Dystrophin gene exon deletion using modified nucleases

Abstract

To provide methods for treating patients with Duchenne muscular dystrophy.SOLUTION: Disclosed is a method for treating Duchenne muscular dystrophy in a subject in need thereof, the method comprising contacting the DNA of a muscle cell of the patient to a first nuclease that cleaves a first recognition sequence and a second nuclease that cleaves a second recognition sequence, where the first recognition sequence is upstream of a first exon of the dystrophin gene, and the second recognition sequence is downstream of the first exon of the dystrophin gene, and at least one second exon is removed from the dystrophin gene of the cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] (Related Applications) This application is a continuation of U.S. Provisional Application No. 61 / 951,648, filed March 12, 2014. The benefit of this patent is claimed and is incorporated herein by reference in its entirety.

[0002] The present invention relates to the fields of molecular biology and recombinant nucleic acid technology. In particular, the present invention relates to modified By using a nuclease to remove at least one exon from the dystrophin gene, and (b) administering to a patient a dose of about 100 mg of the compound of formula (I) to the patient. [Background technology]

[0003] Duchenne muscular dystrophy is rare, affecting approximately 3,500 people worldwide. It is an X-linked muscular degenerative disorder that affects approximately 1 in 20 males. It is caused by a mutation in the genetic dystrophin (DMD) gene. The MD spanned 2.2 Mb of the X chromosome and contained a 14-kb gene derived from 79 exons. It primarily encodes a full-length transcript expressed in skeletal, smooth and cardiac muscle cells. The dystrophin protein is 3685 amino acids and has a molecular weight of 427 kD. The severe Duchenne phenotype generally involves the loss of full-length dystrophin from skeletal and cardiac muscles. This is associated with loss of lophin protein, which leads to muscle degeneration and ultimately heart disease. Many different DMD mutations have been described, ranging from severe Duchenne muscular dystrophy to Many of these have been described, resulting in milder forms of Becker muscular dystrophy. The University of Pennsylvania Medical Center maintains a database of mutations in the DMD gene ( tp: / / www.dmd.nl).

[0004] There are several therapeutic strategies that have been pursued for the treatment of Duchenne muscular dystrophy. First, the "gene replacement" strategy is an active area of ​​research (Oshima, et al. (2009) J of the Am.Soc.of Gene Ther.17 :73-80;Liu,et al.(2005)Mol.Ther.11 :245- 256;Lai,et al.(2006)Hum Gene Ther.17:103 6-1042;Odom et al.(2008) Mol.Ther.16:153 This approach uses a viral delivery vector, typically an adeno-associated virus (AAV). This involves using an autosomal recessive encephalopathy (AAV) to deliver a functional copy of the DMD gene to the patient. While the large size of the DMD gene limits its delivery by common viral vectors, This is because many of the repeated central parts of genes are unable to produce minimally functional proteins. It requires the use of a "micro-dystrophin" gene that is removed to leave only the tissue. However, it is unclear whether expression of "microdystrophin" is sufficient for clinical benefit. In addition, this approach involves inserting genes into the patient's genome, which may lead to insertional mutagenesis. Concerns remain over the possibility of random gene integration and potential immune responses to the delivery vector. To be fooled.

[0005] The second approach to Duchenne muscular dystrophy involves synthesizing healthy muscle fibers into the patient's muscle tissue. including transplantation of progenitor cells (Peault et al. (2007) Mol. Ther. 15:867-877;Skuk,et al.(2007)Neuromuscul. Disord. 17:38-46). This approach addresses the inefficiency of transplanted myoblasts. It suffers from the potential for migration and immune rejection by the patient.

[0006] A third approach involves suppression of nonsense mutations using PTC124 (Wel ch, et al. (2007) Nature 447:87-91). However, This requires lifelong administration of the drug, and the approach has not demonstrated sufficient clinical benefit. Not yet.

[0007] The fourth and more promising potential treatment for Duchenne muscular dystrophy is This is called "exon skipping" (Williams, et al. (2008) BM C Biotechnol.8:35;Jearawiriyapaisarn et al.(2008)Mol Ther.16:1624-1629;Yokota,et al.(2007)Acta Myol.26:179-184;van Deute kom et al.(2001)Hum.Mol.Gen.10:1547-1554 ;Benedetti et al.(2013)FEBS J.280:4263-8 0;Rodino-Klapac(2013)Curr Neurol Neurosc i Rep.13:332;Verhaart and Aartsma-Rus(20 12) Curr Opin Neurol. 25: 588-96). In general, dystrophic The N- and C-terminal regions of the ribosome-binding protein (RIP) encode a “foot protein” that maintains membrane integrity in muscle fibers. While essential for the mitochondrial function as a field protein, the 24 spectrin-like repeats The central "rod domain" containing the barbs is, at least in part, dispensable. The pathological Duchenne phenotype is due to the introduction of a frameshift and / or a premature stop codon, Dystrophin, which essentially lacks the C-terminal domain, resulting in a truncated form of the protein Typically associated with mutations in the lophin gene. Mutations in the lead domain ensure that the C-terminal domain of the protein is intact. Maintaining the coding frame typically results in a milder Becker phenotype.

[0008] Duchenne muscular dystrophy results in a reading frame shift, 1 or more It is most frequently caused by a deletion of an entire exon or exons in the chromosome 45. is frequently deleted in Duchenne patients. Exon 45 cannot be divided into three Since the exon is 176 bp long, deleting the exon may result in a misleading linkage of exons 46-79. The same is true for exon 44, which is 148 bp long. However, if exons 44 and 45 are deleted, the total size of the deletion is 324 bp, which can be divided into three parts. Thus, the deletion of both exons results in a leading frame. These exons are non-essential exons of the dystrophin protein. Since they encode parts of the rod domain, deleting them from the protein is a mild alternative. Thus, deletion of one or more exons (multiple Patients with Duchenne phenotype due to deletion of a gene that can be used to restore the reading frame This could potentially be treated by deleting one or more adjacent exons to eliminate the The oligonucleotide identified was a splice acceptor in the dystrophin pre-mRNA. This is the principle behind "exon skipping," which is used to block exon sites. As a result, one or more specific exons are absent from the processed transcript. Skipping exon 23 harbors a disease-inducing sense mutation in the mdx mouse lineage It has been used to restore dystrophin gene expression in a model of dystrophinemia (Mann, et al.(2001)Proc.Nat.Acad.Sci.USA 98:42- 47) Oligonucleotide analogs that induce exon 51 skipping also induce early It has shown promise in human clinical trials (Benedetti et al. 13)FEBSJ.280:4263-80). The main limitations with this approach are (1 ) The exon skipping process is unhelpful, resulting in relatively low levels of functional dystrophin and (2) the exon skipping oligonucleotide is relatively short. The half-life of these drugs is short, resulting in transient effects and the need for repeated and lifelong dosing. Thus, the whole exon skipping approach has shown some promise in clinical trials. However, improvements in disease progression have been minimal and variable. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] J.of the Am.Soc.of Gene Ther.17:73-80(2009) [Non-Patent Document 2] Mol. Ther. 11:245-256(2005) [Non-Patent Document 3] Hum Gene Ther.17:1036-1042(2006) [Non-Patent Document 4] Mol. Ther. 16:1539-1545(2008) [Non-Patent Document 5] Mol. Ther. 15:867-877(2007) [Non-Patent Document 6] Neuromuscul.Disord.17:38-46(2007) [Non-Patent Document 7] Nature 447:87-91(2007) [Non-Patent Document 8] BMC Biotechnol.8:35(2008) [Non-Patent Document 9] Mol Ther.16:1624-1629(2008) [Non-Patent Document 10] Acta Myol.26:179-184(2007) [Non-Patent Document 11] Hum.Mol.Gen.10:1547-1554(2001) [Non-Patent Document 12] FEBS J.280:4263-80(2013) [Non-Patent Document 13] Curr Neurol Neurosci Rep.13:332(2013) [Non-Patent Document 14] Curr Opin Neurol.25:588-96(2012) [Non-Patent Document 15] Proc.Nat.Acad.Sci.USA 98:42-47(2001) [Non-Patent Document 16] FEBSJ.280:4263-80(2013) Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention focuses on correcting gene expression at the level of genomic DNA rather than pre-mRNA. This improves on current exon skipping approaches. Deletion of specific exons from the DMD coding sequence using site-specific endonucleases The treatment for Duchenne muscular dystrophy is permanent, involving the removal of exons and adjacent By targeting sites in the endonuclease region with such a pair of endonucleases, This allows for the permanent removal of the intervening exon-containing fragment from the genome. The resulting cells, and their progeny, contain a portion of the non-essential spectrin repeat domain. Mutant dystrophin with essential N- and C-terminal domains intact, but with the deletions will be manifested.

