DYSTROPHIN GENE EXON DELETION USING ENGINEERED NUCLEASES

Abstract

The invention relates to the field of molecular biology and recombinant nucleic acid technology. In particular, the invention relates to a method of treating a patient with Duchenne Muscular Dystrophy comprising the removal of at least one exon from the dystrophin gene using engineered nucleases.

Claims

1. A method for treating Duchenne Muscular Dystrophy associated with a reading frame shift in a dystrophin gene in a subject in need thereof, the method comprising contacting the DNA of a muscle cell of the subject with a first nuclease that cuts a first recognition sequence and a second nuclease that cuts a second recognition sequence; wherein said first recognition sequence is within an intron that is adjacent to and 5 upstream of a first exon in said dystrophin gene of said muscle cell; wherein said second recognition sequence is within an intron that is adjacent to and 3 downstream of said first exon in said dystrophin gene of said muscle cell; wherein said first recognition sequence and said second recognition sequence have complementary overhangs when cut by said first nuclease and said second nuclease; wherein said first exon is removed from said dystrophin gene in said muscle cell; and wherein the normal reading frame of said dystrophin gene is restored.

2-4. (canceled)

5. The method of claim 1, wherein said first exon is Exon 44.

6. The method of claim 1, wherein said first exon is Exon 45.

7. The method of claim 1, wherein said first exon is Exon 51.

8. The method of claim 5, wherein: (a) said first recognition sequence is SEQ ID NO: 2, and wherein said second recognition sequence is SEQ ID NO: 38; (b) said first recognition sequence is selected from SEQ ID NOs: 3, 11, 17, 21, and 27, and said second recognition sequence is selected from SEQ ID NOs: 29 and 41; (c) said first recognition sequence is SEQ ID NO: 5, and said second recognition sequence is SEQ ID NO: 31; (d) said first recognition sequence is selected from SEQ ID NOs: 8 and 12, and said second recognition sequence is selected from SEQ ID NOs: 32, 37, 43, and 44; (e) said first recognition sequence is selected from SEQ ID NOs: 14 and 26, and said second recognition sequence is SEQ ID NO: 34; (f) said first recognition sequence is SEQ ID NO: 18, and said second recognition sequence is SEQ ID NO: 36; (g) said first recognition sequence is SEQ ID NO: 19, and said second recognition sequence is SEQ ID NO: 42; (h) said first recognition sequence is SEQ ID NO: 20, and said second recognition sequence is SEQ ID NO: 40; (i) said first recognition sequence is SEQ ID NO: 23, and said second recognition sequence is selected from SEQ ID NOs: 30 and 35; or (j) said first recognition sequence is SEQ ID NO: 28, and said second recognition sequence is SEQ ID NO: 33.

9. The method of claim 6, wherein: (a) said first recognition sequence is selected from SEQ ID NOs: 50, 51, 53, and 56, and wherein said second recognition sequence is SEQ ID NO: 73; (b) said first recognition sequence is selected from SEQ ID NOs: 54 and 60, and wherein said second recognition sequence is SEQ ID NO: 67; (c) said first recognition sequence is SEQ ID NO: 55, and wherein said second recognition sequence is SEQ ID NO: 66; or (d) said first recognition sequence is SEQ ID NO: 62, and wherein said second recognition sequence is SEQ ID NO: 74.

10. The method of claim 7, wherein: (a) said first recognition sequence is SEQ ID NO: 76, and wherein said second recognition sequence is SEQ ID NO: 134; (b) said first recognition sequence is selected from SEQ ID NOs: 78 and 83, and wherein said second recognition sequence is selected from SEQ ID NOs: 110, 111, and 117; (c) said first recognition sequence is selected from SEQ ID NOs: 79 and 82, and wherein said second recognition sequence is SEQ ID NO: 119; (d) said first recognition sequence is selected from SEQ ID NOs: 85 and 99, and wherein said second recognition sequence is selected from SEQ ID NOs: 120, 124, and 131; (e) said first recognition sequence is SEQ ID NO: 87, and wherein said second recognition sequence is SEQ ID NO: 126; (f) said first recognition sequence is selected from SEQ ID NOs: 88, 93, and 103, and wherein said second recognition sequence is SEQ ID NO: 114 (g) said first recognition sequence is selected from SEQ ID NOs: 89 and 100, and wherein said second recognition sequence is SEQ ID NO: 128 (h) said first recognition sequence is SEQ ID NO: 91, and wherein said second recognition sequence is selected from SEQ ID NOs: 108, 127, and 133; (i) said first recognition sequence is SEQ ID NO: 92, and wherein said second recognition sequence is selected from SEQ ID NOs: 116 and 129; (j) said first recognition sequence is SEQ ID NO: 96, and wherein said second recognition sequence is selected from SEQ ID NOs: 112, 123, and 130; (k) said first recognition sequence is SEQ ID NO: 97, and wherein said second recognition sequence is SEQ ID NO: 118; or (l) said first recognition sequence is SEQ ID NO: 105, and wherein said second recognition sequence is selected from SEQ ID NOs: 106 and 107.

11. The method of claim 8, wherein said first nuclease is SEQ ID NO: 135 and said second nuclease is SEQ ID NO: 136.

12. The method of claim 9, wherein said first nuclease is SEQ ID NO: 137 and said second nuclease is SEQ ID NO: 138.

13. The method of claim 1, wherein the genes encoding said first nuclease and said second nuclease are delivered to said muscle cell using a recombinant adeno-associated virus (AAV).

14-19. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0020] FIG. 1. Structure of the DMD gene. 79 exons are drawn to indicate reading frame. The essential Actin-binding and Dystroglycan-binding domains, which span approximately Exons 2-8 and 62-70, respectively, are indicated.

[0021] FIG. 2A-2E. Strategies for deleting exons from the DMD gene using different types of nucleases. FIG. 2A) Strategy for deleting an exon using a pair of CRISPRs. A pair of guide RNAs (gRNAs) are used which are complementary to a pair of recognition sites flanking the exon of interest. As drawn in this figure, the gRNAs can be complementary to recognition sequences that are distal to the conserved GG motif and the site of Cas9 DNA cleavage. In this orientation, the CRISPR recognition sequences are largely conserved following DNA cleavage, excision of the intervening fragment of genomic DNA, and re-joining of the chromosome ends. FIG. 2B) An alternative scheme for deleting an exon using a pair of CRISPRs in which the gRNAs are complementary to recognition sequences that are proximal to the exon. In this orientation, the CRISPR recognition sequences are largely deleted following DNA cleavage, excision of the intervening fragment of genomic DNA, and re-joining of the chromosome ends. It is contemplated in the invention could also comprise a hybrid of the schemes shown in FIG. 2A and FIG. 2B. FIG. 2C) Strategy for deleting an exon using a pair of compact TALENs (cTALENs). A pair of TAL effector DNA-binding domains (TALEs) are used which bind to a pair of recognition sites flanking the exon of interest. As drawn in this figure, the TALEs can bind to recognition sequences that are distal to the conserved CNNNG motif that is recognized and cut by the I-TevI cleavage domain (TevI-CD). In this orientation, the cTALEN recognition sequences are largely conserved following DNA cleavage, excision of the intervening fragment of genomic DNA, and re-joining of the chromosome ends. Also, the cTALENs in this figure are shown with the TALE and TevI-CD domains in an N- to C-orientation. It is also possible to generate cTALENs with these two domains in a C- to N-orientation. FIG. 2D) An alternative scheme for deleting an exon using a pair of cTALENS in which the TALE domains bind to recognition sequences that are proximal to the exon. In this orientation, the cTALEN recognition sequences are largely deleted following DNA cleavage, excision of the intervening fragment of genomic DNA, and re-joining of the chromosome ends. Also, the cTALENs in this figure are drawn with the TALE and TevI-CD domains in a C- to N-orientation. It is contemplated in the invention could also comprise a hybrid of the schemes shown in FIG. 2C and FIG. 2D.

[0022] FIG. 2E) Strategy for deleting an exon from the DMD gene using a pair of single-chain meganucleases. The meganucleases are drawn as two-domain proteins (MGN-N: the N-terminal domain; and MGN-C: the C-terminal domain) joined by a linker. In the figure, the C-terminal domain is drawn as binding to the half of the recognition sequence that is closest to the exon. In some embodiments, however, the N-terminal domain can bind to this half of the recognition sequence. The central four basepairs of the recognition sequence are shown as NNNN. These four basepairs become single-strand 3 overhangs following cleavage by the meganuclease. The subset of preferred four basepair sequences that comprise this region of the sequence are identified in WO/2010/009147. DNA cleavage by the pair of meganucleases generates a pair of four basepair 3 overhangs at the chromosome ends. If these overhangs are complementary, they can anneal to one another and be directly re-ligated, resulting in the four basepair sequence being retained in the chromosome following exon excision. Because meganucleases cleave near the middle of the recognition sequence, half of each recognition sequence will frequently be retained in the chromosome following excision of the exon. The other half of each recognition sequence will removed from the genome with the exon.

[0023] FIG. 3A-3C. Excision of DMD Exon 44 using the DYS-1/2 and DYS-3/4 meganucleases. FIG. 3A) Sequence of DMD Exon 44 and flanking regions. The Exon sequence is underlined. Recognition sites for the DYS-1/2 and DYS-3/4 meganucleases are shaded in gray with the central four basepairs (which become the 3 overhang following cleavage by the meganuclease) in bold. Annealing sites for a pair of PCR primers used for analysis are italicized. FIG. 3B) Agarose gel electrophoresis analysis of HEK-293 cells co-expressing DYS-1/2 and DYS-3/4. Genomic DNA was isolated from the cells and evaluated by PCR using the primers indicated in (FIG. 3A). PCR products were resolved on an agarose gel and it was found that HEK-293 cells co-expressing the two meganucleases yielded a pair of PCR bands whereas wild-type HEK-293 cells yielded only the larger band. FIG. 3C) sequences from three plasmids harboring the smaller PCR product from (FIG. 3B). The three sequences are shown aligned to the wild-type human sequence. The locations of the DYS-1/2 and DYS-3/4 recognition sequences are shaded in gray with the central four basepairs in bold.

[0024] FIG. 4A-4C. Excision of DMD Exon 45 using the DYS-5/6 and DYS-7/8 meganucleases. FIG. 4A) Sequence of DMD Exon 45 and flanking regions. The Exon sequence is underlined. Recognition sites for the DYS-5/6 and DYS-7/8 meganucleases are shaded in gray with the central four basepairs (which become the 3 overhang following cleavage by the meganuclease) in bold. Annealing sites for a pair of PCR primers used for analysis are italicized. FIG. 4B) Agarose gel electrophoresis analysis of HEK-293 cells co-expressing DYS-5/6 and DYS-7/8. Genomic DNA was isolated from the cells and evaluated by PCR using the primers indicated in (FIG. 4A). PCR products were resolved on an agarose gel and it was found that HEK-293 cells co-expressing the two meganucleases yielded a pair of PCR bands whereas wild-type HEK-293 cells yielded only the larger band. FIG. 4C) sequences from 16 plasmids harboring the smaller PCR product from (FIG. 4B). The sequences are shown aligned to the wild-type human sequence. The locations of the DYS-5/6 and DYS-7/8 recognition sequences are shaded in gray with the central four basepairs in bold.

[0025] FIG. 5A-5C. Evaluation of the MDX-1/2 and MDX-13/14 meganucleases in a reporter assay in CHO cells. FIG. 5A) Schematic of the assay. For each of the two meganucleases, we produced a CHO cell line in which a reporter cassette was integrated stably into the genome of the cell. The reporter cassette comprised, in 5 to 3 order: an SV40 Early Promoter; the 5 2/3 of the GFP gene; the recognition site for either MDX-1/2 (SEQ ID NO: 149) or the recognition site for MDX-13/14 (SEQ ID NO: 150); the recognition site for the CHO-23/24 meganuclease (WO/2012/167192); and the 3 2/3 of the GFP gene. Cells stably transfected with this cassette did not express GFP in the absence of a DNA break-inducing agent. When a DNA break was induced at either of the meganuclease recognition sites, however, the duplicated regions of the GFP gene recombined with one another to produce a functional GFP gene. The percentage of GFP-expressing cells could then be determined by flow cytometry as an indirect measure of the frequency of genome cleavage by the meganucleases. FIG. 5B and FIG. 5C) The two CHO reporter lines were transfected with mRNA encoding the MDX-1/2 (A), MDX-13/14 (FIG. 5B), or CHO-23/34 (FIG. 5A and FIG. 5B) meganucleases. 1.5e6 CHO cells were transfected with 1e6 copies of mRNA per cell using a Lonza Nucleofector 2 and program U-024 according to the manufacturer's instructions. 48 hours post-transfection, the cells were evaluated by flow cytometry to determine the percentage of GFP-positive cells compared to an untransfected (Empty) negative control. The assay was performed in triplicate and standard deviations are shown. The MDX-1/2 and MDX-13/14 meganucleases were found to produce GFP+ cells in their respective cell lines at frequencies significantly exceeding both the negative (Empty) control and the CHO-23/24 positive control, indicating that the nucleases are able to efficiently recognize and cut their intended target sequences in a cell.

