Production of large-sized quasi-dystrophin using overlapping AAV vectors

Abstract

The present invention relates to quasi-dystrophin (QD), which has the structure CH1CH2H1R1R2R3H2R8R9 in its N-terminal part and advantageously further comprises the R16 and R17 rod domains, as well as to a dual AAV vector system making it possible to produce it.

Description

[Technical field]

[0001] The present invention relates to gene therapy vectors useful in the treatment or prevention of dystrophic diseases, in particular Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).

[0002] This application reports that the use of overlapping AAV vectors allows the production of large amounts of active truncated dystrophin that displays important functional domains. [Background technology]

[0003] Duchenne muscular dystrophy (DMD) is the most common progressive muscle degenerative disease, affecting approximately 1 in every 3,500-5,000 male newborns. DMD is caused by deletions or mutations in the gene encoding dystrophin, located on the X chromosome. Dystrophin is required for the assembly of the dystrophin-glycoprotein complex, providing a mechanical and functional link between the cytoskeleton of muscle fibers and the extracellular matrix. The absence of functional dystrophin leads to fiber degeneration, inflammation, necrosis, and replacement of muscle with scarring and adipose tissue, resulting in progressive muscle weakness and premature death from respiratory and cardiac failure during the teenage to 30th decades of life (Moser, H., Hum Genet, 1984. 66(1): 17-40).

[0004] A milder form of the disease is called Becker muscular dystrophy (BMD), which is distinguished from DMD by its delayed onset, later need for wheelchair support, and relatively long life span. BMD usually corresponds to reading frame-preserving mutations (Muntoni F et al., Lancet Neurol, 2003. 2(12): 731-40).

[0005] There is no available care or effective treatment for DMD (Rodino-Klapac, LR, et al., Curr Neurol Neurosci Rep, 2013. 13(3): 332) or BMD. Conventional therapies are limited to supportive care, which partially alleviate signs and symptoms but do not directly target disease mechanisms or reverse the phenotype.

[0006] Currently, there are several therapeutic strategies under development for DMD, such as in vivo gene therapy, cell transplantation therapy, pharmacological rescue of DMD nonsense mutations and exon skipping, or gene editing strategies to restore the reading frame of the dystrophin gene. All of these strategies have problems to overcome, such as efficiency, targeting different muscle groups, optimizing delivery, long-term expression of the transgene, and possible immune responses (Jarmin et al., Expert Opin Biol Ther, 2014. 14(2): 209-30).

[0007] Different gene transfer approaches for DMD aim to compensate for the functional deficiency of dystrophin, offering the possibility of treating all patients using a single drug therapy. Approximately 30% of the normal levels of dystrophin protein are likely required to prevent muscle degeneration.

[0008] The dystrophin gene is the largest known gene in the human genome, spanning over 2.5 Mb in humans, or about 2% of the entire X chromosome. It consists of 79 exons (full length cDNA: 11,1 kb) that encode the 427 kD dystrophin protein of 3685 amino acids. The dystrophin protein is defined by four structural regions (Figure 1A). These are the NH 2 These are the actin-binding domains at the termini (exons 1–8), the central rod domain (24 spectrin-like repeats R1–24 and four hinge regions H1–4; exons 9–62), the cysteine-rich (CR) domain (exons 63–69), and the carboxy-terminal (CT) domain (exons 70–79).

[0009] The size of the cDNA is too large to fit into known gene therapy vector systems, especially the adeno-associated virus (AAV) vector, which is one of the promising candidates for efficient gene transfer into various muscle groups depending on the tropism of the AAV serotype. AAV vectors have the potential to show long-term gene transduction in both dividing (myofibrous and cardiomyocytes) and non-dividing (mature myotube) muscle cells.

[0010] Indeed, a major limitation of AAV is that its cargo capacity appears to be restricted to the size of the parental viral genome, approximately 5 kb (Wu Z. et al., Mol Ther., 2010, 18(1): 80-86; Lai Y. et al., Mol Ther., 2010, 18(1): 75-79; Wang Y. et al., Hum Gene Ther Methods, 2012, 23(4): 225-33). Larger vector genomes have resulted in truncated packaged genomes, a heterogeneous population of genomes with a broad size distribution, and lower expression efficiency (Wu Z. et al., Mol Ther., 2010, 18(1): 80-86).