[0011] Methods for producing modified site-specific endonucleases are known in the art. For example, zinc finger nucleases (ZFNs) recognize specific sites in the genome. ZFNs can be engineered to cleave the nuclease domain of the FokI restriction enzyme. It is a chimeric protein that contains a zinc finger DNA that binds to a domain fused to a phosphodiesterase. The zinc finger domain binds to a predetermined DNA sequence of approximately 18 base pairs in length. F can be redesigned through rational or experimental means to produce proteins that bind to the Fusion of the okI nuclease with this engineered protein domain results in specific genomic expression ZFNs can be used to target DNA breaks in a wide range of eukaryotic organisms. , widely used for targeting removal and replacement (S. Durai et al. , Nucleic Acids Res 33, 5978 (2005). TAL-effector nucleases (TALENs) target specific sites in genomic DNA. Like ZFNs, TALENs can be produced to cleave the FokI nuclease. It contains a modified site-specific DNA binding domain fused to the domain (Mak, et al. (2 013) See Curr Opin Struct Biol.23:93-9. However, in this case, the DNA binding domain is a tandem of the TAL-effector domain. The present invention is directed to the practice of the present invention, which comprises a set of nucleic acid sequences, each of which specifically recognizes a single DNA base pair. The limitation of ZFNs and TALENs is that they are heterodimers and therefore cannot be expressed in cells. Production of a single functional nuclease in Escherichia coli requires coexpression of two protein monomers The present invention requires two nucleases, one that is capable of cleaving the associated exon. The goal of this study was to cut at one side of the gene, which is the same as the first ZFN or TALEN gene cut in the same cell. Conventional gene delivery vectors have limited delivery capacity. This poses a significant challenge in gene delivery because the monomers The aim of the present invention is to identify unintended dimeric endonuclease species that can recognize and cleave target sites in the genome. It also introduces the possibility of "mis-dimerization," which occurs when the four monomers form The Folk nuclease domain preferentially dimerizes with its intended partner monomer. This can potentially be minimized by differentially engineered orthogonal obligate heterodimers.

[0012] Compact TALENs are an alternative endonuclease structure that avoids the need for dimerization. (Beurdeley, et al. (2013) Nat Commun. 4:17 62) Compact TALENs are nucleotides derived from the I-TevI ​​homing endonuclease. Engineered site-specific TAL-effector DNA-binding domain fused to a clease domain Unlike FokI, I-TevI ​​dimerizes to generate double-stranded DNA breaks. Compact TALENs are functional as monomers, since they do not require polymerization. Therefore, it is possible to co-express two compact TALENs in the same cell to carry out the present invention. It is possible.

[0013] Engineered endonucleases based on the CRISPR / Cas9 system are known (Ran, et al.(2013)Nat Protoc.8:2281-2308;Mali et al. (2013) Nat Methods.10:957-63). CRISP R endonucleases are two-component (1) caspase effector nucleases, typically (1) a microorganism that targets the nuclease to the relevant location in the genome; and (2) a 2-fold protein that targets the nuclease to the relevant location in the genome. Each contains a short "guide RNA" that contains a 0 nucleotide target sequence. By expressing multiple guide RNAs in the same cell, It is possible to simultaneously target DNA breaks at multiple sites in a CR ISPR / Cas9 nucleases are suitable for the present invention. The main weakness is its reported off-targets, which limit the utility of the system to treat human patients. The high frequency of DNA breaks (Fu, et al. (2013) Nat Biotech hnol.31:822-6). [Means for solving the problem]

[0014] In a preferred embodiment of the present invention, the DNA break inducing agent is a modified homing endonuclease. Homing endonucleases are enzymes that bind to the target protein and are known as "meganucleases." is a 15-40 base pair break commonly found in plant and fungal genomes. They are a group of naturally occurring nucleases that recognize the 5'-terminal end of the 5'-terminal nucleotide. They are group 1 self-splicing They are often associated with parasitic DNA elements such as introns and inteins. Infects the host genome by generating double-strand breaks in chromosomes that recruit cellular DNA repair mechanisms naturally promotes homologous recombination or gene insertion at specific sites in d(2006), Q.Rev.Biophys.38:49-95). Denucleases are generally classified into four families: the LAFLIDADG family, the GIY- Groups include the YIG family, His-Cys box family, and HNH family. These families are divided into structural groups that influence catalytic activity and recognition sequences. For example, members of the LAGLIDADF family are or two conserved LAGLIDADG motifs. (Chevalier et al. (2001), Nucleic Acid s Res.29(18):3757-3774). LAGAIDADG motif LAGLIDADG homing endonucleases with a single copy of whereas members with two copies of the LAGLIDADG motif form monomers. It is found as.

[0015] I-CreI (SEQ ID NO: 1) is a gene encoding 22 chloroplast chromosomes of the alga Chlamydomonas reinhardtii. LAGLIDAG, a homing endonuclease that recognizes and cleaves base-pair recognition sequences. It is a member of the family. The genetic selection technique was used to select wild-type I-CreI cleavage site preferred It is used to modify the lens (Sussman et al. (2004), J. Mol.Biol.342:31-41;Chames et al.(2005), Nucleic Acids Res.33:el78;Seligman et al (2002), Nucleic Acids Res.30:3870-9;Arno uld et al. (2006), J. Mol. Biol. 355:443-58). More recently, a wide range of genomes have been developed, including mammalian, yeast, plant, bacterial and viral genomes. I-CreI and other homing endonucleases targeting branching DNA sites Rational synthesis of mono-LAGLIDADG homing endonucleases that can be comprehensively redesigned A method for redesigning the same has been disclosed (International Publication WO2007 / 047859).

[0016] As first described in International Publication WO 2009 / 059195, I-Cr eI and its modified derivatives are normally dimeric, but consist of a C-terminus of the first subunit and a C-terminus of the second subunit. The subunits are fused to a single peptide using a short peptide linker connecting the N-termini (Li, et al. (2009) Nucleic Acids Res. 37 :1650-62;Grizot,et al.(2009)Nucleic Acid s Res.37:5405-19.) Thus, functional "single-stranded" meganuclei The enzymes are expressed from a single transcript. By delivering a gene that cleaves at two different sites, it becomes possible to cleave the gene at two different sites simultaneously. Combined with the extremely low frequency of off-target cleavage observed with the engineered meganucleases, this This makes them preferred endonucleases of the present invention.