[0026] FIG. 6. Sequence alignments from 20 C2C12 mouse myoblast clones in which a portion of the DMD gene was deleted by co-transfection with the MDX-1/2 and MDX-13/14 meganucleases. The location of DMD Exon 23 is shown as are the locations and sequences of the MDX-1/2 and MDX-13/14 target sites. Each of the 20 sequences (SEQ ID NO: 153-172) was aligned to a reference wild-type DMD sequence and deletions relative to the reference are shown as hollow bars.

[0027] FIG. 7. Vector map of the pAAV-MDX plasmid. This packaging plasmid was used in conjunction with an Ad helper plasmid to produce AAV virus capable of simultaneously delivering the genes encoding the MDX-1/2 and MDX-13/14 meganucleases.

DETAILED DESCRIPTION OF THE INVENTION

1.1 References and Definitions

[0028] The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The issued U.S. patents, allowed applications, published foreign applications, and references, including GenBank database sequences, that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.

[0029] As used herein, the term meganuclease refers to an endonuclease that is derived from I-CreI. The term meganuclease, as used herein, refers to an engineered variant of I-CreI that has been modified relative to natural I-CreI with respect to, 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). A meganuclease may bind to double-stranded DNA as a homodimer, as is the case for wild-type I-CreI, or it may bind to DNA as a heterodimer. A meganuclease may also be a single-chain meganuclease in which a pair of DNA-binding domains derived from I-CreI are joined into a single polypeptide using a peptide linker.

[0030] As used herein, the term single-chain meganuclease refers to a polypeptide comprising a pair of meganuclease subunits joined by a linker. A single-chain meganuclease has the organization: N-terminal subunit-Linker-C-terminal subunit. The two meganuclease subunits, each of which is derived from I-CreI, will generally be non-identical in amino acid sequence and will recognize non-identical DNA sequences. Thus, single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. A single chain meganuclease may be referred to as a single-chain heterodimer or single-chain heterodimeric meganuclease although it is not, in fact, dimeric. For clarity, unless otherwise specified, the term meganuclease can refer to a dimeric or single-chain meganuclease.

[0031] As used herein, the term Compact TALEN refers to an endonuclease comprising a DNA-binding domain with 16-22 TAL domain repeats fused in any orientation to any portion of the I-TevI homing endonuclease.

[0032] As used herein, the term CRISPR refers to a caspase-based endonuclease comprising a caspase, such as Cas9, and a guide RNA that directs DNA cleavage of the caspase by hybridizing to a recognition site in the genomic DNA.

[0033] As used herein, with respect to a protein, the term recombinant means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids which encode the protein, and cells or organisms which express the protein. With respect to a nucleic acid, the term recombinant means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally-occurring protein, but produced by cloning and expression in a heterologous host, is not considered recombinant.

[0034] As used herein, the term wild-type refers to any naturally-occurring form of a meganuclease. The term wild-type is not intended to mean the most common allelic variant of the enzyme in nature but, rather, any allelic variant found in nature. Wild-type homing endonucleases are distinguished from recombinant or non-naturally-occurring meganucleases.

[0035] As used herein, the term recognition sequence refers to a DNA sequence that is bound and cleaved by an endonuclease. In the case of a meganuclease, a recognition sequence comprises a pair of inverted, 9 basepair half sites which are separated by four basepairs. In the case of a single-chain meganuclease, the N-terminal domain of the protein contacts a first half-site and the C-terminal domain of the protein contacts a second half-site. Cleavage by a meganuclease produces four basepair 3 overhangs. Overhangs, or sticky ends are short, single-stranded DNA segments that can be produced by endonuclease cleavage of a double-stranded DNA sequence. In the case of meganucleases and single-chain meganucleases derived from I-CreI, the overhang comprises bases 10-13 of the 22 basepair recognition sequence. In the case of a Compact TALEN, the recognition sequence comprises a first CNNNGN sequence that is recognized by the I-TevI domain, followed by a non-specific spacer 4-16 basepairs in length, followed by a second sequence 16-22 bp in length that is recognized by the TAL-effector domain (this sequence typically has a 5 T base). Cleavage by a Compact TALEN produces two basepair 3 overhangs. In the case of a CRISPR, the recognition sequence is the sequence, typically 16-24 basepairs, to which the guide RNA binds to direct Cas9 cleavage. Cleavage by a CRISPR produced blunt ends.

[0036] As used herein, the term target site or target sequence refers to a region of the chromosomal DNA of a cell comprising a recognition sequence for a meganuclease.

[0037] As used herein, the term homologous recombination or HR refers to the natural, cellular process in which a double-stranded DNA-break is repaired using a homologous DNA sequence as the repair template (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). The homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid that was delivered to the cell.

[0038] As used herein, the term non-homologous end-joining or NHEJ refers to the natural, cellular process in which a double-stranded DNA-break is repaired by the direct joining of two non-homologous DNA segments (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). DNA repair by non-homologous end-joining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair.

[0039] As used herein, the term re-ligation refers to a process in which two DNA ends produced by a pair of double-strand DNA breaks are covalently attached to one another with the loss of the intervening DNA sequence but without the gain or loss of any additional DNA sequence. In the case of a pair of DNA breaks that are produced with single-strand overhangs, re-ligation can proceed via annealing of complementary overhangs followed by covalent attachment of 5 and 3 ends by a DNA ligase. Re-ligation is distinguished from NHEJ in that it it does not result in the untemplated addition or removal of DNA from the site of repair.

[0040] As used herein, unless specifically indicated otherwise, the word or is used in the inclusive sense of and/or and not the exclusive sense of either/or.

2.1 Principle of Exon Deletion

[0041] The present invention is based, in part, on the hypothesis that certain deletions in the DMD gene that give rise to the Duchenne phenotype can be compensated for by deleting (an) additional exon(s) immediately up- or downstream of the mutation. The DMD-Leiden Database indicates that most of the mutations that cause Duchenne Muscular Dystrophy are deletions of one or more whole exons that cause a shift in reading frame. In many cases, the reading frame can be restored by eliminating the exon immediately before or after the mutation. As shown in Table 1, 29 different Duchenne-causing mutations, representing 65% of patients, can be compensated for by deleting a single exon adjacent to the mutation. For example, a patient with disease due to the deletion of DMD Exon 45, which occurs in approximately 7% of patients, can be treated with a therapeutic that deletes Exon 46. Notably, a therapeutic capable of deleting Exon 51 or Exon 45 could be used to treat 15% and 13% of patients, respectively.

TABLE-US-00001 TABLE 1 Exon(s) deleted Additional Exon Frequency in DMD-Leiden in patient to delete Database (%) 44, 44-47 43 5 35-43, 45, 45-54 44 8 18-44, 44, 46-47, 45 13 46-48, 46-49, 46-51, 46-53 45 46 7 51, 51-55 50 5 50, 45-50, 51 15 48-50, 49-50, 52, 52-63 51, 53, 53-55 52 3 45-52, 48-52, 53 9 49-52, 50-52, 52

2.2 Nucleases for Deleting Exons

[0042] It is known in the art that it is possible to use a site-specific nuclease to make a DNA break in the genome of a living cell and that such a DNA break can result in permanent modification of the genome via mutagenic NHEJ repair or via HR with a transgenic DNA sequence. The present invention, however, involves co-expression of a pair of nucleases in the same cell. Surprisingly, we found that a pair of nucleases targeted to DNA sites in close proximity to one another (less than 10,000 basepairs apart) can excise the intervening DNA fragment from the genome. Also surprisingly, we found that DNA excision using a pair of nucleases frequently proceeds via a mechanism involving the single-stranded DNA overhangs generated by the nucleases. In experiments involving a pair of meganucleases that generate complementary (i.e. identical) DNA overhangs, it was found that the overhang sequence was frequently conserved following fragment excision and repair of the resulting chromosome ends (see Examples 1 and 2). The mechanism of DNA repair, in this case, appears to direct re-ligation of the broken ends, which has not been observed in mammalian cells. Such precise deletion and re-ligation was not observed when using a pair of meganucleases that generated non-identical overhangs (see Example 3). Thus, in a preferred embodiment, the pair of nucleases used for DMD exon excision are selected to generate complementary overhangs.

[0043] To excise an exon efficiently, the pair of nuclease cut sites need to be relatively close together. In general, the closer the two sites are to one another, the more efficient the process will be. Thus, the preferred embodiment of the invention uses a pair of nucleases that cut sequences that are less than 10,000 basepairs or, more preferably, 5,000 basepairs or, still more preferably, less than 2,500 basepairs, or, most preferably, less than 1,500 basepairs apart.

[0044] As shown in FIG. 2, a variety of different types of nuclease are useful for practicing the invention. FIGS. 2A and 2B show examples of how the invention can be practiced using a pair of CRISPR nucleases. In this case, the invention can be practiced by delivering three genes to the cell: one gene encoding the Cas9 protein and one gene encoding each of the two guide RNAs. CRISPRs cleave DNA to leave blunt ends which are not generally re-ligated cleanly such that the final product will generally have additional insertion and/or deletion (indel) mutations in the sequence. In an alternative embodiment, a CRISPR Nickase may be used, as reported in Ran, et al. (2013) Cell. 154:1380-9. To practice this embodiment, it is necessary to express four guide RNAs in the cell, two of which are complementary to the sequence upstream of the exon and two of which are complementary to the sequence downstream of the exon. In this embodiment, the two pairs of guide RNAs hybridize with complementary strands in the target region and each member of the pair produces a single strand DNA nick on one of the strands. The result is a pair of nicks (equivalent to a double-strand break) that can be off-set from one another to yield a single-strand overhang that is advantageous for practicing the invention. Methods for making CRISPRs and CRISPR Nickases that recognize pre-determined DNA sites are known in the art, for example Ran, et al. (2013) Nat Protoc. 8:2281-308.

[0045] In alternative embodiments, as diagrammed in FIGS. 2C and 2D, the nuclease pair can be Compact TALENs. A compact TALEN comprises a TAL-effector DNA-binding domain (TALE) fused at its N- or C-terminus to the cleavage domain from I-TevI, comprising at least residues 1-96 and preferably residues 1-182 of I-TevI. The I-TevI cleavage domain recognizes and cuts DNA sequences of the form 5-CNbNNtG-3, where b represents the site of cleavage of the bottom strand and t represents the site of cleavage of the top strand and whereN is any of the four bases. A Compact TALEN, thus, cleaves to produce two basepair 3 overhangs. In a preferred embodiment, the Compact TALEN pair used for exon excision is selected to have complementary overhangs that can directly re-ligate. Methods for making TALE domains that bind to pre-determined DNA sites are known in the art, for example Reyon et al. (2012) Nat Biotechnol. 30:460-5.

[0046] In the preferred embodiment, as diagrammed in FIG. 2E, the nucleases used to practice the invention are a pair of Single-Chain Meganucleases. A Single-Chain Meganuclease comprises an N-terminal domain and a C-terminal domain joined by a linker peptide. Each of the two domains recognizes half of the Recognition Sequence and the site of DNA cleavage is at the middle of the Recognition Sequence near the interface of the two subunits. DNA strand breaks are offset by four basepairs such that DNA cleavage by a meganuclease generates a pair of four basepair, 3 single-strand overhangs. In a preferred embodiment, single-chain meganucleases are selected which cut Recognition Sequences with complementary overhangs, as in Examples 1 and 2. Example recognition sequences for DMD Exons 44, 45, and 51 are listed in Tables 2-7. To excise Exon 44, for example, a first meganuclease can be selected which cuts a Recognition Sequence from Table 2, which lists Recognition Sequences upstream of Exon 44. A second meganuclease can then be selected which cuts a Recognition sequences from Table 3, which lists Recognition Sequences downstream of Exon 44. Co-expression of the two meganucleases in the same cell will thus excise Exon 44. Preferably, meganucleases are selected which cut DNA to leave complementary single strand overhangs. For example, SEQ ID NO: 19, if cut by a meganuclease, leaves the overhang sequence: 5-GTAC-3. Likewise, SEQ ID NO: 42 if cut by a meganuclease, leaves the overhang sequence: 5-GTAC-3. Thus, co-expressing a first meganuclease which cleaves SEQ ID NO: 19 with a second meganuclease which cleaves SEQ ID NO:42 will excise DMD Exon 44 from the genome of a human cell such that complementary overhangs are produced which can be repaired via direct re-ligation.

