36 site. Reference amplicons were generated, including through reference amplicon assays using primers 66, 68, and 69. Amplification was multiplexed in 20 μL of reactant containing 1× ddPCR Supermix for Probes (without dUTP, BioRad), 250 nM probes, 900 nM primers, 5 U of HindIII-HF, and approximately 50 ng of cellular gDNA. Droplets were generated using a QX100 droplet generator (BioRad). The cycling conditions were as follows: 1 cycle at 95°C (2°C / s ramp) for 10 minutes, 44 cycles at 95°C (1°C / s ramp) for 30 seconds, 59°C (1°C / s ramp) for 45 seconds (see annealing temperature per target site below), 72°C (0.2°C / s ramp) for 2 minutes, and 1 cycle at 98°C, held at 4°C for 10 minutes. Droplets were analyzed using a QX200 droplet reader (BioRad), and data were acquired and analyzed using QuantaSoft analysis software (BioRad). Indel frequencies were calculated by dividing the number of positive copies of the binding site probe by the number of positive copies of the reference probe and comparing the loss of FAM+ copies in nuclease-treated cells with that of pseudo-transfected cells.

[0256] [Table 3]

[0257] Quadriceps femoris tissue sections from mice treated with nucleases were also subjected to IHC analysis to visualize meganuclease protein expression and Pax7, a marker for muscle satellite cells. Briefly, quadriceps femoris tissue was dewaxed and treated with HIER (Heat-Induced Epitope Retrieval) with ER1 on BOND RX for 40 minutes. Slides were blocked for 1 hour at room temperature in 10% NGS PBST (PBS containing 0.1% Tween20) containing MoM (Mouse on Mouse Blocking reagent, VECTOR) blocking reagent, and then incubated overnight at 4°C in a humid chamber in PBST containing 2% NGS with rabbit monoclonal anti-meganuclease antibody (PBI, Rab54) and mouse monoclonal anti-Pax7 antibody (DSHB, supernatant, 1:5) at a dilution of 1:1500. The following day, the samples were incubated with secondary antibodies (goat anti-mouse IgG1 Alexa647 (Invitrogen), goat anti-rabbit Alexa555 (Invitrogen), 1:500) at room temperature for 1 hour, followed by counterstaining with DAPI nuclei for 5 minutes. Excess BOND RX wash buffer was removed, and the coverslips were mounted using VectaShield Vibrance Antifade Mounting Medium. Imaging was performed using Zeiss Apotome 2.0.

[0258] Tissue samples were prepared for histopathological diagnosis using the H&E staining method and sent to a CRO for histopathological analysis. Tissue sections were placed on microscope slides, deparaffinized, and rehydrated (three times with xylene, three times with 100% ethanol, once each with 95% and 80% ethanol, and once with deionized water). Next, the slides were immersed in hematoxylin stain for 3 minutes, rinsed with deionized water, and fixed by immersion in tap water for 5 minutes. The slides were destained by rapid immersion in acidic ethanol about 10 times and rinsed with deionized water for 2 minutes. The slides were stained with eosin for about 30 seconds, rinsed three times with 95% ethanol, then with 100% ethanol (5 min / rinse), and rinsed three times with xylene (15 min / rinse). Cover slips were then placed on the slides using Permount® and allowed to dry overnight. After staining, the slides were visualized under a light microscope by a Study Pathologist. The severity scores for histopathological findings were minor, mild, moderate, and prominent.