[0017] The use of engineered meganucleases for the treatment of Duchenne muscular dystrophy has previously been This is disclosed in International Publication WO2011 / 141820 ('820 application). In this application, the authors propose a novel method to correct the defect in the DMD gene through three different mechanisms. have discussed the possibility of using engineered meganucleases for this purpose (International Publication WO 201 (See Figure 1 in Publication No. 1 / 141820.) First, the authors 2. Inserting a Transfected Copy of the DMD or Micro-DMD Gene into the “Bar” Locus The present invention contemplates the use of engineered meganucleases to achieve a novel therapeutic target that does not affect endogenous gene expression. Second, the authors demonstrated that meganucleases are expressed constitutively in DMD. This DNA break can then be used to break the genome at a site near the deleterious mutation in the The genome mutations DMD and trans are corrected so that the mutations in the genome are corrected. We propose that a gene that is delivered in a genomic DNA sequence can stimulate homozygous recombination between healthy copies of the gene. Third, the authors of the '820 application contend that the engineered meganuclease does not incorporate an intron into the DMD gene. It is possible that such meganucleases could insert foreign DNA into the nucleases and cause disease. Insertion of the essential C-terminal domain of dystrophin upstream of an early intron of the dystrophin mutation Importantly, we propose that the use of megakaryin to manipulate the DMD gene may be used to In considering the myriad uses of nucleases, the authors of the '820 application The use of two meganucleases at the same time is not contemplated, and they do not Nor does it propose removing DNA.

[0018] Finally, Ousterout et al. demonstrated that DNA breaks induce DM using TALENs. D coding sequence and disruptions are frequently repaired via the mutagenic heterologous end-joining pathway. Small insertions and / or deletions ("inserts") that may change the reading frame of a gene This resulted in the introduction of the “Del” (Ousterout et al. (2013) Mol T Her.21:1718-26). They demonstrated that DNA breaks occur next to exons. Rapidly transported into the mutant and into the reading frame in some proportion of cells DMD in a fraction of mutant cells by relying on mutagenic DNA repair to recover This approach demonstrated the feasibility of restoring gene expression. It contains a single nuclease and is targeted to the coding sequence of a gene.

[0019] The present invention relates to a pair of engineered nucleases, or genetically encoded engineered nucleases, Two nucleases were used to restore the normal reading frame of the DMD gene. The present invention relates to a method for treating Duchenne muscular dystrophy, comprising delivering a gene encoding an exon of The cells so treated express central spectrin receptor tyrosine kinase (CRT) and are therefore suitable for use in treating trophoblastic disorders. A dimer in which part of the return domain is absent except for the N- and C-terminal domains being complete. In many cases, this is due to the disease. This would reduce the severity of the disease to a milder Becker phenotype.

[0020] Thus, in one embodiment, the present invention provides a method for the production of ducins using a pair of nucleases. In another embodiment, a general method for treating Hesse muscular dystrophy is provided, comprising: The present invention provides engineered meganucleases suitable for carrying out the method. In the present invention, a modified compact TALEN suitable for carrying out the method is provided. In a fourth embodiment, the present invention provides a CRISPR for carrying out the method. In a fifth embodiment, the method includes a vector and a method for delivering the modified nuclease to cells of a patient. Provides services and technology. [Brief description of the drawings]

[0021] [Figure 1] Structure of the DMD gene. The 79 exons are depicted to indicate the reading frame. The essential actin-binding and dystroglycan-binding domains, spanning approximately exons 2-8 and 62-70, respectively, are shown. [Diagram 2]Strategies for deleting exons from the DMD gene using different types of nucleases. 2A) Strategy for exon deletion using a pair of CRISPRs. A pair of "guide RNAs" ("gRNAs") are used that complement a pair of recognition sites that flank the relevant exons. As depicted in this figure, the gRNAs can complement a protected "GG" motif and a recognition sequence that is distal to the site of Cas9 DNA cleavage. In this orientation, the CRISPR recognition sequence is mostly preserved after DNA cleavage, cleavage of the intervening genomic DNA fragment, and rejoining of the chromosomal end. 2B) An alternative scheme for deleting exons using a pair of CRISPRs where the gRNA complements a recognition sequence that is proximal to the exon. In this orientation, the CRISPR recognition sequence is mostly preserved after DNA cleavage, deletion of the intervening genomic DNA fragment, and rejoining of the chromosomal end. It is contemplated that the present invention may also include a hybrid of the schemes shown in 2A and 2B. 2C) Strategy for exon deletion using a pair of compact TALENs (cTALENs). A pair of TAL effector DNA binding domains ("TALEs") are used that bind to a pair of recognition sites that strip the relevant exon. As shown in this figure, the TALEs can bind to a recognition sequence that is distal to a "CNNNG" motif that is recognized and cleaved by an I-TevI ​​cleavage domain ("TevI-CD"). In this orientation, the cTALEN recognition sequence is mostly preserved after DNA cleavage, deletion of an intervening fragment of genomic DNA, and rejoining of the chromosomal end. Additionally, the cTALEN in this figure is 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. 2D) An alternative scheme to delete an exon using a pair of cTALENs in which the TALE domains bind to a recognition sequence proximal to the exon. In this orientation, the cTALEN recognition sequence is mostly preserved after DNA cleavage, deletion of an intervening fragment of genomic DNA, and rejoining of the chromosomal end. The cTALEN in this figure is also depicted with the TALE and TrevI-CD domains in the C- to N-orientation.It is contemplated that the invention may also include hybrids of the schemes shown in 2C and 2D. 2E) Strategy for deleting exons from the DMD gene using a pair of single-stranded meganucleases. The meganucleases are shown as two domain proteins (MGN-N: N-terminal domain and MGN-C: C-terminal domain) connected by a linker. In the figure, the C-terminal domain is depicted as binding to the half of the recognition sequence closest to the exon. However, in some embodiments, the N-terminal domain may bind to this half of the recognition sequence. The central four base pairs of the recognition sequence are shown as "NNNN". These four base pairs become a single-stranded 3' "overhang" after cleavage by the meganuclease. A preferred four base pair subset that includes this region of the sequence is identified in WO / 2010 / 009147. DNA cleavage by a pair of meganucleases generates a pair of four base pair 3' overhangs at the chromosomal ends. If these overhangs are complementary, they can anneal to each other and directly recombine, resulting in a four base pair sequence that remains in the chromosome after exon cleavage. Because meganucleases cleave near the center of the recognition sequence, half of each recognition sequence can frequently be retained in the chromosome after exon cleavage. The other half of each recognition sequence can be removed from the genome along with the exon. [Diagram 3]Deletion of DMD exon 44 using DYS-1 / 2 and DYS-3 / 4 meganucleases. 3A) Sequence of DMD exon 44 and sequence region. Exon sequence is underlined. The recognition sites for DYS-1 / 2 and DYS-3 / 4 meganucleases are shaded in grey with the central four base pairs in bold (resulting in the 3' overhang after cleavage by the meganucleases). Annealing sites of a pair of PCR primers for use in the analysis are in italics. 3B) Agarose gel electrophoresis analysis of HEK-293 cells co-expressing DYS-1 / 2 and DYS-3 / 4. Genomic DNA is isolated from cells and assessed by PCR using the primers shown in (3A). The PCR products were resolved in an agarose gel and found that HEK-293 cells co-expressing the two meganucleases gave a pair of PCR bands, whereas wild-type HEK-293 cells gave only the larger band. 3C) Sequences from three plasmids harboring the smaller PCR product from (3B). The three sequences are shown oriented to the wild-type human sequence. The positions of the DYS-1 / 2 and DYS-3 / 4 recognition sequences are shaded grey with the central four base pairs in bold. [Figure 4]Deletion of DMD exon 45 using DYS-5 / 6 and DYS-7 / 8 meganucleases. 4A) Sequence of DMD exon 45 and sequence region. Exon sequence is underlined. Recognition sites for DYS-5 / 6 and DYS-7 / 8 meganucleases are shaded in grey with the central four base pairs in bold (resulting in the 3' overhang after cleavage by the meganuclease). Annealing sites of a pair of PCR primers for use in the analysis are in italics. 4B) Agarose gel electrophoresis analysis of HEK-293 cells co-expressing DYS-5 / 6 and DYS-7 / 8. Genomic DNA is isolated from cells and assessed by PCR using the primers shown in (4A). The PCR products were resolved in an agarose gel and found that HEK-293 cells co-expressing the two meganucleases gave a pair of PCR bands, whereas wild-type HEK-293 cells gave only the larger band. 4C) Sequences from 16 plasmids harboring the smaller PCR product from (4B). Sequences are shown oriented to the wild-type human sequence. The positions of the DYS-5 / 6 and DYS-7 / 8 recognition sequences are shaded grey with the central four base pairs in bold. [Diagram 5]Evaluation of MDX-1 / 2 and MDX-13 / 14 meganucleases in a CHO cell receptor assay. A) Diagram of the assay. For each of the two meganucleases, we generated CHO cell lines in which a receptor cassette was stably integrated into the genome of the cells. The receptor cassette contained, in 5' to 3' order, the SV40 early promoter, the 5'2 / 3 of the GFP gene, one of the recognition sites for MDX-1 / 2 (SEQ ID NO: 149) or MDX-13 / 14 (SEQ ID NO: 150), the recognition site for CHO-23 / 24 meganuclease (WO / 2012 / 167192), and the 3'2 / 3 of the GFP gene. Cells transfected with this cassette did not express GFP in the absence of a DNA break-inducing agent. However, when a DNA break is induced at one of the meganuclease recognition sites, the duplicated regions of the GFP gene recombine with each other to produce a functional GFP gene. The percentage of GFP-expressing transgenic cells can then be determined by flow cytometry as an indirect measure of the frequency of genome cleavage by the meganuclease. B, C) Two CHO recipient lines were transfected with mRNA encoding MDX-1 / 2 (A), MDX-13 / 14 (B), or CHO-23 / 34 (A and B) meganuclease. 1.5e6 CHO cells were transfected with 1e6 copies of mRNA per cell using Lonza Nucleofector 2 and program U-024 according to the manufacturer's instructions. After 48 hours of transfection, cells were assessed by flow cytometry to determine the percentage of GFP-positive cells compared to the non-transfected (empty) negative control. Assays were 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 greatly exceeding both the negative (blank) control and the CHO-23 / 24 positive control, indicating that the nucleases can efficiently recognize and cleave the intended target sequence in the cells. [Figure 6]Alignment of sequences from 20C2C12 mouse myoblast clones in which portions of the DMD gene have been deleted by co-transfection with 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 NOs: 153-172) is aligned against a reference wild-type DMD sequence, with deletions relative to the reference shown as hollow bars. [Figure 7] Vector map of pAAV-MDX plasmid. This "packaging" plasmid is used with an Ad helper plasmid to generate AAV viruses that can simultaneously deliver genes encoding MDX-1 / 2 and MDX-13 / 14 meganucleases. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] 1.1 References and Definitions The patent and scientific literature referred to herein constitutes knowledge available to those skilled in the art. Any issued U.S. patents, patent applications, or other publications, including GenBank database sequences, cited in the specification Each approved application, published foreign application, and reference is expressly and individually incorporated herein by reference. Where reference is indicated, the same is hereby incorporated by reference to the same extent. do.