TABLE-US-00002 TABLE2 ExampleMeganucleaseRecognitionSequences UpstreamofDMDExon44 SEQID RecognitionSequence NO: Overhang TTCTCTGTGGTGAGAAAATTTA 2 GTGA TTCACTATTTTGAAATATACAG 3 TTGA TATTTTGAAATATACAGCACAA 4 ATAT TAACTTTGTTCATATTACTATG 5 TCAT ACTTTGTTCATATTACTATGCA 6 ATAT CATATTACTATGCAATAGAACA 7 ATGC CACTAGAACTTATTACTCCTTT 8 TTAT TTTCAGTTGATGAACAGGCAGT 9 ATGA AGTTTTGGATCAAGAATAATAT 10 TCAA AAAAATATTTTGAAAGGGAATA 11 TTGA CCAAATAATTTATTACAATGTT 12 TTAT ATCTTTCTTTTAATCAATAAAT 13 TTAA TTTTAATCAATAAATATATTCA 14 ATAA ACCTTCCATTTAAAATCAGCTT 15 TTAA TCAGCTTTTATATTGAGTATTT 16 ATAT GCTTTTATATTGAGTATTTTTT 17 TTGA TAAAATGTTGTGTGTACATGCT 18 GTGT ATGTTGTGTGTACATGCTAGGT 19 GTAC GCTAGGTGTGTATATTAATTTT 20 GTAT ATTTGTTACTTGAAACTAAACT 21 TTGA CTAAACTCTGCAAATGCAGGAA 22 GCAA GTGATATCTTTGTCAGTATAAC 23 TTGT AAAAAATATACGCTATATCTCT 24 ACGC ATCTGTTTTACATAATCCATCT 25 ACAT CTGTTTTACATAATCCATCTAT 26 ATAA CTATTTTTCTTGATCCATATGC 27 TTGA CATATGCTTTTACCTGCAGGCG 28 TTAC

TABLE-US-00003 TABLE3 ExampleMeganucleaseRecognitionSequences DownstreamofDMDExon44 SEQID RecognitionSequence NO: Overhang AAATTACTTTTGACTGTTGTTG 29 TTGA TGACTGTTGTTGTCATCATTAT 30 TTGT TTGTTGTCATCATTATATTACT 31 TCAT TTGTCATCATTATATTACTAGA 32 TTAT ATCATTATATTACTAGAAAGAA 33 TTAC AAAATTATCATAATGATAATAT 34 ATAA ATGGACTTTTTGTGTCAGGATG 35 TTGT GGACTTTTTGTGTCAGGATGAG 36 GTGT GGAGCTGGTTTATCTGATAAAC 37 TTAT ATTGAATCTGTGACAGAGGGAA 38 GTGA AGGGAAGCATCGTAACAGCAAG 39 TCGT GGGCAGTGTGTATTTCGGCTTT 40 GTAT TATATTCTATTGACAAAATGCC 41 TTGA TAATTGTTGGTACTTATTGACA 42 GTAC TGTTGGTACTTATTGACATTTT 43 TTAT TTTTATGGTTTATGTTAATAGG 44 TTAT

TABLE-US-00004 TABLE4 ExampleMeganucleaseRecognitionSequences UpstreamofDMDExon45 SEQID RecognitionSequence NO: Overhang AGTTTTTTTTTAATACTGTGAC 45 TTAA TTTAATACTGTGACTAACCTAT 46 GTGA TTTCACCTCTCGTATCCACGAT 47 TCGT TCACCTCTCGTATCCACGATCA 48 GTAT CTCGTATCCACGATCACTAAGA 49 ACGA CCAAATACTTTGTTCATGTTTA 50 TTGT GGAACATCCTTGTGGGGACAAG 51 TTGT AATTTGCTCTTGAAAAGGTTTC 52 TTGA CTAATTGATTTGTAGGACATTA 53 TTGT TTCCCTGACACATAAAAGGTGT 54 ACAT CCCTGACACATAAAAGGTGTCT 55 ATAA CTTTCTGTCTTGTATCCTTTGG 56 TTGT ATCCTTTGGATATGGGCATGTC 57 ATAT TGGATATGGGCATGTCAGTTTC 58 GCAT GATATGGGCATGTCAGTTTCAT 59 ATGT GAAATTTTCACATGGAGCTTTT 60 ACAT TTTCTTTCTTTGCCAGTACAAC 61 TTGC TCTTTGCCAGTACAACTGCATG 62 GTAC TTTGGTATCTTACAGGAACTCC 63 TTAC

TABLE-US-00005 TABLE5 ExampleMeganucleaseRecognitionSequences DownstreamofDMDExon45 SEQID RecognitionSequence NO: Overhang AAGAATATTTCATGAGAGATTA 64 TCAT GAATATTTCATGAGAGATTATA 65 ATGA TGAGAGATTATAAGCAGGGTGA 66 ATAA AAGGCACTAACATTAAAGAACC 67 ACAT TCAACAGCAGTAAAGAAATTTT 68 GTAA TTCTTTTTTTCATATACTAAAA 69 TCAT CTAAAATATATACTTGTGGCTA 70 ATAC TGAATATCTTCAATATATTTTA 71 TCAA CAATTATAAATGATTGTTTTGT 72 ATGA ATGATTGTTTTGTAGGAAAGAC 73 TTGT TCATATTTTGTACAAAATAAAC 74 GTAC

TABLE-US-00006 TABLE6 ExampleMeganucleaseRecognitionSequences UpstreamofDMDExon51 RecognitionSequence SEQIDNO: Overhang ATACGTGTATTGCTTGTACTAC 75 TTGC GTATTGCTTGTACTACTCACTG 76 GTAC ACTGAATCTACACAACTGCCCT 77 ACAC TGAATCTACACAACTGCCCTTA 78 ACAA CAACTGCCCTTATGACATTTAC 79 TTAT GGTAAATACATGAAAAATGCTT 80 ATGA TTGCCTTGCTTACTGCTTATTG 81 TTAC GCTTACTGCTTATTGCTAGTAC 82 TTAT TAGTACTGAACAAATGTTAGAA 83 ACAA ACTGAACAAATGTTAGAACTGA 84 ATGT AAGATTTATTTAATGACTTTGA 85 TTAA CAGTATTTCATGTCTAAATAGA 86 ATGT GGTTTTTCTTCACTGCTGGCCA 87 TCAC CAATCTGAAATAAAAAGAAAAA 88 ATAA CTGCTCCCAGTATAAAATACAG 89 GTAT AAGAACGTTTCATTGGCTTTGA 90 TCAT ACTTCCTATTCAAGGGAATTTT 91 TCAA TGTTTTTTCTTGAATAAAAAAA 92 TTGA TTTTCTTGAATAAAAAAAAAAT 93 ATAA TTGTTTTCTTTACCACTTCCAC 94 TTAC ACAATGTATATGATTGTTACTG 95 ATGA TGTATATGATTGTTACTGAGAA 96 TTGT CTTGTCCAGGCATGAGAATGAG 97 GCAT TGTCCAGGCATGAGAATGAGCA 98 ATGA AATCGTTTTTTAAAAAATTGTT 99 TTAA TTCTACCATGTATTGCTAAACA 100 GTAT TACCATGTATTGCTAAACAAAG 101 TTGC TATAATGTCATGAATAAGAGTT 102 ATGA ATGTCATGAATAAGAGTTTGGC 103 ATAA TTTTCCTTTTTGCAAAAACCCA 104 TTGC TTCCTTTTTGCAAAAACCCAAA 105 GCAA

TABLE-US-00007 TABLE7 ExampleMeganucleaseRecognitionSequences DownstreamofDMDExon51 RecognitionSequence SEQIDNO: Overhang AGTTCTTAGGCAACTGTTTCTC 106 GCAA TCTCTCTCAGCAAACACATTAC 107 GCAA TAAGTATAATCAAGGATATAAA 108 TCAA AGTAGCCATACATTAAAAAGGA 109 ACAT AGGAAATATACAAAAAAAAAAA 110 ACAA AGAAACCTTACAAGAATAGTTG 111 ACAA CAAGAATAGTTGTCTCAGTTAA 112 TTGT ATCTATTTTATACCAAATAAGT 113 ATAC TTATACCAAATAAGTCACTCAA 114 ATAA TTTGTTTTGGCACTACGCAGCC 115 GCAC TAAGGATAATTGAAAGAGAGCT 116 TTGA AGAAAAGTAACAAAACATAAGA 117 ACAA TTAAAGTTGGCATTTATGCAAT 118 GCAT AGTTGGCATTTATGCAATGCCA 119 TTAT AACATGTTTTTAATACAAATAG 120 TTAA TACATTGATGTAAATATGGTTT 121 GTAA ATATCTTTTATATTTGTGAATG 122 ATAT CTTTTATATTTGTGAATGATTA 123 TTGT TGTGAATGATTAAGAAAAATAA 124 TTAA AATTGTTATACATTAAAGTTTT 125 ACAT AAAGTTTTTTCACTTGTAACAG 126 TCAC TAACAGCTTTCAAGCCTTTCTA 127 TCAA GGTATTTAGGTATTAAAGTACT 128 GTAT TACTACCTTTTGAAAAAACAAG 129 TTGA GGAATTTCTTTGTAAAATAAAC 130 TTGT AACCTGCATTTAAAGGCCTTGA 131 TTAA TGAGCTTGAATACAGAAGACCT 132 ATAC TGATTGTGGTCAAGCCATCTCT 133 TCAA CTATTCTGAGTACAGAGCATAC 134 GTAC

2.3 Methods for Delivering and Expressing Nucleases

[0047] Treating Duchenne Muscular Dystrophy using the invention requires that a pair of nucleases be expressed in a muscle cell. The nucleases can be delivered as purified protein or as RNA or DNA encoding the nucleases. In one embodiment, the nuclease proteins or mRNA or vector encoding the nucleases are supplied to muscle cells via intramuscular injection (Maltzahn, et al. (2012) Proc Natl Acad Sci USA. 109:20614-9) or hydrodynamic injection (Taniyama et al. (2012) Curr Top Med Chem. 12:1630-7; Hegge, et al. (2010) Hum Gene Ther. 21:829-42). To facilitate cellular uptake, the proteins or nucleic acid(s) can be coupled to a cell penetrating peptide to facilitate uptake by muscle cells. Examples of cell pentrating peptides known in the art include poly-arginine (Jearawiriyapaisarn, et al. (2008) Mol Ther. 16:1624-9), TAT peptide from the HIV virus (Hudecz et al. (2005), Med. Res. Rev. 25: 679-736), MPG (Simeoni, et al. (2003) Nucleic Acids Res. 31:2717-2724), Pep-1 (Deshayes et al. (2004) Biochemistry 43: 7698-7706, and HSV-1 VP-22 (Deshayes et al. (2005) Cell Mol Life Sci. 62:1839-49. Alternatively, cell penetration can be facilitated by liposome encapsulation (see, e.g., Lipofectamine, Life Technologies Corp., Carlsbad, Calif.). The liposome formulation can be used to facilitate lipid bilayer fusion with a target cell, thereby allowing the contents of the liposome or proteins associated with its surface to be brought into the cell.

[0048] In some embodiments, the genes encoding a pair of nucleases are delivered using a viral vector. Such vectors are known in the art and include lentiviral vectors, adenoviral vectors, and adeno-associated virus (AAV) vectors (reviewed in Vannucci, et al. (2013 New Microbiol. 36:1-22). In some embodiments, the viral vectors are injected directly into muscle tissue. In alternative embodiments, the viral vectors are delivered systemically. Example 3 describes a preferred embodiment in which the muscle is injected with a recombinant AAV virus encoding a pair of single-chain meganucleases. It is known in the art that different AAV vectors tend to localize to different tissues. Muscle-tropic AAV serotypes include AAV1, AAV9, and AAV2.5 (Bowles, et al. (2012) Mol Ther. 20:443-55). Thuse, these serotypes are preferred for the delivery of nucleases to muscle tissue.

[0049] If the nuclease genes are delivered in DNA form (e.g. plasmid) and/or via a viral vector (e.g. AAV) they must be operably linked to a promoter. In some embodiments, this can be a viral promoter such as endogenous promoters from the viral vector (e.g. the LTR of a lentiviral vector) or the well-known cytomegalovirus- or SV40 virus-early promoters. In a preferred embodiment, the nuclease genes are operably linked to a promoter that drives gene expression preferentially in muscle cells. Examples of muscle-specific promoters include C5-12 (Liu, et al. (2004) Hum Gene Ther. 15:783-92), the muscle-specific creatine kinase (MCK) promoter (Yuasa, et al. (2002) Gene Ther. 9:1576-88), or the smooth muscle 22 (SM22) promoter (Haase, et al. (2013) BMC Biotechnol. 13:49-54). In some embodiments, the nuclease genes are under the control of two separate promoters. In alternative embodiments, the genes are under the control of a single promoter and are separated by an internal-ribosome entry site (IRES) or a 2A peptide sequence (Szymczak and Vignali (2005) Expert Opin Biol Ther. 5:627-38).