[0259] 2.Results To determine the differences between muscle-specific promoters in driving meganuclease protein expression, meganuclease levels were measured using MSD. Meganuclease expression was measured using MSD in mice treated with meganuclease injected with either AAV9 or AAVrh74 into the heart and quadriceps femoris muscle, as described above. Meganuclease protein measured in the quadriceps femoris muscle ranged from approximately 40–102 ng (MHCK7 promoter) and 16–42 ng (tMCK promoter) per 1 mg of total protein containing the AAV transgene delivered by AAV9. Meganuclease protein measured in the heart ranged from approximately 26–206 ng (MHCK7 promoter) and 3–9 ng (tMCK promoter) per 1 mg of total protein containing the AAV transgene delivered by AAV9 (Figure 1A). Meganuclease proteins measured in the quadriceps femoris muscle ranged from approximately 20–56 ng (MHCK7 promoter) and 12–28 ng (tMCK promoter) per 1 mg of total protein containing the AAV transgene delivered by AAVrh74. Meganuclease proteins measured in the heart ranged from approximately 88–142 ng (MHCK7 promoter) and 5–9.5 ng (tMCK promoter) per 1 mg of total protein containing the AAV transgene delivered by AAVrh74 (Figure 1B).

[0260] To determine the differences between muscle-specific promoters in the recovery of dystrophin protein expression, dystrophin protein levels were measured in cardiac and quadriceps femoris tissue using WES. Dystrophin recovery was quantified by comparing dystrophin protein from protein standards to corresponding tissues in hDMD mice using a standard curve from WES protein analysis. Based on the standard curve, AAV9 MHCK7-treated mice were found to have an average of 4.4% dystrophin in the heart and 15% dystrophin in the quadriceps femoris. AAV9 tMCK-treated mice were found to have an average of 7% dystrophin in the heart and 20% dystrophin in the quadriceps femoris (Figure 2A). AAVrh74 MHCK7-treated mice were found to have an average of 5% dystrophin in the heart and 17% dystrophin in the quadriceps femoris. AAVrh74 tMCK-treated mice were found to have an average of 13% dystrophin in the heart and 22% dystrophin in the quadriceps femoris muscle (Figure 2B).

[0261] To determine the differences between muscle-specific promoters in large-scale excision of exons 45–55, total ligation was measured in cardiac and quadriceps femoris tissue by ddPCR. In AAV9 MHCK7 mice, the average total ligation event rate was 20% in the quadriceps femoris and 2.4% in the heart. In AAV9 tMCK mice, the average total ligation event rate was 16% in the quadriceps femoris and 2.8% in the heart (Figure 3A). In AAVrh74 MHCK7 mice, the average total ligation event rate was 12% in the quadriceps femoris and 2% in the heart. In AAVrh74 tMCK mice, the average total ligation event rate was 15.5% in the quadriceps femoris and 5.8% in the heart (Figure 3B). On average, 6% of all ligation events occurred in the liver in AAV9 and AAVrh74 MHCK7 mice, while on average, approximately 2% of all ligation events occurred in the liver in AAV9 and AAVrh74 tMCK mice (Figure 4).

[0262] Immunofluorescence staining of quadriceps femoris tissue sections from nuclease-treated animals showed co-staining of a population of Pax7-positive cells indicating nuclease expression in muscle satellite cells (Figures 5A and 5B). Immunofluorescence staining of quadriceps femoris tissue sections from PBS-treated animals showed minimal background staining for nucleases (green) and clear Pax7 staining of satellite cells (red) (Figure 5C).

[0263] To determine the role of WPRE in the restoration of dystrophin protein expression, dystrophin protein levels were measured in cardiac and quadriceps femoris tissue using WES. Dystrophin restoration was quantified by comparing dystrophin proteins from protein standards to corresponding tissues in hDMD mice using a standard curve from WES protein analysis. Based on the standard curve, AAV9 tMCK WPRE-treated mice were found to have, on average, 7–10% (1e14 and 3e14VG / Kg dose levels) and 2% (1e13VG / Kg dose level) dystrophin in the heart, and 20% (1e14 dose level), 15% (3e14VG / Kg dose level), and 6.6% (1e13VG / Kg dose level) dystrophin in the quadriceps femoris (Figure 6A). Mice treated with AAV9 tMCK but not with WPRE showed, on average, 2% (1e14 and 3e14VG / kg dose levels) and 0.5% (1e13VG / kg dose level) of dystrophin in the heart, and 15% (1e14 dose level), 20% (3e14VG / kg dose level), and 5% (1e13VG / kg dose level) of dystrophin in the quadriceps femoris muscle (Figure 6B).