[0023] As used herein, the term "meganuclease" refers to a nuclease derived from I-CreI. The term meganuclease as used herein refers to an endonuclease derived from a For example, DNA binding specificity, DNA cleavage activity, DNA binding affinity, or dimerization may be performed. Engineered variants of I-CreI that have been modified relative to the native I-CreI in terms of their somatization properties were Methods for producing such modified variants of I-CreI are known in the art. (For example, International Publication WO2007 / 047859). Meganucleases are can bind duplex DNA as a homodimer, as in the case of wild-type I-CreI, or It can bind to DNA as a heterodimer. Meganucleases consist of a pair of I-CreI The derived DNA binding domains are linked into a single polypeptide using a peptide linker. This could result in a "single-chain meganuclease" that can be used to

[0024] As used herein, the term "single-chain meganuclease" refers to a single-chain meganuclease that is linked by a linker. A polypeptide that contains a pair of linked meganuclease subunits. The lyase has the structure: N-terminal subunit-linker-C-terminal subunit. The two meganuclease subunits are each derived from I-CreI and have the amino acid sequence They may be largely non-identical in sequence and may recognize non-identical DNA sequences. Single-stranded meganucleases typically cleave pseudo-patterned or non-patterned recognition sequences. The ganucleases are referred to as "single-chain heterodimers" or "single-chain heterodimeric meganucleases." Although it may be shown as a dimer, it is not actually a dimer. For clarity, unless stated explicitly, The term "meganuclease" can refer to dimeric or single-chain meganucleases.

[0025] As used herein, the term "compact TALEN" refers to a TALEN that can be used in any orientation. 16-22 fused to any part of the I-TevI ​​homing endonuclease Refers to an endonuclease that contains a DNA-binding domain with TAL domain repeats .

[0026] As used herein, the term "CRISPR" refers to a caspases such as Cas9. and caspase recognition in genomic DNA by hybridization Refers to a caspase-based endonuclease that contains a guide RNA that targets the target site.

[0027] As used herein, with respect to a protein, the term "recombinant" refers to a protein that is The modification techniques are directed to the nucleic acid encoding the protein, and to the cells or organisms expressing the protein. As used herein, the term "amino acid sequence" refers to a sequence that is modified by the application of the term "amino acid sequence" to a nucleic acid. "Recombinant" means having an altered nucleic acid sequence as a result of the application of genome modification techniques. Genetic modification techniques include, but are not limited to, PCR and DNA cloning techniques, transcription Infection, transformation, and other transfer techniques, homogeneous recombination This definition includes recombinant gene products, site-directed mutagenesis, and gene fusion. Identical to the native protein, but produced by cloning and expression in a heterologous host Proteins having an amino acid sequence similar to that described above are not considered recombinant.

[0028] As used herein, the term "wild type" refers to any of the meganucleases. The term "wild type" refers to the most common allelic variant of an enzyme found in nature. It is not intended to refer to alleles, but rather to any alleles found in nature. A native homing endonuclease refers to a recombinant or non-naturally occurring variant of a native homing endonuclease. Distinguished from developmental meganucleases.

[0029] As used herein, the term "recognition sequence" refers to a sequence that is bound by an endonuclease. In the case of meganucleases, the recognition sequence is a sequence of four bases. It contains a pair of inverted nine base pair "half sites" separated by a base pair. In the case of lyase, the N-terminal domain of the protein contacts the first half-site and The C-terminal domain of the nuclease contacts the second half-site. Cleavage by meganucleases occurs via base-pairing. Produces a 3' "overhang." "Overhang" or "sticky end" are short single-stranded DNA segments that can be produced by endonuclease cleavage of double-stranded DNA sequences. I-CreI-derived meganuclease and single-chain meganuclease In the case of the enzyme, the overhang comprises bases 10-13 of the 22 base pair recognition sequence. In the case of express TALENs, the recognition sequence is a I-TevI ​​domain followed by a non-specific spacer 4-16 base pairs in length, followed by the TAL-effector domain (this sequence is characterised by a 5' T base). The first CNN recognized by the second sequence length 16-22bp (typically including Contains the NGN sequence. Cleavage by Compact TALEN produces two 3' overhangs. In the case of CRISPR, the recognition sequence allows the guide RNA to target Cas9 cleavage to the sequence. CRISPR is a sequence that binds to a target gene, typically 16-24 base pairs in length. Cleavage by produces blunt ends.