[0050] It is envisioned that a single treatment will permanently delete exons from a percentage of patient cells. In preferred embodiments, these cells will be myoblasts or other muscle precurser cells that are capable of replicating and giving rise to whole muscle fibers that express functional (or semi-functional) dystrophin. If the frequency of exon deletion is low, however, it may be necessary to perform multiple treatments on each patient such as multiple rounds of intramuscular injections.

EXAMPLES

[0051] This invention is further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are intended to be encompassed in the scope of the claims that follow the examples below.

Example 1

Deletion of DMD Exon 44 Using a Pair of Engineered, Single-Chain Meganucleases

[0052] 1. Meganucleases that Recognize SEQ ID NO: 19 and SEQ ID NO: 42

[0053] An engineered meganuclease (SEQ ID NO: 135) was produced which recognizes and cleaves SEQ ID NO: 19. This meganuclease is called DYS-1/2. A second engineered meganuclease (SEQ ID NO: 136) was produced which recognizes and cleaves SEQ ID NO: 42. This meganuclease is called DYS-3/4 (FIG. 3A). Each meganuclease comprises an N-terminal nuclease-localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit.

2. Deletion of DMD Exon 44 in HEK-293 Cells

[0054] Human embryonic kidney (HEK-293) cells were co-transfected with mRNA encoding DYS-1/2 and DYS-3/4. mRNA was prepared by first producing a PCR template for an in vitro transcription reaction (SEQ ID NO: 139 and SEQ ID NO: 140. Each PCR product included a T7 promoter and 609 bp of vector sequence downstream of the meganuclease gene. The PCR product was gel purified to ensure a single template. Capped (m7G) RNA was generated using the RiboMAX T7 kit (Promega) according to the manufacturer's instructions and. Ribo m7G cap analog (Promega) was included in the reaction and 0.5 ug of the purified meganuclease PCR product served as the DNA template. Capped RNA was purified using the SV Total RNA Isolation System (Promega) according to the manufacturer's instructions.

[0055] 1.510.sup.6HEK-293 cells were nucleofected with 1.510.sup.12 copies of DYS-1/2 mRNA and 1.510.sup.12 copies of DYS-3/4 mRNA (210.sup.6 copies/cell) using an Amaxa Nucleofector II device (Lonza) according to the manufacturer's instructions. 48 hours post-transfection, genomic DNA was isolated from the cells using a FlexiGene kit (Qiagen) according to the manufacturer's instructions. The genomic DNA was then subjected to PCR using primers flanking the DYS-1/2 and DYS-3/4 cut sites (SEQ ID NO: 141 and SEQ ID NO:142). When PCR products were resolved by agarose gel electrophoresis, it was apparent that cells co-expressing DYS-1/2 and DYS-3/4 yielded two PCR products with apparent lengths of 1079 basepairs and 233 basepairs whereas genomic DNA from untransfected HEK-293 cells yielded only the larger product (FIG. 3B). The larger product is consistent with the expected size of a PCR fragment from cells with intact DMD Exon 44. The smaller product is consistent with the expected size of a PCR fragment from cells in which Exon 44 has been excised from the DMD gene.

[0056] The smaller PCR product was isolated from the gel and cloned into a bacterial plasmid (pUC-19) for sequence analysis. Three plasmid clones were sequenced, all of which were found to have Exon 44 deleted (FIG. 3C). Surprisingly, two of the three plasmids carried PCR products from cells in which the deletion consisted precisely of the region intervening the expected DYS-1/2 and DYS-3/4-induced DNA breaks. It appears that the two meganucleases cleaved their intended recognition sites, leaving compatible 5-GTAC-3 overhangs, the intervening fragment comprising Exon 44 was lost, and the two chromosome ends were then re-ligated. The third plasmid clone carried a PCR product from a cell in which the region intervening the two cleavage sites was excised along with 10 additional bases.

3. Conclusions

[0057] We have demonstrated that it is possible to use a pair of engineered single-chain meganucleases to excise a fragment from the human genome in a cultured cell line. The DNA removal and repair process appears to have proceeded via a mechanism that involves the 3 overhangs produced by the nucleases, suggesting that the process is more efficient when the overhangs are complementary and able to anneal to one another.

Example 2

Deletion of DMD Exon 45 Using a Pair of Engineered, Single-Chain Meganucleases

[0058] 1. Meganucleases that Recognize SEQ ID NO: 62 and SEQ ID NO: 74

[0059] An engineered meganuclease (SEQ ID NO: 137) was produced which recognizes and cleaves SEQ ID NO: 62. This meganuclease is called DYS-5/6. A second engineered meganuclease (SEQ ID NO: 138) was produced which recognizes and cleaves SEQ ID NO: 74. This meganuclease is called DYS-7/8 (FIG. 4A). Each meganuclease comprises an N-terminal nuclease-localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit.

2. Deletion of DMD Exon 45 in HEK-293 Cells

[0060] Human embryonic kidney (HEK-293) cells were co-transfected with mRNA encoding DYS-5/6 and DYS-7/8. mRNA was prepared by first producing a PCR template for an in vitro transcription reaction (SEQ ID NO: 143(20) and SEQ ID NO: 144(21). Each PCR product included a T7 promoter and 609 bp of vector sequence downstream of the meganuclease gene. The PCR product was gel purified to ensure a single template. Capped (m7G) RNA was generated using the RiboMAX T7 kit (Promega) according to the manufacturer's instructions and. Ribo m7G cap analog (Promega) was included in the reaction and 0.5 ug of the purified meganuclease PCR product served as the DNA template. Capped RNA was purified using the SV Total RNA Isolation System (Promega) according to the manufacturer's instructions.

[0061] 1.510.sup.6HEK-293 cells were nucleofected with 1.510.sup.12 copies of DYS-5/6 mRNA and 1.510.sup.12 copies of DYS-7/8 mRNA (210.sup.6 copies/cell) using an Amaxa Nucleofector II device (Lonza) according to the manufacturer's instructions. 48 hours post-transfection, genomic DNA was isolated from the cells using a FlexiGene kit (Qiagen) according to the manufacturer's instructions. The genomic DNA was then subjected to PCR using primers flanking the DYS-5/6 and DYS-7/8 cut sites (SEQ ID NO: 145 and SEQ ID NO:146). When PCR products were resolved by agarose gel electrophoresis, it was apparent that cells co-expressing DYS-5/6 and DYS-7/8 yielded two PCR products with apparent lengths of 1384 basepairs and 161 basepairs whereas genomic DNA from untransfected HEK-293 cells yielded only the larger product (FIG. 4B). The larger product is consistent with the expected size of a PCR fragment from cells with intact DMD Exon 45. The smaller product is consistent with the expected size of a PCR fragment from cells in which Exon 45 has been excised from the DMD gene.

[0062] The smaller PCR product was isolated from the gel and cloned into a bacterial plasmid (pUC-19) for sequence analysis. 16 plasmid clones were sequenced, all of which were found to have Exon 45 deleted (FIG. 4C). Surprisingly, 14 of the 16 plasmids carried PCR products from cells in which the deletion consisted precisely of the region intervening the expected DYS-5/6 and DYS-7/8-induced DNA breaks. It appears that the two meganucleases cleaved their intended recognition sites, leaving compatible 5-GTAC-3 overhangs, the intervening fragment comprising Exon 45 was lost, and the two chromosome ends were then re-ligated. The two remaining plasmid clones carried PCR product from cells in which the region intervening the two cleavage sites was excised along with 36 additional bases.

3. Conclusions

[0063] We have demonstrated that it is possible to use a pair of engineered single-chain meganucleases to excise a fragment from the human genome in a cultured cell line. The DNA removal and repair process appears to have proceeded via a mechanism that involves the 3 overhangs produced by the nucleases, suggesting that the process is more efficient when the overhangs are complementary and able to anneal to one another.

Example 3

Deletion of DMD Exon 23 in a Mouse Using AAV-Delivered Meganucleases

1. Development of Nucleases to Delete Mouse DMD Exon 23

[0064] The standard mouse model of DMD is the mdx mouse, which has a point mutation in Exon 23 that introduces a premature stop codon (Sicinski et al. (1989) Science. 244:1578-80). In the mouse, DMD Exon 23 is 213 basepairs, equivalent to 71 amino acids. Thus, we reasoned that it should be possible to delete Exon 23 in its entirety and thereby remove the stop codon while maintaining the reading frame of the DMD gene. To this end, we developed a pair of single-chain meganucleases called MDX-1/2 (SEQ ID NO: 147) and MDX-13/14 (SEQ ID NO: 148). The former recognizes a DNA sequence upstream of mouse DMD Exon 23 (SEQ ID NO: 149) while the latter recognizes a DNA sequence downstream of mouse DMD Exon 23 (SEQ ID NO: 150). The nucleases were tested, initially, using a reporter assay called iGFFP in CHO cells as shown in FIG. 5. Both nucleases were found to efficiently cut their intended DNA sites using this assay.

2. Deletion of Mouse DMD Exon 23 in Mouse Myoblast Cells

[0065] A mouse myoblast cell line (C2C12) was co-transfected with in vitro transcribed mRNA encoding the MDX-1/2 and MDX-13/14 nucleases. mRNA was produced using the RiboMAX T7 kit from Promega. 1e6 C2C12 cells were Nucleofected with a total of 2e6 copies/cell of mRNA encoding each MDX enzyme pairs (1e6 copies of each mRNA) using an Amaxa 2b device and the B-032 program. After 96 hours, cells were cloned by limiting dilution in 96-well plates. After approximately 2 weeks growth, cells were harvested and genomic DNA was isolated using a FlexiGene kit from Qiagen. A PCR product was then generated for each clone using a forward primer in DMD Intron 22 (SEQ ID NO: 151) and a reverse primer in Intron 23 (SEQ ID NO: 152). 60 of the PCR products were then cloned and sequenced. 20 of the sequences had deletions consistent with meganuclease-induced cleavage of the DMD gene followed by mutagenic DNA repair (FIG. 6, SEQ ID NO:153-172). 11 of the sequences were missing at least a portion of the MDX-1/2 and MDX-13/14 recognition sites, as well as Exon 23 (SEQ ID NO:153-163). These sequences were likely derived from cells in which both nucleases cut their intended sites and the intervening sequence was deleted. 4 of the sequences were missing Exon 23 but had an intact MDX-1/2 recognition sequence (SEQ ID NO:164-167). These appear to be due to DNA cleavage by MDX-13/14 alone followed by the deletion of a large amount of sequence. Five of the sequences had an intact MDX-1/2 recognition site and all or a portion of Exon 23 but were missing all or a portion of the MDX-13/14 recognition site (SEQ ID NO:168-172). These sequences appear to be due to DNA cleavage by MDX-13/14 alone followed by the deletion of a smaller amount of sequence insufficient to eliminate all of Exon 23. In stark contrast to the experiments in Examples 1 and 2, we did not obtain a consistent DNA sequence following the deletion of DMD Exon 23 in the mouse cells. This is likely because the two MDX meganucleases do not generate DNA breaks with compatible 3 overhangs. MDX-1/2 generates an overhang with the sequence 5-GTGA-3 and MDX-13/14 generates an overhang with the sequence 5-ACAC-3. Thus, we conclude that the consistent sequence results obtained in Examples 1 and 2 are due to the compatibility of the 3 overhangs generated by the pair of meganucleases.

3. Generation of Recombinant AAV Vectors for Delivery of a Pair of Engineered Nucleases.

[0066] To produce AAV vectors for simultaneous delivery of MDX-1/2 and MDX-13/14 genes, we first produced a packaging plasmid called pAAV-MDX (FIG. 7, SEQ ID NO. 173) comprising a pair of inverted terminal repeat (ITR) sequences from AAV2, as well as the gene coding sequences for the MDX-1/2 and MDX-13/14 meganucleases, each under the control of a CMV Early promoter. This vector was used to produce recombinant AAV2 virus by co-transfection of HEK-293 cells with an Ad helper plasmid according to the method of Xiao, et al (Xiao, et al. (1998) J. Virology 72:2224-2232). Virus was then isolated by cesium-chloride gradient centrifugation as described by Grieger and Samulski (Grieger and Samulski (2012) Methods Enzymol. 507:229-254). To confirm that the resulting virus particles were infectious and capable of expressing both engineered meganucleases, they were added to cultured iGFFP CHO cells carrying reporter cassettes for either MDX-1/2 or MDX-13/14 (see FIG. 5A). The addition of recombinant virus particles to the CHO line carrying a reporter cassette for MDX-1/2 resulted in GFP gene expression in 7.1% of the cells. The addition of virus to the CHO line carrying a reporter for MDX-13/14 resulted in GFP gene expression in 10.2% of cells. Thus, we conclude that the virus was able to transduce CHO cells and that transduced cells expressed both nucleases.