[0264] As shown in Figure 1, the expression construct utilizing the MHCK7 promoter resulted in substantially higher meganuclease expression in the heart compared to the tMCK promoter. Therefore, cardiac histological analysis was completed to investigate the potential adverse effects of higher meganuclease expression in the heart. As shown in Figure 7, the MHCK7 promoter was associated with a higher degree of necrosis, vacuolation, inflammatory cell infiltration, and atrial thrombosis. These higher levels of cardiac lesions were significantly less prevalent in mice treated with expression constructs utilizing either the AAV9 or rh74 AAV capsid tMCK promoter compared to mice treated with the MHCK7-containing expression cassette.

[0265] 3. Conclusion Meganuclease expression in the quadriceps femoris muscle was highest in AAV9 compared to AAVrh74 with a transgene containing the MHCK7 promoter. Meganuclease expression was significantly lower in both serotype groups with transgenes containing the tMCK promoter. Importantly, there was a more than 10-fold increase in meganuclease expression in the heart using the expression cassette utilizing the MHCK7 promoter. These high levels of expression were associated with an increase in cardiac lesions. Constructs utilizing the tMCK promoter resulted in higher levels of dystrophin recovery in the heart and significantly less cardiac lesions, despite lower overall meganuclease expression. Similarly, but to a lesser extent, there was a greater average dystrophin recovery in the quadriceps femoris muscle using the tMCK promoter compared to the MHCK7 promoter, despite lower meganuclease expression levels. Dystrophin editing (excision of exons 45–55), detected by ddPCR assays of all ligations across serotypes and promoters, was measured within a similar range, contradicting the significant differences in meganuclease expression observed in the same tissues, particularly evident in the heart. These data suggest that overexpression of meganuclease driven by the MHCK7 promoter is significantly excessive in the heart, leading to decreased meganuclease activity and increased cardiac complications. Furthermore, off-tissue editing in the liver was significantly reduced compared to that utilizing the MHCK7 promoter, regardless of serotypes possessing AAV transgenes containing the tMCK promoter.

[0266] Further evidence of transduction of muscle satellite cells, as demonstrated by IHC, was found, with nuclei positive for Pax7 and meganuclease expression markers. Finally, removal of WPRE from the expression cassette resulted in a significant decrease in dystrophin recovery as measured in the heart, suggesting the need for post-transcriptional regulatory elements such as WPRE in myocardial tissue, whereas it had minimal effect on dystrophin recovery in the quadriceps femoris muscle.

[0267] Therefore, these data as a whole suggest significant advantages of using tMCK promoters in conjunction with post-transcriptional regulatory elements (e.g., WPRE) for selective editing of the dystrophin gene in skeletal muscle and cardiomyocyte tissue, regardless of AAV serotype.

[0268] Example 2 Dose-dependent editing of the dystrophin gene in vivo in hDMD mouse studies