[0030] As used herein, the term "target site" or "target sequence" refers to a meganuclease. The term refers to a region of a cell's chromosomal DNA that contains a recognition sequence for a lyase.

[0031] As used herein, the term "homologous recombination" or "HR" refers to the synthesis of a double-stranded DNA fragment. This refers to the natural cellular process by which DNA disruptions are repaired using homologous DNA sequences as repair templates ( Cahill et al.(2006),Front.Biosci.11:1958 Homologous DNA sequences can be endogenous chromosomal sequences or exogenous sequences delivered to a cell. The nucleic acid may be a nucleic acid.

[0032] As used herein, the term "non-homologous end joining" or "NHEJ" refers to a bipartite A natural cellular process in which heavy-strand DNA breaks are repaired by direct ligation of two nonhomologous DNA segments. This refers to the process (Cahill et al. (2006), Front. Biosci. 11:1958-1976). DNA repair by non-homologous end joining is error-prone and often This results in an unintended conversion or deletion of the DNA sequence at the repair site.

[0033] As used herein, the term "religation" refers to the recombination of a single double-stranded DNA break. The two resulting DNA ends are covalently linked together with the loss of the intervening DNA sequence. Refers to a process in which no additional DNA sequence is gained or lost. In the case of paired DNA breaks produced in followed by covalent joining of the 5' and 3' ends by DNA ligase. Religation is called NHEJ, which does not result in the unintended addition or deletion of DNA from the site of repair. is different.

[0034] As used herein, unless expressly stated otherwise, the word "or" means "and" It is used in the inclusive sense of "and / or" and not in the exclusive sense of "either / or."

[0035] 2.1 Principle of exon deletion The present invention relates, in part, to a specific defect in the DMD gene that causes the Duchenne phenotype. The deletion is due to the immediate deletion of an additional exon(s) upstream or downstream of the mutation. The DMD-Leiden database is based on the hypothesis that Duchenne Most mutations that cause muscular dystrophies affect the sequence of the reading frame. Describes a deletion of one or more whole exons that causes a deficiency in the gene. The matching frame can be restored by excluding the exon immediately before or after the mutation. Table 1 As shown in Figure 1, 29 different Duchenne-inducing mutations, representing approximately 65% ​​of patients, The mutation can be complemented by deleting a single exon adjacent to the mutation. Approximately 7% of patients with DMD exon 45 deletion disease have a deletion of exon 46. Of note, the exon 51 or exon 45 deletions can be treated with Possible treatments could be used to treat 15% and 13% of patients, respectively.

[0036] [Table 1]

[0037] 2.2 Nucleases for exon deletion Using site-specific nucleases to make DNA breaks in the genome of living cells and that such DNA breaks can be used to induce mutagenic NHEJ repair or or transfected DNA sequences can result in permanent genome alterations via HR. However, the present invention relates to a pair of nuclei in the same cell. Surprisingly, we found that the co-expression of the two genes at DNA sites close to each other (10,000 s A pair of nucleases targeted to the intervening DNA fragments (separated by less than a base pair) removes the intervening DNA fragments from the genome. Surprisingly, we found that it is possible to excise a pair of nuclei. DNA deletion using nucleases contains single-stranded DNA overhangs generated by nucleases They found that the mechanism frequently proceeds via the synthesis of complementary (i.e., identical) DNA over In experiments involving a pair of meganucleases that generate hangovers, It has been found to be frequently preserved after fragment excision and the resulting chromosome ends ( (See Examples 1 and 2). In this case, the DNA repair mechanism is not observed in mammalian cells. Such precise deletions and recombinations appear to direct religation of undetected broken ends. Ligation was observed when using a pair of meganucleases that generate non-identical overhangs. (See Example 3.) Thus, in a preferred embodiment, The pair of nucleases used for the excision of the nucleotide sequence produce complementary overhangs. is selected.

[0038] To efficiently excise an exon, a pair of nuclease excision sites must be relatively close to each other. Generally, the closer the two sites are to each other, the more efficient the process. Therefore, the preferred embodiment of the present invention is A base pair separation of less than 5,000, more preferably 2,500, and most preferably less than 1,500. The method uses a pair of nucleases that excise the inserted sequence.

[0039] As shown in FIG. 2, a variety of different nucleases are useful in the practice of the present invention. 2A and 2B show how the present invention uses a pair of CRISPR nucleases to Here is an example of how it can be implemented. In this case, the invention is directed to delivering three genes to a cell. The method can be carried out by: The final product generally differs in sequence. CRISPR can be used to engineer insertion and / or deletion ("indel") mutations that , excising the DNA to leave blunt ends that generally do not religate correctly. The morphology is reported in Ran, et al. (2013) Cell. 154:1380-9. As disclosed, "CRISPR nickase" may be used. To achieve this, cells must express four guide RNAs, two of which are exogenous. One is complementary to the sequence upstream of the exon, and the other two 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. The pair is then split into two strands, with each member of the pair generating a single-stranded DNA nick in one of the strands. A pair of nucleotides that can be offset from each other to create advantageous single-stranded overhangs for application to the nucleic acid. A notch (equivalent to a double-strand break) is generated by a CRIS that recognizes a predetermined DNA site. Methods for producing PR and CRISPR nickases are described, for example, in Ran, et al. 2013) Nat Protoc. 8:2281-308, which are known in the art. .

[0040] In an alternative embodiment, as depicted in Figures 2C and 2D, the nuclease pair is Compact TALENs can be small TALENs that have at least one N- or C-terminus. at least residues 1-96 of I-TevI, preferably residues 1-182 of I-TevI; TAL-effector DNA binding domain (TAL) fused to the cleavage domain from evI E). The I-TevI ​​cleavage domain cleaves DNA in the form 5'-CNbNNtG-3'. It recognizes and excises sequences, where "b" represents the cleavage site of the bottom strand and "t" represents the cleavage site of the top strand. The 'N' represents one of the four bases. N cleaves to produce a two base pair 3' overhang. In this study, the compact TALEN pairs used for exon excision can be directly religated It is selected to have a complementary overhang that binds to a predetermined DNA site. Methods for producing TALE domains are described, for example, in Reyon et al. (2012) N at Biotechnol. 30:460-5.

[0041] In a preferred embodiment, a 3D printer is used to practice the present invention, as shown in FIG. The nuclease is a pair of single-chain meganucleases. Each of the two domains has an N-terminus and a C-terminus connected by a peptide. It recognizes half of the recognition sequence and the site of DNA cleavage is near the interface of the two subunits. The recognition sequence is located in the middle of the DNA strand break. A pair of four base pairs is added to produce a 3' single-stranded overhang. In a preferred embodiment, as in Examples 1 and 2, a supplementary overlay is set. A single-chain meganuclease that cuts the recognition sequence at the hang is selected. DMD exon 44 Exemplary recognition sequences for exon 44, 45 and 51 are listed in Tables 2 to 7. The first meganuclease cuts the recognition sequence to remove the exon 44. The second meganuclease that cuts the recognition sequence can be selected from Table 2, which lists the upstream , can be selected from Table 3, which lists the recognition sequence downstream of exon 44. Co-expression of the two meganucleases in cells deletes exon 44. A meganuclease is selected that cuts the DNA to leave targeted single-stranded overhangs. For example, when SEQ ID NO: 19 is cleaved by a meganuclease, the overhang sequence Similarly, SEQ ID NO:42 is cleaved by meganuclease to leave 5'-GTAC-3'. If the overhang sequence is 5'-GTAC-3', then the overhang sequence is left. Co-expressing a first meganuclease that cleaves SEQ ID NO:19 with a second meganuclease that cleaves This would allow the deletion of DMD exon 44 from the genome of human cells, resulting in a direct Complementary overhangs are produced that can be repaired through religation.