4. Deletion of DMD Exon 23 in Mouse Muscle Following AAV Delivery of a Pair of Meganuclease Genes.

[0067] Recombinant AAV1 virus particles carrying the MDX-1/2 and MDX-13/14 genes were produced as described above. Three hindlimb TA muscles from a pair of mdx mice were injected with virus as described in Xiao, et al (Xiao, et al. (1998) J. Virology 72:2224-2232). One muscle from one mouse was not injected as a negative control. Muscles from the two mice were harvested at 4 days or 7 days post-injection and genomic DNA was isolated from the muscle tissue. The genomic region surrounding DMD Exon 23 was amplified by PCR using a first primer pair (SEQ ID NO:151 and SEQ ID NO: 152). This reaction was then used to template a second PCR reaction using a nested primer pair (SEQ ID NO:174 and SEQ ID NO: 175) to eliminate non-specific PCR products. PCR products were then visualized on an agarose gel and it was found that genomic DNA from the three AAV1 injected muscles, but not the un-injected control muscle, yielded smaller PCR products that were consistent in size with the product expected following deletion of DMD Exon 23 by the MDX-1/2 and MDX-13/14 meganucleases. The smaller PCR products were then cloned and sequenced. Three unique sequences were obtained, each of which comprised a portion of the mouse DMD gene including part of Intron 22 and Intron 23 but lacking Exon 23 and all of the sequence intervening the cut sites for the MDX-1/2 and MDX-13/14 meganucleases (SEQ ID NO: 176-178). Thus, we have demonstrated that a pair of meganucleases delivered by AAV can be used to delete a portion of the DMD gene in vivo from mouse muscle.

5. Conclusions

[0068] We have demonstrated that the genes encoding a pair of engineered single-chain meganucleases can be delivered to cells and organisms using recombinant AAV vectors and that meganucleases so delivered are able to cleave genomic DNA in the cell and delete fragments of DNA from the genome. We have further demonstrated that a pair of meganuclease-induced DNA breaks that do not generate compatible overhangs will not re-ligate to yield a defined sequence outcome following removal of the intervening sequence. Thus, for therapeutic applications in which a defined sequence outcome is desirable, it is preferable to use a pair of nucleases that generate identical overhangs.