[0269] 1. Method In vivo studies were conducted in hDMDdel52 / mdx (hDMD) mice to investigate potential dose-response editing and restoration of truncated, modified human dystrophin protein induced by delivery of meganuclease pairs DMD 19-20L.329 and DMD 35-36L.349. Mice were injected by post-orbital systemic injection with one construct encapsulated in AAV9 (2e14VG / kg, 1e14VG / kg, and 2e13VG / kg) and another construct encapsulated in AAVrh74 (2e14VG / kg, 1e14VG / kg, and 2e13VG / kg). The AAV9 inclusion product contained a viral genome including Sequence ID No. 79, from 5' to 3', which included a muscle-specific promoter tMCK, a first SV40 NLS coding sequence, a DMD 19-20L.329 meganuclease coding sequence, a first c-myc NLS coding sequence, a furin GSG P2A cleavage sequence, a second SV40 NLS coding sequence, a DMD 35-36L.349 meganuclease coding sequence, a second c-myc NLS coding sequence, a WPRE element, and an SV40 polyadenylation signal. The AAVrh74 inclusion product contained a viral genome including SEQ ID NO: 79, from 5' to 3', which included a muscle-specific promoter tMCK, a first SV40 NLS coding sequence, a DMD 19-20L.329 meganuclease coding sequence, a first c-myc NLS coding sequence, a furin GSG P2A cleavage sequence, a second SV40 NLS coding sequence, a DMD 35-36L.349 meganuclease coding sequence, a second c-myc NLS coding sequence, a WPRE element, and an SV40 polyadenylation signal. The nucleic acid sequence of DMD 35-36L.349 meganuclease was codon-modified as described in Example 1. Twenty-eight days after injection, mice were sacrificed and tissue sections derived from skeletal muscle (quadriceps femoris), heart, diaphragm, gastrocnemius, tibialis anterior (TA), and liver were collected for molecular, protein, and histological analysis. As shown in Example 1, the frequency (indel %) of large deletions was determined using droplet digital PCR, employing primer pairs and probes extending to the junctions with DMD nucleases at 19-20 target sites and corresponding 35-36 sites.In addition, samples were analyzed using the WES® system (Protein Simple) for the identification and quantification of dystrophin protein, following the same method outlined in Example 1. Furthermore, AAV9-treated samples were analyzed for meganuclease protein expression via the MSD platform, referring to the method highlighted in Example 1.

[0270] The editing of satellite cells was measured using the Basescope method.

[0271] Slides containing target quadriceps femoris tissue were baked at 60°C for 1 hour and dewaxed using a washing solution of xylene and alcohol (100%). The tissue was then covered with RNAscope® hydrogen peroxide solution for 10 minutes at room temperature (RT). Target retrieval was performed by placing the slides in a container filled with RNAscope® 1X Target Retrieval Reagent (ACD / 322000) in a steamer at 95°C for 15 minutes. Tissue sections were covered with RNAscope® Protease IV and incubated in a HybEZ® oven (ACD / 321711) at 40°C for 30 minutes. Probes for edited DMD44-56 (ACD / 1064961-C2) and mPax7 (ACD / 1070841-C1) were mixed and added to the sections, and incubated in a HybEZ® oven at 40°C for 2 hours. Tissue sections were washed twice with RNAScope washing buffer and then stored overnight in 5X saline-sodium citrate (SSC). After overnight incubation, slides were washed twice with washing buffer and incubated at 40°C with the Hybridize BaseScope™ Duplex AMP reagent series (ACD / 323810) (reagents 1-8) in HybEZ™, washing twice with washing buffer between each reagent. A mixture of Duplex Fast Red-B and Duplex Fast Red-A was added to the slides at room temperature for 10 minutes. Once a red signal was observed using an upright microscope (Fisherbrand™ Research Grade Upright Microscope), the slides were washed twice with washing buffer and then washed at 40°C with Hybridize BaseScope™ Duplex AMP reagents 9-12 in HybEZ™, washing twice with washing buffer between each AMP reagent. A mixed solution of Duplex Green-B and Duplex Green-A was added to the ...

Claims

1. A polynucleotide comprising a muscle-specific expression cassette containing the nucleic acid sequence of Sequence ID No. 86, The aforementioned polynucleotide is the recombinant adenovirus 9 (AAV9) viral genome. The muscle-specific expression cassette is adjacent to the 5' inverted terminal sequence (ITR) and the 3' ITR, and A polynucleotide in which the 5'ITR comprises a first D sequence containing the nucleic acid sequence of SEQ ID NO: 94, and the 3'ITR comprises a second D sequence containing the nucleic acid sequence of SEQ ID NO:

95.

2. A recombinant AAV comprising the polynucleotide described in claim 1 and having an AAV9 capsid.

3. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the polynucleotide described in claim 1.

4. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the recombinant AAV described in claim 2.

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