[0042] [Table 2]

[0043] [Table 3]

[0044] [Table 4]

[0045] [Table 5]

[0046] [Table 6]

[0047] [Table 7]

[0048] 2.3 Nuclease delivery and expression methods The treatment of Duchenne muscular dystrophy using the present invention involves the binding of a pair of nucleases to muscle fibers. The nucleases may be expressed in cells as purified proteins or as nucleic acid fragments. The enzyme may be delivered as RNA or DNA. , a vector encoding a nuclease protein or mRNA or a nuclease, Intramuscular injection (Maltzahn, et al. (2012) Proc Natl Aca d Sci USA.109:20614-9) or hydrodynamic injection (Ta niyama et al. (2012) Curr Top Med Chem.12: 1630-7;Hegge,et al.(2010)Hum Gene Ther.2 1:829-42) to muscle cells. To achieve this, the protein or nucleic acid(s) is / are delivered to the muscle cell via a cellular transporter. Examples of cell-penetrating peptides known in the art include polyamino acids, Luginine (Jearawiriyapaisarn, et al. (2008) Mol Ther. 16:1624-9), TAT peptide from HIV virus (Hudecz et al. (2005),Med.Res.Rev.25:679-736), MP G((Simeoni,et al.(2003)Nucleic Acids Res .31:2717-2724), Pep-1 (Deshayes et al. (200 4) Biochemistry 43:7698-7706) and HSV-1 VP- 22(Deshayes et al.(2005)Cell Mol Life Sc i.62:1839-49). Alternatively, cell penetration may be achieved by liposomal encapsulation. This can be easily achieved (e.g., Lipofectamine™, Life Technologies, (See Biotechnology Corp., Carlsbad, CA.) Liposomal formulations include: They can be used to facilitate lipid bilayer fusion with target cells, thereby allowing the formation of surface-associated These liposomes allow the delivery of the contents of the liposome or protein to the cell.

[0049] In some embodiments, the pair of nuclease-encoding genes are derived from a viral vector. Such vectors are known in the art and include lentiviral vectors. vectors, adenovirus vectors and adeno-associated viruses (AAV) (Vann ucci, et al. (2013 New Microbiol.36:1-22) In some embodiments, the virus is injected directly into muscle tissue. In an alternative embodiment, the viral vector is delivered systemically. A recombinant AAV virus encoding a pair of single-chain meganucleases is injected into the muscle. A preferred embodiment is shown. Different AAV vectors tend to be locally restricted to different tissues. It is known in the art that muscle-tropic AAV serotypes include AAV1, AAV9 and and AAV2.5 (Bowles, et al. (2012) Mol Ther.20:4 43-55) Thus, these serotypes may be involved in the transport of nucleases into muscle tissue. This is favorable for shipping.

[0050] In the form of DNA (e.g., plasmids) and / or viral vectors (e.g., AAV When the nuclease genes are delivered via the In some embodiments, this is from a viral vector. The endogenous promoter of the gene (e.g., lentiviral vector) or a well-known cytometric promoter Viral promoters such as the garovirus or SV40 virus early promoter. In a preferred embodiment, the nuclease gene is a gene that prioritizes gene expression in muscle cells. An example of a muscle-specific promoter is C5- 12(Liu,et al.(2004)Hum Gene Ther.15:783- 92), muscle-specific creatine kinase (MCK) promoter (Yuasa, et al. (2002) Gene Ther. 9:1576-88), or lubricating muscle 22 (SM2 2) Promoter (Haase, et al. (2013) BMC Biotechno I.13:49-54). In some embodiments, the nuclease gene comprises: In an alternative embodiment, the gene is under the control of a single It is under the control of the 2A promoter and is regulated by an internal ribosome entry site (IRES) or 2A Peptide sequence (Szymczak and Vignali (2005) Expert Opin Biol Ther.5:627-38).

[0051] It is envisioned that a single treatment will result in permanent deletion of the exon in a percentage of patient cells. In a preferred embodiment, the cells contain functional (or semi-functional) dystrophin Myoblasts or other muscle precursor cells that can replicate and give rise to whole muscle fibers expressing However, in cases where the frequency of exon deletions is low, multiple intramuscular It may be necessary to administer multiple treatments, such as injections. EXAMPLES

[0052] This invention is further illustrated by the following examples which should not be construed as limiting. Those of ordinary skill in the art will be able to, using no more than routine experimentation, develop various combinations of the specific materials and procedures described herein. Many equivalents will be recognized or may be identified. Such equivalents are described in the following examples. It is intended that the present invention be covered by the appended claims.

[0053] Example 1: Deletion of DMD exon 44 using a pair of engineered single-chain meganucleases 1. Meganuclease recognizing SEQ ID NO: 19 and SEQ ID NO: 42 An engineered meganuclease (SEQ ID NO:135) was prepared that recognizes and cleaves SEQ ID NO:19. This meganuclease is called "DYS-1 / 2". It recognizes and cleaves SEQ ID NO:42. A second modified meganuclease (SEQ ID NO: 136) was prepared, which has the following structure: These meganucleases are called "DYS-3 / 4" (Figure 3A). Each meganuclease is derived from SV40. The N-terminal nuclease localization signal, the first meganuclease subunit, and the linker -sequence, and a second meganuclease subunit.

[0054] 2. Deletion of DMD exon 44 in HEK-293 cells Human embryonic kidney (HEK-293) cells express DYS-1 / 2 and DYS-3 / 4. The mRNA was co-transfected with the mRNA encoding the The PCR template for the nucleotide sequence (SEQ ID NO: 139 and SEQ ID NO: 140) was first generated. Each PCR product was inserted under the vector sequence of the T7 promoter and meganuclease gene. The PCR product was gel purified to ensure a single template. m7G RNA was prepared using the RiboMAX T7 kit (Promega) according to the manufacturer's instructions. a) was used. Ribom7G cap analog (prmega) was included in the reaction. 0.5 μg of purified meganuclease PCR product served as DNA template. ChAP RNA was isolated using SV Total RNA Isolation according to the manufacturer's instructions. The purified product was purified using the Promega System.

[0055] 1.5×10 6 HEK-293 cells were cultured using Amaxa Nucleo DYS-1 / 2 mRNA was transfected at 1.5 × 100 μl using the Infector II apparatus (Lonza). 0 12 and 1.5 × 10 copies of DYS-3 / 4 mRNA. 12 Copy (2×10 6 Copy 48 hours post-transfection, the cells were nucleofected with the manufacturer's Genomic DNA was extracted from cells using the FlexiGene kit (Qiagen) according to the instructions. Genomic DNA was then cloned for the DYS-1 / 2 and DYS-3 / 4 cleavage sites. PCR was performed using primers flanking (SEQ ID NO: 141 and SEQ ID NO: 142). The PCR products were separated by agarose gel electrophoresis, and DYS-1 / 2 and Cells co-expressing DYS-3 / 4 had apparent lengths of 1079 and 233 base pairs. The untransfected HEK-29 gene gave rise to two PCR products, It is clear that genomic DNA from the three cells yielded only the larger product (Figure 3 B) The larger product is a PCR flag from cells with an intact DMD exon 44. The smaller product corresponds to the predicted size of the cleavage ... This is consistent with the predicted size of the PCR fragment from the cells deleted from the offspring.