TABLE-US-00008 SEQUENCELISTING SEQIDNO:1(wild-typeI-CreI,GenbankAccession#PO5725) 1 MNTKYNKEFLLYLAGFVDGDGSIIAQIKPNQSYKFKHQLSLAFQVTQKTQRRWFLDKLVD 61 EIGVGYVRDRGSVSDYILSEIKPLHNFLTQLQPFLKLKQKQANLVLKIIWRLPSAKESPD 121 KFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKKKSSP SEQIDNO:135(DYS-1/2) 1 MAPKKKRKVHMNTKYNKEFLLYLAGFVDGDGSIYAWISPSQTCKFKHRLMLRFIVSQKTQ 61 RRWFLDKLVDEIGVGYVQDCGSVSEYRLSEIKPLHNFLTQLQPFLKLKQKQANLVLKIIE 121 QLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSA 181 ASSASSSPGSGISEALRAGAGSGTGYNKEFLLYLAGFVDGDGSIYACILPTQRQKFKHGL 241 TLYFRVTQKTQRRWFLDKLVDEIGVGYVLDFGSVSCYSLSQIKPLHNFLTQLQPFLKLKQ 301 KQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKK 361 KSSP SEQIDNO:136(DYS-3/4) 1 MAPKKKRKVHMNTKYNKEFLLYLAGFVDGDGSIFASIRPRQTSKFKHALALFFVVGQKTQ 61 RRWFLDKLVDEIGVGYVYDRGSVSVYQLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIE 121 QLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSA 181 ASSASSSPGSGISEALRAGAGSGTGYNKEFLLYLAGFVDGDGSIIACIRPHQAYKFKHQL 241 CLSFCVYQKTQRRWFLDKLVDEIGVGYVTDAGSVSSYRLSEIKPLHNFLTQLQPFLKLKQ 301 KQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKK 361 KSSP SEQIDNO:137(DYS-5/6) 1 MAPKKKRKVHMNTKYNKEFLLYLAGFVDGDGSIFACIQPDQRAKFKHTLRLSFEVGQKTQ 61 RRWFLDKLVDEIGVGYVNDSGSVSKYRLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIE 121 QLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSA 181 ASSASSSPGSGISEALRAGAGSGTGYNKEFLLYLAGFVDGDGSIYATIQPTQCAKFKHQL 241 TLRFSVSQKTQRRWFLDKLVDEIGVGYVCDKGSVSEYMLSEIKPLHNFLTQLQPFLKLKQ 301 KQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKK 361 KSSP SEQIDNO:138(DYS-7/8) 1 MAPKKKRKVHMNTKYNKEFLLYLAGFVDGDGSIYACILPVQRCKFKHGLSLRFMVSQKTQ 61 RRWFLDKLVDEIGVGYVYDCGSVSEYRLSEIKPLHNFLTQLQPFLKLKQKQANLVLKIIE 121 QLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSA 181 ASSASSSPGSGISEALRAGAGSGTGYNKEFLLYLAGFVDGDGSIFASIVPDQRSKFKHGL 241 ALRFNVVQKTQRRWFLDKLVDEIGVGYVYDQGSVSEYRLSEIKPLHNFLTQLQPFLKLKQ 301 KQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKK 361 KSSP SEQIDNO:139(DYS-1/2PCRTemplateformRNA) 1 CACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTC 61 ACTATAGGCTAGCCTCGAGCCGCCACCATGGCACCGAAGAAGAAGCGCAAGGTGCATATG 121 AATACAAAATATAATAAAGAGTTCTTACTCTACTTAGCAGGGTTTGTAGACGGTGACGGT 181 TCCATCTATGCCTGGATCAGTCCTTCGCAAACGTGTAAGTTCAAGCACAGGCTGATGCTC 241 CGGTTCATTGTCTCGCAGAAGACACAGCGCCGTTGGTTCCTCGACAAGCTGGTGGACGAG 301 ATCGGTGTGGGTTACGTGCAGGACTGTGGCAGCGTCTCCGAGTACCGGCTGTCCGAGATC 361 AAGCCTTTGCATAATTTTTTAACACAACTACAACCTTTTCTAAAACTAAAACAAAAACAA 421 GCAAATTTAGTTTTAAAAATTATTGAACAACTTCCGTCAGCAAAAGAATCCCCGGACAAA 481 TTCTTAGAAGTTTGTACATGGGTGGATCAAATTGCAGCTCTGAATGATTCGAAGACGCGT 541 AAAACAACTTCTGAAACCGTTCGTGCTGTGCTAGACAGTTTACCAGGATCCGTGGGAGGT 601 CTATCGCCATCTCAGGCATCCAGCGCCGCATCCTCGGCTTCCTCAAGCCCGGGTTCAGGG 661 ATCTCCGAAGCACTCAGAGCTGGAGCAGGTTCCGGCACTGGATACAACAAGGAATTCCTG 721 CTCTACCTGGCGGGCTTCGTCGACGGGGACGGCTCCATCTATGCCTGTATCCTTCCGACT 781 CAGCGTCAGAAGTTCAAGCACGGGCTGACGCTCTATTTCCGGGTCACTCAGAAGACACAG 841 CGCCGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGTGTGGGTTACGTGCTGGACTTT 901 GGCAGCGTCTCCTGTTACTCTCTGTCCCAGATCAAGCCTCTGCACAACTTCCTGACCCAG 961 CTCCAGCCCTTCCTGAAGCTCAAGCAGAAGCAGGCCAACCTCGTGCTGAAGATCATCGAG 1021 CAGCTGCCCTCCGCCAAGGAATCCCCGGACAAGTTCCTGGAGGTGTGCACCTGGGTGGAC 1081 CAGATCGCCGCTCTGAACGACTCCAAGACCCGCAAGACCACTTCCGAAACCGTCCGCGCC 1141 GTTCTAGACAGTCTCTCCGAGAAGAAGAAGTCGTCCCCCTAAACAGTCTCTCCGAGAAGA 1201 AGAAGTCGTCCCCCTAGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTG 1261 GACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTA 1321 TTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTC 1381 ATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCT 1441 ACAAATGTGGTAAAATCGATAAGATCTTGATCCGGGCTGGCGTAATAGCGAAGAGGCCCG 1501 CACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGC 1561 GGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGC 1621 GCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTT 1681 CCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCAC 1741 CTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCG SEQIDNO:140(DYS-3/4PCRTemplateformRNA) 1 CACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTC 61 ACTATAGGCTAGCCTCGAGCCGCCACCATGGCACCGAAGAAGAAGCGCAAGGTGCATATG 121 AATACAAAATATAATAAAGAGTTCTTACTCTACTTAGCAGGGTTTGTAGACGGTGACGGT 181 TCCATCTTTGCCTCTATCCGGCCTCGGCAAACGAGTAAGTTCAAGCACGCGCTGGCTCTC 241 TTTTTCGTGGTCGGGCAGAAGACACAGCGCCGTTGGTTCCTCGACAAGCTGGTGGACGAG 301 ATCGGTGTGGGTTACGTGTATGACCGTGGCAGCGTCTCCGTGTACCAGCTGTCCCAGATC 361 AAGCCTTTGCATAATTTTTTAACACAACTACAACCTTTTCTAAAACTAAAACAAAAACAA 421 GCAAATTTAGTTTTAAAAATTATTGAACAACTTCCGTCAGCAAAAGAATCCCCGGACAAA 481 TTCTTAGAAGTTTGTACATGGGTGGATCAAATTGCAGCTCTGAATGATTCGAAGACGCGT 541 AAAACAACTTCTGAAACCGTTCGTGCTGTGCTAGACAGTTTACCAGGATCCGTGGGAGGT 601 CTATCGCCATCTCAGGCATCCAGCGCCGCATCCTCGGCTTCCTCAAGCCCGGGTTCAGGG 661 ATCTCCGAAGCACTCAGAGCTGGAGCAGGTTCCGGCACTGGATACAACAAGGAATTCCTG 721 CTCTACCTGGCGGGCTTCGTCGACGGGGACGGCTCCATCATTGCCTGTATCCGGCCTCAT 781 CAAGCTTATAAGTTCAAGCACCAGCTGTGTCTCTCTTTCTGTGTCTATCAGAAGACACAG 841 CGCCGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGTGTGGGTTACGTGACGGACGCT 901 GGCAGCGTCTCCTCTTACCGGCTGTCCGAGATCAAGCCTCTGCACAACTTCCTGACCCAG 961 CTCCAGCCCTTCCTGAAGCTCAAGCAGAAGCAGGCCAACCTCGTGCTGAAGATCATCGAG 1021 CAGCTGCCCTCCGCCAAGGAATCCCCGGACAAGTTCCTGGAGGTGTGCACCTGGGTGGAC 1081 CAGATCGCCGCTCTGAACGACTCCAAGACCCGCAAGACCACTTCCGAAACCGTCCGCGCC 1141 GTTCTAGACAGTCTCTCCGAGAAGAAGAAGTCGTCCCCCTAAACAGTCTCTCCGAGAAGA 1201 AGAAGTCGTCCCCCTAGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTG 1261 GACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTA 1321 TTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTC 1381 ATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCT 1441 ACAAATGTGGTAAAATCGATAAGATCTTGATCCGGGCTGGCGTAATAGCGAAGAGGCCCG 1501 CACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGC 1561 GGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGC 1621 GCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTT 1681 CCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCAC 1741 CTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCG SEQIDNO:141(Exon44ForwardPCRprimer) 1 GAAAGAAAATGCCAATAGTCCAAAATAGTTG SEQIDNO:142(Exon44ReversePCRprimer) 1 CATATTCAAAGGACACCACAAGTTG SEQIDNO:143(DYS-5/6PCRTemplateformRNA) 1 CACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTC 61 ACTATAGGCTAGCCTCGAGCCGCCACCATGGCACCGAAGAAGAAGCGCAAGGTGCATATG 121 AATACAAAATATAATAAAGAGTTCTTACTCTACTTAGCAGGGTTTGTAGACGGTGACGGT 181 TCCATCTTTGCCTGTATCCAGCCTGATCAAAGGGCGAAGTTCAAGCACACGCTGCGGCTC 241 TCTTTCGAGGTCGGGCAGAAGACACAGCGCCGTTGGTTCCTCGACAAGCTGGTGGACGAG 301 ATCGGTGTGGGTTACGTGAATGACTCTGGCAGCGTCTCCAAGTACAGGCTGTCCCAGATC 361 AAGCCTTTGCATAATTTTTTAACACAACTACAACCTTTTCTAAAACTAAAACAAAAACAA 421 GCAAATTTAGTTTTAAAAATTATTGAACAACTTCCGTCAGCAAAAGAATCCCCGGACAAA 481 TTCTTAGAAGTTTGTACATGGGTGGATCAAATTGCAGCTCTGAATGATTCGAAGACGCGT 541 AAAACAACTTCTGAAACCGTTCGTGCTGTGCTAGACAGTTTACCAGGATCCGTGGGAGGT 601 CTATCGCCATCTCAGGCATCCAGCGCCGCATCCTCGGCTTCCTCAAGCCCGGGTTCAGGG 661 ATCTCCGAAGCACTCAGAGCTGGAGCAGGTTCCGGCACTGGATACAACAAGGAATTCCTG 721 CTCTACCTGGCGGGCTTCGTCGACGGGGACGGCTCCATCTATGCCACTATCCAGCCTACT 781 CAATGTGCGAAGTTCAAGCACCAGCTGACTCTCCGTTTCTCGGTCTCTCAGAAGACACAG 841 CGCCGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGTGTGGGTTACGTGTGTGACAAG 901 GGCAGCGTCTCCGAGTACATGCTGTCCGAGATCAAGCCTCTGCACAACTTCCTGACCCAG 961 CTCCAGCCCTTCCTGAAGCTCAAGCAGAAGCAGGCCAACCTCGTGCTGAAGATCATCGAG 1021 CAGCTGCCCTCCGCCAAGGAATCCCCGGACAAGTTCCTGGAGGTGTGCACCTGGGTGGAC 1081 CAGATCGCCGCTCTGAACGACTCCAAGACCCGCAAGACCACTTCCGAAACCGTCCGCGCC 1141 GTTCTAGACAGTCTCTCCGAGAAGAAGAAGTCGTCCCCCTAAACAGTCTCTCCGAGAAGA 1201 AGAAGTCGTCCCCCTAGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTG 1261 GACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTA 1321 TTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTC 1381 ATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCT 1441 ACAAATGTGGTAAAATCGATAAGATCTTGATCCGGGCTGGCGTAATAGCGAAGAGGCCCG 1501 CACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGC 1561 GGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGC 1621 GCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTT 1681 CCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCAC 1741 CTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCG SEQIDNO:144(DYS-7/8PCRTemplateformRNA) 1 CACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTC 61 ACTATAGGCTAGCCTCGAGCCGCCACCATGGCACCGAAGAAGAAGCGCAAGGTGCATATG 121 AATACAAAATATAATAAAGAGTTCTTACTCTACTTAGCAGGGTTTGTAGACGGTGACGGT 181 TCCATCTATGCCTGTATCTTGCCGGTGCAGCGTTGTAAGTTCAAGCACGGGCTGTCTCTC 241 CGATTCATGGTCAGTCAGAAGACACAGCGCCGTTGGTTCCTCGACAAGCTGGTGGACGAG 301 ATCGGTGTGGGTTACGTGTATGACTGTGGCAGCGTCTCCGAGTACAGGCTGTCCGAGATC 361 AAGCCTTTGCATAATTTTTTAACACAACTACAACCTTTTCTAAAACTAAAACAAAAACAA 421 GCAAATTTAGTTTTAAAAATTATTGAACAACTTCCGTCAGCAAAAGAATCCCCGGACAAA 481 TTCTTAGAAGTTTGTACATGGGTGGATCAAATTGCAGCTCTGAATGATTCGAAGACGCGT 541 AAAACAACTTCTGAAACCGTTCGTGCTGTGCTAGACAGTTTACCAGGATCCGTGGGAGGT 601 CTATCGCCATCTCAGGCATCCAGCGCCGCATCCTCGGCTTCCTCAAGCCCGGGTTCAGGG 661 ATCTCCGAAGCACTCAGAGCTGGAGCAGGTTCCGGCACTGGATACAACAAGGAATTCCTG 721 CTCTACCTGGCGGGCTTCGTCGACGGGGACGGCTCCATCTTTGCCTCTATCGTGCCGGAT 781 CAGCGTAGTAAGTTCAAGCACGGTCTGGCTCTCAGGTTCAATGTCGTTCAGAAGACACAG 841 CGCCGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGTGTGGGTTACGTGTATGACCAG 901 GGCAGCGTCTCCGAGTACAGGCTGTCCGAGATCAAGCCTCTGCACAACTTCCTGACCCAG 961 CTCCAGCCCTTCCTGAAGCTCAAGCAGAAGCAGGCCAACCTCGTGCTGAAGATCATCGAG 1021 CAGCTGCCCTCCGCCAAGGAATCCCCGGACAAGTTCCTGGAGGTGTGCACCTGGGTGGAC 1081 CAGATCGCCGCTCTGAACGACTCCAAGACCCGCAAGACCACTTCCGAAACCGTCCGCGCC 1141 GTTCTAGACAGTCTCTCCGAGAAGAAGAAGTCGTCCCCCTAAACAGTCTCTCCGAGAAGA 1201 AGAAGTCGTCCCCCTAGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTG 1261 GACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTA 1321 TTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTC 1381 ATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCT 1441 ACAAATGTGGTAAAATCGATAAGATCTTGATCCGGGCTGGCGTAATAGCGAAGAGGCCCG 1501 CACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGC 1561 GGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGC 1621 GCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTT 1681 CCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCAC 1741 CTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCG SEQIDNO:145(Exon45ForwardPCRprimer) 1 CTAACCGAGAGGGTGCTTTTTTC SEQIDNO:146(Exon45ReversePCRprimer) 1 GTGTTTAGGTCAACTAATGTGTTTATTTTG SEQIDNO:147(MDX-1/2Meganuclease) 1 MAPKKKRKVHMNTKYNKEFLLYLAGFVDGDGSIFACIHPSQAYKFKHRLTLHFTVTQKTQ 61 RRWFLDKLVDEIGVGYVQDVGSVSQYRLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIE 121 QLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSA 181 ASSASSSPGSGISEALRAGAGSGTGYNKEFLLYLAGFVDGDGSISATIAPAQYGKFKHYL 241 GLRFYVSQKTQRRWFLDKLVDEIGVGYVSDQGSVSRYCLSQIKPLHNFLTQLQPFLKLKQ 301 KQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKK 361 KSSP SEQIDNO:148(MDX-13/14Meganuclease) 1 MAPKKKRKVHMNTKYNKEFLLYLAGFVDGDGSIYACIRPTQSVKFKHDLLLCFDVSQKTQ 61 RRWFLDKLVDEIGVGYVYDRGSVSSYRLSEIKPLHNFLTQLQPFLKLKQKQANLVLKIIE 121 QLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSA 181 ASSASSSPGSGISEALRAGAGSGTGYNKEFLLYLAGFVDGDGSIWASIEPRQQSKFKHQL 241 RLGFSVYQKTQRRWFLDKLVDEIGVGYVRDTGSVSCYCLSQIKPLHNFLTQLQPFLKLKQ 301 KQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKK 361 KSSP SEQIDNO:149(MDX-1/2RecognitionSequence) 1 TTCTGTGATGTGAGGACATATA SEQIDNO:150(MDX-13/14RecognitionSequence) 1 ACTAATGAAACACCACTCCACA SEQIDNO:151(MouseDMDIntron22ForwardPrimer) 1 GTCTTATCAGTCAAGAGATCATATTG SEQIDNO:152(MouseDMDIntron23ReversePrimer) 1 GTGTCAGTAATCTCTATCCCTTTCATG SEQIDNO:153(MutantSequencefromMouseDMDGene) 1 AGAATTTAAATATTAACAAACTATAACACTATGATTAAATGCTTGATATTGAGTAGTTAT 61 TTTAATAGCCTAAGTCTGGAAATTAAATACTAGTAAGAGAAACTTCTAGAATTTAAATAT 121 TAACAAACTATAACACTATGATTAAATGCTTGATATTGAGTAGTTATTTTAATAGCCTAA 181 GTCTGGAAATTAAATACTAGTAAGAGAAACTTCT SEQIDNO:154(MutantSequencefromMouseDMDGene) 1 TTTAATAGCCTAAGTCTGGAAATACTCCACAGGTGATTTCAGCCACTTTATGAACTGCTG 61 GAAGCAAAAATGAGATCTTT SEQIDNO:155(MutantSequencefromMouseDMDGene) 1 TTAGTTAGAATTTAAATATTAACAAACTATAACACTATGATTAAATGCTTGATATTGAGT 61 AGTTATTTTAATAGCCTAAGTCTGGAAATTAAATACTAGTAAGAGAAACTTCTGTGATGT 121 GACCACTCCACAGGTGATTTCAGCCACTTTATGAACTGCTGGAAGCAAAAATGAGATCTT 181 T SEQIDNO:156(MutantSequencefromMouseDMDGene) 1 TATAACACTATGATTAAATGCTTGATATTGAGTAGTTATTTTATGTGTCATACCTTCTTG 61 GATTGTCTGTATAAATGAATTGATTTTTTTTCACCAACTCCAAGTATACTTAACATTTTA 121 ACATAATAATTTAAAATATCCTTATTCCATTATGTTCATTTTTTAAGTTGTAGATATGAT 181 TTAGCTCACAGCATACATATATACACATGTATTACATATGCATATATTATATATATGGCA 241 GACATATGTTTTCACTACCATATTTCACTTTTGAATTATGAATATATGTTTAATTTCTGC 301 CATATTTCCTTCCCTACATTGACTTCTATTAATTTAGTATTTCAGTAGTTCTAACACATT 361 AATAATAACCTAGACTCAATACAGTAATCTAACAATTATATTTGTGCCTGTAATTCTAAG 421 TTAGTTAAATTCATAGGTTGTGTTTCTCATAGTTGGCCATTTGTGAAATATAATAATATC 481 CGAAAAGAAAGTTCAAAAATGTCATGACTTCATATAGAGTTATTGAAACAGTGCCCTTAC 541 TTTCATTCTGGCCATGCTAGTGACTTGATCATTCTTGTATTTTACAGCTAAAACACTACC 601 AAAAGTGTCAAATCCATGATCTACATGTTTGACCACTCCACAGGTGATTTCAGCCACTTT 661 ATGAACTGCTGGAAGCAAAAATGAGATCTTT SEQIDNO:157(MutantSequencefromMouseDMDGene) 1 TTGAGTAGTTATTTTAATAGCCTAAGTCTGGAAATTAAATACTAGTAAGAGAAACTTCTG 61 TGATGTGCACAGGTGATTTCAGCCACTTTATGAACTGCTGGAAGCAAAAATG SEQIDNO:158(MutantSequencefromMouseDMDGene) 1 GATATTGAGTAGTTATTTTAATAGCCTAAGTCTGGAAATTAAATACTAGTAGATTTCAGC 61 CACTTTATGAACTGCTGGAAGCAAAAATGA SEQIDNO:159(MutantSequencefromMouseDMDGene) 1 AATACTAGTAAGAGAAACTTCTGTGATGTGAGGACTCCACAGGTGATTTCAGCCACTTTA 61 TGAACTGCTGGAAGCAAAAATGAGATCTTTGCAACATGAAGCAGTTGCTCAGTTCATTAA 121 ACTGTGTTCAATATTTCAGCCATAACATACATTAGAGAATGATTTATATTGTTCAAACAT 181 TT SEQIDNO:160(MutantSequencefromMouseDMDGene) 1 AATACTAGTAAGAGAAACTTCTGTGATGTGAGGACATTTCAGCCACTTTATGAACTGCTG 61 GAAGCAAAAATGAGATCTTTGCAACATGAAGCAGTTGCTCAGTTCATTAAACTGTGTTCA 121 ATATTTCAGCCATAACATACATTAGAGAATGATTTATATTGTTCAAACATTT SEQIDNO:161(MutantSequencefromMouseDMDGene) 1 AATACTAGTAAGAGAAGATTTCAGCCACTTTATGAACTGCTGGAAGCAAAAATGAGATCT 61 TTGCAACATGAAGCAGTTGCTCAGTTCATTAAACTGTGTTCAATATTTCAGCCATAACAT 121 ACATTAGAGAATGATTTATATTGTTCAAACATTT SEQIDNO:162(MutantSequencefromMouseDMDGene) 1 TTTAATAGCCTAAGTCTGGAAATTAAATACTAGTAAGAGAGTGATTTCAGCCACTTTATG 61 AACTGCTGGAAGCAAAAATGA SEQIDNO:163(MutantSequencefromMouseDMDGene) 1 TTAGTTAGAATTTAAATATTAACAAACTATAACACTATGATTAAATGCTTGATATTGAGT 61 AGTTATTTTAATAGCCTAAGTCTGGAAATTAAATACTAGTTCAGCCACTTTATGAACTGC 121 TGGAAGCAAAAATGAGATCTCATTAAACTGTGTTCAATATTTCAGCCATAACATACATTA 181 GAGAATGATTTATATTGTTCAAACATTTGGTGCTCTATTTTTGCATGACGTGGGA SEQIDNO:164(MutantSequencefromMouseDMDGene) 1 TTAGTTAGAATTTAAATATTAACAAACTATAACACTATGATTAAATGCTTGATATTGAGT 61 AGTTATTTTAATAGCCTAAGTCTGGAAATTAAATACTAGTAAGAGAAACTTCTGTGATGT 121 GAGGACATATAAAGACTAATTTTTTTGTTGATTCTAAAAATCCACAGGTGATTTCAGCCA 181 CTTTATGAACTGCTGGAAGCAAAAATGAGATCTTTGCAACATGAAGCAGTTGCTCAGTTC 241 ATTAAACTGTGTTCAATATTTCAGCCATAACATACATTAGAGAATGATTTATATTGTTCA 301 AACATTTGGTGCTCTATTTTTGCATGACGTGGGA SEQIDNO:165(MutantSequencefromMouseDMDGene) 1 TTAGTTAGAATTTAAATATTAACAAACTATAACACTATGATTAAATGCTTGATATTGAGT 61 AGTTATTTTAATAGCCTAAGTCTGGAAATTAAATACTAGTAAGAGAAACTTCTGTGATGT 121 GAGGACATATAAAGACTAATTTTTTCACTCCACAGGTGATTTCAGCCACTTTATGAACTG 181 CTGGAAGCAAAAATGAGATCTTT SEQIDNO:166(MutantSequencefromMouseDMDGene) 1 TTATTTTAATAGCCTAAGTCTGGAAATTAAATACTAGTAAGAGAAACTTCTGTGATGTGA 61 GGACATATAAAGACTAATTTTTTTGTTGATTCTAAAAATCCCATGTTGTATACTTATTCT 121 TTTTAAATCTGAAAATATATTAATCATATATTGCCTAAATGTCTTAATAATGTTTCACTG 181 TAGGTAAGTTAAAATGTATCACATATATAATAAACATAGTTATTAATGCATAGATATTCA 241 GTAAAATTATGACTTCTAAATTTCTGTCTAAATATAATATGCCCTGTAATATAATAGAAA 301 TTATTCATAAGAATACATATATATTGCTTTATCAGATATTCTACTTTGTTTAGATCTCTA 361 AATTACATAAACTTTTATTTACCTTCTTCTTGATATGAATGAAACTCATCAAATATGCGT 421 GTTAGTGTAAATGAACTTCTATTTAAACTCCACAGGTGATTTCAGCCACTTTATGAAC SEQIDNO:167(MutantSequencefromMouseDMDGene) 1 TTAGTTAGAATTTAAATATTAACAAACTATAACACTATGATTAAATGCTTGATATTGAGT 61 AGTTATTTTAATAGCCTAAGTCTGGAAATTAAATACTAGTAAGAGAAACTTCTGTGATGT 121 GAGGACATATAAAGACTAATTTTTTTGTTGATTCTAAAAATCCCATGTTGTATACTTATT 181 CTTTTTAAATCTGAAAATATATTAATCATATATTGCCTAAATGTCTTAATAATGTTTCAC 241 TGTAGGTAAGTTAAAATGTATCACATATATAATAAACATAGTTATTAATGCATAGATATT 301 CAGTAAAATTATGACTTCTAAATTTCTGTCTAAATATAATATGCCCTGTAATATAATAGA 361 AATTATTCATAAGAATACATATATATTGCTTTATCAGATATTCTACTTTGTTTAGATCTC 421 TAAATTACATAAACTTTTATTTACCTTCTTCTTGATATGAATGAAACTCATCAAATATGC 481 GTGTTAGTGTAAATGAACTTCTATTTAATTTTGAGGCTCTGCAAAGTTCTCCACAGGTGA 541 TTTCAGCCACTTTATGAACTGCTGGAAGCAAAAATGAGATCTTTGCAACATGAAGCAGTT 601 GCTCAGTTCATTAAACTGTGTTCAATATTTCAGCCATAACATACATTAGAGAATGATTTA 661 TATTGTTCAAACATTTGGTGCTCTATTTTTGCATGACGTGGGA SEQIDNO:168(MutantSequencefromMouseDMDGene) 1 AATACTAGTAAGAGAAACTTCTGTGATGTGAGGACATATAAAGACTAATTTTTTTGTTGA 61 TTCTAAAAATCCCATGTTGTATACTTATTCTTTTTAAATCTGAAAATATATTAATCATAT 121 ATTGCCTAAATGTCTTAATAATGTTTCACTGTAGGTAAGTTAAAATGTATCACATATATA 181 ATAAACATAGTTATTAATGCATAGATATTCAGTAAAATTATGACTTCTAAATTTCTGTCT 241 AAATATAATATGCCCTGTAATATAATAGAAATTATTCATAAGAATACATATATATTGCTT 301 TATCAGATATTCTACTTTGTTTAGATCTCTAAATTACATAAACTTTTATTTACCTTCTTC 361 TTGATATGAATGAAACTCATCAAATATGCGTGTTAGTGTAAATGAACTTCTATTTAATTT 421 TGAGGCTCTGCAAAGTTCTTTGAAAGAGCAACAAAATGGCTTCACCACTCCACAGGTGAT 481 TTCAGCCACTTTATGAACTGCTGGAAGCAAAAATGAGATCTTTGCAACATGAAGCAGTTG 541 CTCAGTTCATTAAACTGTGTTCAATATTTCAGCCATAACATACATTAGAGAATGATTTAT 601 ATTGTTCAAACATTT SEQIDNO:169(MutantSequencefromMouseDMDGene) 1 TTAGTTAGAATTTAAATATTAACAAACTATAACACTATGATTAAATGCTTGATATTGAGT 61 AGTTATTTTAATAGCCTAAGTCTGGAAATTAAATACTAGTAAGAGAAACTTCTGTGATGT 121 GAGGACATATAAAGACTAATTTTTTTGTTGATTCTAAAAATCCCATGTTGTATACTTATT 181 CTTTTTAAATCTGAAAATATATTAATCATATATTGCCTAAATGTCTTAATAATGTTTCAC 241 TGTAGGTAAGTTAAAATGTATCACATATATAATAAACATAGTTATTAATGCATAGATATT 301 CAGTAAAATTATGACTTCTAAATTTCTGTCTAAATATAATATGCCCTGTAATATAATAGA 361 AATTATTCATAAGAATACATATATATTGCTTTATCAGATATTCTACTTTGTTTAGATCTC 421 TAAATTACATAAACTTTTATTTACCTTCTTCTTGATATGAATGAAACTCATCAAATATGC 481 GTGTTAGTGTAAATGAACTTCTATTTAATTTTGAGGCTCTGCAAAGTTCTTTGAAAGAGC 541 AACAAAATGGCTTCAACTATCTGAGTGACACTGTGAAGGAGATGGCCAAGAAAGCACCTT 601 CAGAAATATGCCATTTCAGCCACTTTATGAACTGCTGGAAGCAAAAATGAGATCTTTGCA 661 ACATGAAGCAGTTGCTCAGTTCATTAAACTGTGTTCAATATTTCAGCCATAACATACATT 721 AGAGAATGATTTATATTGTTCAAACATTTGGTGCTCTATTTTTGCATGACGTGGGA SEQIDNO:170(MutantSequencefromMouseDMDGene) 1 GTCTGGAAATTAAATACTAGTAAGAGAAACTTCTGTGATGTGAGGACATATAAAGACTAA 61 TTTTTTTGTTGATTCTAAAAATCCCATGTTGTATACTTATTCTTTTTAAATCTGAAAATA 121 TATTAATCATATATTGCCTAAATGTCTTAATAATGTTTCACTGTAGGTAAGTTAAAATGT 181 ATCACATATATAATAAACATAGTTATTAATGCATAGATATTCAGTAAAATTATGACTTCT 241 AAATTTCTGTCTAAATATAATATGCCCTGTAATATAATAGAAATTATTCATAAGAATACA 301 TATATATTGCTTTATCAGATATTCTACTTTGTTTAGATCTCTAAATTACATAAACTTTTA 361 TTTACCTTCTTCTTGATATGAATGAAACTCATCAAATATGCGTGTTAGTGTAAATGAACT 421 TCTATTTAATTTTGAGGCTCTGCAAAGTTCTTTGAAAGAGCAACAAAATGGCTTCAACTA 481 TCTGAGTGACACTGTGAAGGAGATGGCCAAGAAAGCACCTTCAGAAATATGCCAGAAATA 541 TCTGTCAGAATTTGAAGAGATTGAGGGGCACTGGAAGAAACTTTCCTCCCAGTTGGTGGA 601 AAACACCACTCCACAGGTGATTTCAGCCACTTTAT SEQIDNO:171(MutantSequencefromMouseDMDGene) 1 TGGAAATTAAATACTAGTAAGAGAAACTTCTGTGATGTGAGGACATATAAAGACTAATTT 61 TTTTGTTGATTCTAAAAATCCCATGTTGTATACTTATTCTTTTTAAATCTGAAAATATAT 121 TAATCATATATTGCCTAAATGTCTTAATAATGTTTCACTGTAGGTAAGTTAAAATGTATC 181 ACATATATAATAAACATAGTTATTAATGCATAGATATTCAGTAAAATTATGACTTCTAAA 241 TTTCTGTCTAAATATAATATGCCCTGTAATATAATAGAAATTATTCATAAGAATACATAT 301 ATATTGCTTTATCAGATATTCTACTTTGTTTAGATCTCTAAATTACATAAACTTTTATTT 361 ACCTTCTTCTTGATATGAATGAAACTCATCAAATATGCGTGTTAGTGTAAATGAACTTCT 421 ATTTAATTTTGAGGCTCTGCAAAGTTCTTTGAAAGAGCAACAAAATGGCTTCAACTATCT 481 GAGTGACACTGTGAAGGAGATGGCCAAGAAAGCACCTTCAGAAATATGCCAGAAATATCT 541 GTCAGAATTTGAAGAGATTGAGGGGCACTGGAAGAAACTTTCCTCCCAGTTGGTGGAAAG 601 CTGCCAAAAGCTAGAAGAACATATGAATAAACTTCGAAAATTTCAGGTAAGCCGAGGTTT 661 GGCCTTTAAACTATATTTTTCCACTCCACAGGTGATTTCAGCCACTTTATGAAC SEQIDNO:172(MutantSequencefromMouseDMDGene) 1 CCTAAGTCTGGAAATTAAATACTAGTAAGAGAAACTTCTGTGATGTGAGGACATATAAAG 61 ACTAATTTTTTTGTTGATTCTAAAAATCCCATGTTGTATACTTATTCTTTTTAAATCTGA 121 AAATATATTAATCATATATTGCCTAAATGTCTTAATAATGTTTCACTGTAGGTAAGTTAA 181 AATGTATCACATATATAATAAACATAGTTATTAATGCATAGATATTCAGTAAAATTATGA 241 CTTCTAAATTTCTGTCTAAATATAATATGCCCTGTAATATAATAGAAATTATTCATAAGA 301 ATACATATATATTGCTTTATCAGATATTCTACTTTGTTTAGATCTCTAAATTACATAAAC 361 TTTTATTTACCTTCTTCTTGATATGAATGAAACTCATCAAATATGCGTGTTAGTGTAAAT 421 GAACTTCTATTTAATTTTGAGGCTCTGCAAAGTTCTTTGAAAGAGCAACAAAATGGCTTC 481 AACTATCTGAGTGACACTGTGAAGGAGATGGCCAAGAAAGCACCTTCAGAAATATGCCAG 