[0056] The smaller PCR product was isolated from the gel and cloned into a bacterial plasmid (pUC Three plasmid clones were sequenced, all of which were Surprisingly, the three plasmids were found to be lacking gene 44 (Fig. 3C). In two of the cases, the deletions were precisely in line with the expected DYS-1 / 2 and DYS-3 / 4 induction. PCR products were derived from cells containing regions of intervening DNA breaks. The enzymes cleave their intended recognition sites and produce compatible 5'-GTAC-3' overhangs. The intervening fragment containing exon 44 is lost, leaving behind a chromosome fragment with two chromosome ends. The third plasmid clone contains a region between the two cleavage sites. carried the PCR product from cells that was deleted along with 10 additional bases.

[0057] 3. Conclusion We have developed a pair of engineered single-unit mutants to delete fragments from the human genome in cultured cell lines. We demonstrated that it is possible to use a multistrand meganuclease to perform DNA removal and repair processes. The process is mediated by nucleases if the overhangs are complementary and capable of annealing to each other. The process appears to be facilitated by a mechanism involving the formation of 3' overhangs by This suggests that is more effective.

[0058] Example 2: Deletion of DMD exon 45 using a pair of engineered single-chain meganucleases 1. Meganuclease recognizing SEQ ID NO:62 and SEQ ID NO:74 An engineered meganuclease (SEQ ID NO: 137) was prepared that recognizes and cleaves SEQ ID NO: 62. This meganuclease is called "DYS-5 / 6". It recognizes and cleaves SEQ ID NO:74. A second modified meganuclease (SEQ ID NO: 138) was prepared, which has the following structure: The meganucleases are called "DYS-7 / 8" (Figure 4A). Terminal nuclease localization signal, first meganuclease subunit, linker sequence and and a second meganuclease subunit.

[0059] 2. Deletion of DMD exon 45 in HEK-293 cells Human embryonic kidney (HEK-293) cells were cultured using DYS-5 / 6 and DYS-7 / 8. The mRNA was co-transfected with the mRNA encoding the PCR templates for sequence number 143 (20) and sequence number 144 (21) were first generated. Each PCR product was prepared by ligating the vector sequence downstream of the meganuclease gene. The PCR product contained a 609 bp fragment of the ribozyme and a T7 promoter. Capped (m7G) RNA was purified using RiboMAX™ according to the manufacturer's instructions. Ribom7G cap analog (pr meganuclease) was included in the reaction, and 0.5 μg of purified meganuclease PCR product was Capped RNA served as the SV total RNA template according to the manufacturer's instructions. Purification was performed using the Solation system (Promega).

[0060] Amexa Nucleofector II instrument (Lonza) according to the manufacturer's instructions Using 1.5×10 6 HEK-293 cells were incubated with 1.5 × 10 12 and 1.5 × 10 copies of DYS-7 / 8 mRNA. 12 Copy (2×10 6 copy 48 hours post-transfection, nucleofected with 100 µg / cell (100 µg / cell) as per manufacturer's instructions. Genomic DNA was isolated from cells using the FlexiGene kit (Qiagen). Genomic DNA was then cloned into the DYS-5 / 6 and DYS-7 / 8 cleavage sites (SEQ ID NO: PCR was performed using primers flanking the sequences (SEQ ID NO: 145 and SEQ ID NO: 146). The R products were separated by agarose gel electrophoresis, and the DYS-5 / 6 and DYS- Cells co-expressing 7 / 8 expressed 2 The transfected HEK-293 cells produced two PCR products, whereas the untransfected HEK-293 cells produced two PCR products. It was clear that their genomic DNA only yielded the larger product (Fig. 4B). The larger product is a PCR fragment from cells with an intact DMD exon 45. The smaller product is consistent with the expected size of exon 45 from the DMD gene. This is consistent with the expected size of the PCR fragment from the deleted cells.

[0061] The smaller PCR product was isolated from the gel and cloned into a bacterial plasmid (pUC Sixteen plasmid clones were sequenced, all of which Surprisingly, 16 genes were found to be deleted in exon 45 (Fig. 4C). Fourteen of the plasmids contained the exact deletions predicted for DYS-5 / 6 and DYS-7 / PCR products were carried from cells consisting of regions intervening 8-induced DNA breaks. The enzymes cleave their intended recognition sites and produce compatible 5'-GTAC-3' nucleotides. The intervening fragment containing exon 45 is lost, leaving a bar hang, and then two chromosomes The ends appear to have been religated. The two remaining plasmid clones have two cleavage sites. PCR products were carried from cells in which the intervening region was deleted along with 36 additional bases.

[0062] 3. Conclusion We used a pair of modifications to delete fragments from the human genome in cultured cell lines. We demonstrate that mutant single-chain meganucleases can be used to perform DNA deletion and repair. The process is facilitated through a mechanism involving nuclease-generated 3' overhangs. If the overhangs are complementary and can anneal to each other, the process This suggests that it will be more efficient.

[0063] Example 3: Deletion of DMD exon 23 in mice using AAV-delivered meganuclease loss 1. Development of a nuclease that deletes mouse DMD exon 23 The standard mouse model for DMD is the mdx mouse, which introduces a premature stop codon The patient has a point mutation in exon 23 that inserts into the nucleus (Sicinski et al. (1989) Science. 244:1578-80). Chron 23 is 213 base pairs long, equivalent to 71 amino acids. They deleted the entire exon 23, thereby preserving the reading frame of the DMD gene. We reasoned that it would be possible to delete the stop codon while retaining the original DNA sequence. -1 / 2" (SEQ ID NO: 147) and "MDX-13 / 14" (SEQ ID NO: 148). We have developed a pair of single-chain meganucleases that can be used to identify the DMD exon 23 (sequence No. 149), whereas the latter recognizes the DNA sequence upstream of mouse DMD exon 23 (SEQ ID NO: First, as shown in Figure 5, in CHO cells, The nuclease was tested using a receptor assay called "iGFFP." Using the I method, both nucleases efficiently cut their intended DNA sites. It was found that.

[0064] 2. Deletion of mouse DMD exon 23 in mouse myoblasts A mouse germinal cell line (C2C12) was transformed with MDX-1 / 2 and MDX-13 / 14 nucleases. The mice were co-transfected with in vitro transcribed mRNA encoding the α-aspartatease. Prepared using the RiboMAX T7 kit from Promega. The cells were analyzed for each MDX enzyme pair (each mR Nucleofected with a total of 2e6 copies / cell of mRNA encoding NA (1e6 copies of NA). After 96 hours, cells were cloned by limiting dilution in 96-well plates. After approximately 2 weeks of growth, cells were harvested and transfected using the FlexiGene kit from Qiagen. Genomic DNA was isolated using a PCR kit. The PCR product was then amplified to identify the DMD intron 22 (sequence The pre-primer in sequence 151) and the pre-primer in intron 23 (SEQ ID NO: 152) A reverse primer was used to generate a PCR product for each clone. The 60 PCR products were then cloned. The 20 sequences were cloned and sequenced. The deletions are consistent with DNA repair of the mutations in (Figure 6, SEQ ID NOs: 153-172). The sequence of exon 23 (SEQ ID NO: 153-163) as well as MDX-1 / 2 and MD The sequences were missing at least part of the X-13 / 14 recognition site. The enzymes cut their intended sites and are derived from cells in which the intervening sequences are deleted. Four sequences were missing exon 23 but contained the complete MDX-1 / 2 recognition sequence. These were the DNA sequences from MDX-13 / 14 alone (SEQ ID NOs: 164-167). The sequence is likely due to the deletion of many sequences after the A cleavage. It has the MDX-13 / 14 recognition site and all or part of exon 23. These sequences were missing all or part of the recognition site (SEQ ID NOs: 168-172). DNA cleavage by DX-13 / 14 alone is insufficient to remove all of exon 23. This appears to be due to the deletion of a smaller amount of sequence than is sufficient. In stark contrast, we found consistent DNase I after deletion of DMD exon 23 in mouse cells. This is because the two MDX meganucleases have compatible 3' o- This is because MDX-1 / 2 does not cause DNA breaks at the overhang. -GTGA-3', and MDX-13 / 14 produces an overhang with the sequence 5'- This produces an overhang with ACAC-3'. Thus, we have The consistent sequence results obtained in 2 were consistent with the 3' The conclusion is that it depends on the compatibility of the overhangs.

[0065] 3. Generation of Recombinant AAV Vectors for Delivery of a Pair of Engineered Nucleases AAV vectors for co-delivery of MDX-1 / 2 and MDX-13 / 14 genes To prepare the vectors, we first used the vectors M, M Genes encoding sequences for DX-1 / 2 and MDX13 / 14 meganucleases Similarly, “pAAV-MDX” ( A "packaging" plasmid called GFP (Figure 7, SEQ ID NO: 173) was prepared. The target was co-transfecting HEK-293 cells with Ad helper plasmids by the method of Xiao et al. It was used to produce recombinant AAV2 virus by transfection (X iao, et al. (1998) J. Virology 72:2224-2232) The virus was then isolated using the method described by Grieger and Samulski (Grieger and d Samulski(2012)Methods Enzymol 507:229- The resulting proteins were isolated by cesium chloride gradient centrifugation as described by 254. Confirm that the resulting viral particles are infectious and capable of expressing both engineered meganucleases. To achieve this, we used a receptor cassette for either MDX-1 / 2 or MDX-13 / 14. The iGFFP-transducing CHO cells were cultured and added to the cultured CHO cells (see Figure 5A). Addition of the virus to a CHO line carrying the receptor resulted in GFP gene expression in 10.2% of cells. Thus, we demonstrated that the virus can transduce CHO cells and that the transduced We conclude that the cells expressed both nucleases.

[0066] 4. DMD exons in mouse muscle following AAV delivery of paired meganuclease genes 23 deletions Recombinant AAV1 viral particles carrying MDX-1 / 2 and MDX-13 / 14 genes A pair of mdx mice was prepared as described above. The virus was injected into three hind leg TA muscles (Xiao, et al. (1998) JV irology 72:2224-2232). One muscle from one mouse was negative. As a control, no injection was performed. Muscles from two mice were harvested at 4 or 7 days after injection. The genomic region surrounding DMD exon 23 was cloned into the genomic DNA of the first Amplified by PCR using one primer pair (SEQ ID NO: 151 and SEQ ID NO: 152). The reaction was then subjected to a "nested" (n A second PCR was performed using the primer pair (SEQ ID NO: 174 and SEQ ID NO: 175) The PCR products were then visualized on an agarose gel. Genomic DNA from three AAV1-injected muscles, but not from uninjected control muscles, was Following deletion of DMD exon 23 by X-12 and MDX-13 / 14 meganucleases This produced a smaller PCR product that matched the predicted product size. The smaller PCR products were cloned and sequenced. Three unique sequences were obtained, each of which This includes intron 22 and part of intron 23 but lacks exon 23. Part of the D gene and the MDX-1 / 2 and MDX-13 / 14 meganucleases (sequence The cut site for the nucleotide sequence 176-178 is composed of all the intervening sequences. We demonstrated that a pair of meganucleases delivered by AAV can be used to in vivo They concluded that this could be used to delete parts of the DMD gene.

[0067] 5. Conclusion We used a recombinant AAV vector to encode a pair of engineered single-chain meganucleases. The genes that can be delivered to cells and organs can be expressed by the meganuclear The enzyme cleaves genomic DNA in cells and removes fragments of DNA from the genome. We further concluded that it is possible to eliminate the overhangs by using a compatible A pair of meganuclease-induced DNA breaks that do not produce nucleotides are defined after removal of the intervening sequences. It was concluded that the nucleotide sequences would not be religated to produce the desired sequence results. For the desired therapeutic application of a defined sequence outcome, a set of identical overhang-forming It is preferred to use paired nucleases.

Claims

1. A first nuclease that cleaves the first recognition sequence and a second nuclease that cleaves the second recognition sequence. contacting the enzyme with DNA of muscle cells of the subject; the first recognition sequence is upstream of the first exon of the dystrophin gene; the second recognition sequence is downstream of the first exon of the dystrophin gene; and At least one second exon is removed from the dystrophin gene of the cell. A method of treating Duchenne muscular dystrophy in a subject in need thereof.

2. 2. The method of claim 1, wherein each of the first and second nucleases is a meganuclease. Method of posting.

3. 2. The method of claim 1, wherein each of the first and second nucleases is a CRISPR. method.

4. Each of the first and second nucleases is a compact TALEN.

1. The method described in 1.

5. 2. The method of claim 1, wherein the first exon is exon 44.

6. 2. The method of claim 1, wherein the first exon is exon 45.

7. 2. The method of claim 1, wherein the first exon is exon 51.

8. The first recognition sequence is selected from SEQ ID NO: 2 to 28, and the second recognition sequence is selected from SEQ ID NO:

6. The method of claim 5, wherein the amino acid sequence is selected from the group consisting of 29 to 44.

9. The first recognition sequence is selected from SEQ ID NOs: 45 to 63, and the second recognition sequence is selected from SEQ ID NOs: The method according to claim 6, wherein the compound is selected from Nos. 64 to 74.

10. The first recognition sequence is selected from SEQ ID NOs: 75-105, and the second recognition sequence is The method of claim 7, wherein the compound is selected from the group consisting of numbers 106 to 134.

11. The first nuclease is SEQ ID NO: 135 and the second nuclease is SEQ ID NO:

9. The method of claim 8, wherein the molecular weight is 136.

12. The first nuclease is SEQ ID NO: 137 and the second nuclease is SEQ ID NO: 1 10. The method of claim 9, wherein the .mu.m is 38.

13. The genes encoding the first and second nucleases are The method according to any one of claims 1 to 12, wherein the antibody is delivered to the cell using an AAV. 。

14. The first and second recognition sites, when cleaved by the first and second nucleases, In the case of the above, the overhangs are selected to be identical. The method described.

15. A first nuclease that cleaves the first recognition site and a second nuclease that cleaves the second recognition site. contacting the DNA with an enzyme, said first and second recognition sites being Selected to have identical overhangs when cleaved by a second nuclease A method for removing a DNA sequence from the genome of a cell.

16. The genes encoding the first and second nucleases are The method of claim 15, wherein the antibody is delivered to the cell using an AAV virus.

17. 17. The method of claim 15, wherein the first and second nucleases are meganucleases. The method described.

18. 17. The method of claim 15 or 16, wherein the first and second nucleases are CRISPR. How to.

19. 15. The method of claim 14, wherein the first and second nucleases are compact TALENs.

16. The method according to claim 16.

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