541 AAATATCTGTCAGAATTTGAAGAGATTGAGGGGCACTGGAAGAAACTTTCCTCCCAGTTG 601 GTGGAAAGCTGCCAAAAGCTAGAAGAACATATGAATAAACTTCGAAAATTTCAGGTAAGC 661 CGAGGTTTGGCCTTTAAACTATATTTTTTCACATAGCAATTAATTGGAAAATGTGATGGG 721 AAACAGATATTTTACCCAGAGTCCTTCAAAGATATTGATGATATCAAAAGCCAAATCTAT 781 TTCAAAGGATTGCAACTTGCCTATTTTTCCTATGAAAACAGTAATGTGTCATACCTTCTT 841 GGATTGTCTGTATAAATGAATTGATTTTTTTTCACCAACTCCAAGTATACTTAACATTTT 901 AACATAATAATTTAAAATATCCTTATTCCATTATGTTCATTTTTTAAGTTGTAGATATGA 961 TTTAGCTCACAGCATACATATATACACATGTATTACATATGCATATATTATATATATGGC 1021 AGACATATGTTTTCACTACCATATTTCACTTTTGAATTATGAATATATGTTTAATTTCTG 1081 CCATATTTCCTTCCCTACATTGACTTCTATTAATTTAGTATTTCAGTAGTTCTAACACAT 1141 TAATAATAACCTAGACTCAATACAGTAATCTAACAATTATATTTGTGCCTGTAATTCTAA 1201 GTTAGTTAAATTCATAGGTTGTGTTTCTCATAGTTGGCCATTTGTGAAATATAATAATAT 1261 CCGAAAAGAAAGTTCAAAAATGTCATGACTTCATATAGACAGGTGATTTCAGCCACTTTA 1321 TG SEQIDNO:173(pAAV-MDXPlasmid) 1 GGGGGGGGGGGGGGGGGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCG 61 GGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAG 121 CGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCTTCAATATTGGGT 181 ATTAGTCATCGCTATTACCATGATGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATA 241 GCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTT 301 TTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCA 361 AATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCG 421 TCAGATCACTAGAAGCTTTATTGCGGTAGTTTATCACAGTTAAATTGCTAGCGCAGTCAG 481 TGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGCACTGGGCA 541 GGTAAGTATCAAGGTTACAAGACAGGTTTAAGGACACCAATAGAAACTGGGCTTGTCGAG 601 ACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGC 661 CTTTCTCTCCACAGGTAATTGTGAGCGGATAACAATTGATGTCGCACAGGCCACGGATTA 721 GGCACCCCAGGCTTGACACTTTATGCTTCCGGCTCGTATATTGTGTGGAATTGTGAGCGG 781 ATAACAATTTCACACAGGAGATATATATATGGGCTAGGCCACCATGGCACCGAAGAAGAA 841 GCGCAAGGTGCATATGAATACAAAATATAATAAAGAGTTCTTACTCTACTTAGCAGGGTT 901 TGTAGACGGTGACGGTTCCATCTTTGCCTGTATCCATCCTAGTCAAGCGTATAAGTTCAA 961 GCACCGGCTGACTCTCCATTTCACGGTCACTCAGAAGACACAGCGCCGTTGGTTCCTCGA 1021 CAAGCTGGTGGACGAGATCGGTGTGGGTTACGTGCAGGACGTGGGCAGCGTCTCCCAGTA 1081 CCGGCTGTCCCAGATCAAGCCTTTGCATAATTTTTTAACACAACTACAACCTTTTCTAAA 1141 ACTAAAACAAAAACAAGCAAATTTAGTTTTAAAAATTATTGAACAACTTCCGTCAGCAAA 1201 AGAATCCCCGGACAAATTCTTAGAAGTTTGTACATGGGTGGATCAAATTGCAGCTCTGAA 1261 TGATTCGAAGACGCGTAAAACAACTTCTGAAACCGTTCGTGCTGTGCTAGACAGTTTACC 1321 AGGATCCGTGGGAGGTCTATCGCCATCTCAGGCATCCAGCGCCGCATCCTCGGCTTCCTC 1381 AAGCCCGGGTTCAGGGATCTCCGAAGCACTCAGAGCTGGAGCAGGTTCCGGCACTGGATA 1441 CAACAAGGAATTCCTGCTCTACCTGGCGGGCTTCGTCGACGGGGACGGCTCCATCTCTGC 1501 CACTATCGCTCCGGCTCAGTATGGTAAGTTCAAGCACTATCTGGGGCTCCGGTTCTATGT 1561 CAGTCAGAAGACACAGCGCCGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGTGTGGG 1621 TTACGTGAGTGACCAGGGCAGCGTCTCCAGGTACTGTCTGTCCCAGATCAAGCCTCTGCA 1681 CAACTTCCTGACCCAGCTCCAGCCCTTCCTGAAGCTCAAGCAGAAGCAGGCCAACCTCGT 1741 GCTGAAGATCATCGAGCAGCTGCCCTCCGCCAAGGAATCCCCGGACAAGTTCCTGGAGGT 1801 GTGCACCTGGGTGGACCAGATCGCCGCTCTGAACGACTCCAAGACCCGCAAGACCACTTC 1861 CGAAACCGTCCGCGCCGTTCTAGACAGTCTCTCCGAGAAGAAGAAGTCGTCCCCCTAAGG 1921 TACCAGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACA 1981 ACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTT 2041 GTAACCATTATAAGCTGCAATAAACAAGTTGTATTAGTCATCGCTATTACCATGATGATG 2101 CGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGT 2161 CTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCA 2221 AAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAG 2281 GTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTGCGGTA 2341 GTTTATCACAGTTAAATTGCTAGCGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTA 2401 AGCTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTT 2461 TAAGGACACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAG 2521 GCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTAATTGTGAGCGG 2581 ATAACAATTGATGTCGCACAGGCCACGGATTAGGCACCCCAGGCTTGACACTTTATGCTT 2641 CCGGCTCGTATATTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAGATATATAT 2701 ATGGGCTAGGCCACCATGGCACCGAAGAAGAAGCGCAAGGTGCATATGAATACAAAATAT 2761 AATAAAGAGTTCTTACTCTACTTAGCAGGGTTTGTAGACGGTGACGGTTCCATCTATGCC 2821 TGTATCAGGCCGACGCAGAGTGTGAAGTTCAAGCACGATCTGCTGCTCTGTTTCGATGTC 2881 TCTCAGAAGACACAGCGCCGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGTGTGGGT 2941 TACGTGTATGACCGTGGCAGCGTCTCCTCGTACAGGCTGTCCGAGATCAAGCCTTTGCAT 3001 AATTTTTTAACACAACTACAACCTTTTCTAAAACTAAAACAAAAACAAGCAAATTTAGTT 3061 TTAAAAATTATTGAACAACTTCCGTCAGCAAAAGAATCCCCGGACAAATTCTTAGAAGTT 3121 TGTACATGGGTGGATCAAATTGCAGCTCTGAATGATTCGAAGACGCGTAAAACAACTTCT 3181 GAAACCGTTCGTGCTGTGCTAGACAGTTTACCAGGATCCGTGGGAGGTCTATCGCCATCT 3241 CAGGCATCCAGCGCCGCATCCTCGGCTTCCTCAAGCCCGGGTTCAGGGATCTCCGAAGCA 3301 CTCAGAGCTGGAGCAGGTTCCGGCACTGGATACAACAAGGAATTCCTGCTCTACCTGGCG 3361 GGCTTCGTCGACGGGGACGGCTCCATCTGGGCCTCGATCGAGCCTAGGCAACAGTCTAAG 3421 TTCAAGCACCAGCTGCGGCTCGGGTTCTCGGTCTATCAGAAGACACAGCGCCGTTGGTTC 3481 CTCGACAAGCTGGTGGACGAGATCGGTGTGGGTTACGTGCGTGACACTGGCAGCGTCTCC 3541 TGTTACTGTCTGTCCCAGATCAAGCCTCTGCACAACTTCCTGACCCAGCTCCAGCCCTTC 3601 CTGAAGCTCAAGCAGAAGCAGGCCAACCTCGTGCTGAAGATCATCGAGCAGCTGCCCTCC 3661 GCCAAGGAATCCCCGGACAAGTTCCTGGAGGTGTGCACCTGGGTGGACCAGATCGCCGCT 3721 CTGAACGACTCCAAGACCCGCAAGACCACTTCCGAAACCGTCCGCGCCGTTCTAGACAGT 3781 CTCTCCGAGAAGAAGAAGTCGTCCCCCTAAGGTACCAGCGGCCGCTTCGAGCAGACATGA 3841 TAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTA 3901 TTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAG 3961 TTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTT 4021 TTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCTAGGAACCCCTA 4081 GTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCA 4141 AAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGA 4201 GAGGGAGTGGCCAACCCCCCCCCCCCCCCCCCTGCAGCCTGGCGTAATAGCGAAGAGGCC 4261 CGCACCGATCGCCCTTCCCAACAGTTGCGTAGCCTGAATGGCGAATGGCGCGACGCGCCC 4321 TGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTT 4381 GCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCC 4441 GGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTA 4501 CGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCC 4561 TGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTG 4621 TTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATT 4681 TTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAAT 4741 TTTAACAAAATATTAACGTTTACAATTTCCTGATGCGCTATTTTCTCCTTACGCATCTGT 4801 GCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGT 4861 TAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCC 4921 CGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTT 4981 CACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGG 5041 TTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGC 5101 GCGGAACCCCTATTTGTTTATTTTTCTAAATACTTTCAAATATGTATCCGCTCATGAGAC 5161 AATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATT 5221 TCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAG 5281 AAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCG 5341 AACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAA 5401 TGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGC 5461 AAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAG 5521 TCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAA 5581 CCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGC 5641 TAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG 5701 AGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAA 5761 CAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAA 5821 TAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTG 5881 GCTGGTTTATTGCGGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAG 5941 CACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGG 6001 CAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATT 6061 GGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTT 6121 AATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAAC 6181 GTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAG 6241 ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGG 6301 TGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCA 6361 GAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA 6421 ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCA 6481 GTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGC 6541 AGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACA 6601 CCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAA 6661 AGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTC 6721 CAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC 6781 GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGG 6841 CCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTAT 6901 CCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATA SEQIDNO:174(MouseDMDIntron22ForwardPrimer) 1 CATTTCATATTTAGTGACATAAGATATGAAGTATG SEQIDNO:175(MouseDMDIntron23ReversePrimer) 1 GTGTCAGTAATCTCTATCCCTTTCATG SEQIDNO:176(MutantSequencefromMouseDMDGene) 1 CATTTCATATTTAGTGACATAAGATATGAAGTATGATTATTCAGCCACTTTATGAACTGC 61 TGGAAGCAAAAATGAGATCTTTGCAACATGAAGCAGTTGCTCAGTTCATTAAACTGTGTT 121 CAATATTTCAGCCATAACATACATTAGAGAATGATTTATATTGTTCAAACATTTGGTGCT 181 CTATTTTTGCATGACGTGGGATTAAACACAGCACCAACAATCAAACAATTGCAAAGATGT 241 ATTACAAGTATTTTTTCTTTTTAAAACAGGAAAGTATACTTATATTTCCATTGTCCAAAC 301 CATCATGAAAGGGATAGAGATTACTGACAC SEQIDNO:177(MutantSequencefromMouseDMDGene) 1 CATTTCATATTTAGTGACATAAGATATGAAGTATGATTATTAAAATTAAATCACATTATT 61 TTATTATAATTACTTTACTCCACAGGTGATTTCAGCCACTTTATGAACTGCTGGAAGCAA 121 AAATGAGATCTTTGCAACATGAAGCAGTTGCTCAGTTCATTAAACTGTGTTCAATATTTC 181 AGCCATAACATACATTAGAGAATGATTTATATTGTTCAAACATTTGGTGCTCTATTTTTG 241 CATGACGTGGGATTAAACACAGCACCAACAATCAAACAATTGCAAAGATGTATTACAAGT 301 ATTTTTTCTTTTTAAAACAGGAAAGTATACTTATATTTCCATTGTCCAAACCATCATGAA 361 AGGGATAGAGATTACTGACAC SEQIDNO:178(MutantSequencefromMouseDMDGene) 1 CATTTCATATTTAGTGACATAAGATATGAAGTATGATTATTAAAATTAAATCACATTATT 61 TTATTATAATTACTTTACACAGGTGATTTCAGCCACTTTATGAACTGCTGGAAGCAAAAA 121 TGAGATCTTTGCAACATGAAGCAGTTGCTCAGTTCATTAAACTGTGTTCAATATTTCAGC 181 CATAACATACATTAGAGAATGATTTATATTGTTCAAACATTTGGTGCTCTATTTTTGCAT 241 GACGTGGGATTAAACACAGCACCAACAATCAAACAATTGCAAAGATGTATTACAAGTATT 301 TTTTCTTTTTAAAACAGGAAAGTATACTTATATTTCCATTGTCCAAACCATCATGAAAGG 361 GATAGAGATTACTGACAC