[0011] To overcome the DNA packaging limitations of AAV (<5 kb), several research groups have attempted to engineer synthetic truncated but functional dystrophins (MDs, also known as "micro-dystrophins" or "mini-dystrophins"). A series of micro-dystrophins have been designed to encode truncated dystrophins optimized to contain more clinically relevant regions of the protein. Such regions have generally been believed to reside within the N-terminus and cysteine-rich domains of dystrophin.

[0012] Microdystrophin, which contains the first three and the last of the 24 spectrin-like repeats without the C-terminal domain (ΔR4-R23 / ΔCT), designated MD1 (see Figure 1B), exhibits high functional activity in restoring dystrophin and colocalizing syntrophin and dystrobrevin, but fails to recruit nNOS at the sarcolemma in the mdx mouse model (Yue et al., Mol Ther, 2006. 14(1): 79-87).

[0013] Studies using AAV2 / 8 vectors encoding sequence-optimized canine MD1 microdystrophin with expression driven by the muscle-specific spc512 promoter (AAV8-spc512-cMD1) in dystrophic CXMDj dogs (Koo et al., J. Gene Med. 2011.13:497) have proven promising. Localized regional delivery induces high levels of microdystrophin expression in the limb musculature and significant improvement of histological and functional parameters. Systemic intravenous administration in the absence of immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscle and reduces dystrophic symptoms for more than 2 years (Le Guiner et al., Nat Commun. 2017.8:16105). No toxicity or adverse immune consequences of vector administration have been observed.

[0014] However, the relevance of the deleted regions to F-actin in muscle function, e.g., the nNOS binding site R16-R17 and / or the Par1b binding site R8-R9 and / or the binding site R10-R17, remains in question.

[0015] As an alternative strategy, it has been proposed to use recombination events to produce quasi-dystrophin using a double AAV vector system.As known in the art, the two vectors of the double AAV system can be overlapping vectors, trans-splicing AAV vectors, or hybrid trans-splicing AAV vectors (see, for example, Pryadkina et al., Molecular Therapy, 2015, 2, 15009).

[0016] Based on this strategy, Kodippili et al. (Human Gene Therapy, 2018, 29 (3), 299-311) reported the expression of canine ΔH2-R15 mini-dystrophin (SEQ ID NO: 1) using a pair of double AAV9 vectors in a canine model of Duchenne muscular dystrophy (DMD), as illustrated in Figure 1C. More precisely, the first vector contains a cytomegalovirus (CMV) promoter, a flag tag fused to the N-terminus of the dystrophin gene, and the 5' half of the ΔH2-R15 mini-dystrophin gene (including the N-terminal domain, hinges 1 and 3, and part of the spectrin-like repeats R1-R3, R16-R20 and R21). The second vector contained the 3' half of the ΔH2-R15 mini-dystrophin gene (including part of hinge 3, spectrin-like repeats R20-R24, hinge 4, the cysteine-rich domain and the C-terminal domain), a GFP tag fused to the C-terminus and an SV40 polyadenylation signal. A 375 nucleotide dystrophin gene fragment (from the latter half of hinge 3 to the beginning of R21) was common to both vectors. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] WO2016 / 177911 [Patent Document 2] PCT / EP2019 / 053061 [Patent Document 3] US7,282,199 [Patent Document 4] WO2005 / 033321

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[0018]

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[0019] Regardless, there is still a need in the art to produce high levels of active quasidystrophin. [Means for solving the problem]

[0020] The present invention aims to alleviate or cure fatal Duchenne muscular dystrophy (DMD) as well as Becker muscular dystrophy (BMD) by expressing a shorter but functional dystrophin polypeptide called quasidystrophin using a dual AAV vector system.

[0021] To the best of the applicant's knowledge, this invention reports for the first time the genetic tools, i.e., a dual AAV vector system, that allow the production of large amounts of large, active quasi-dystrophin that displays both the R16-R17 nNOS-binding site and the R8-R9 Par1b-binding site, providing a new therapeutic tool for treating dystrophic diseases.

[0022] Over the last few years, it has been revealed that DMD pathology is indeed caused by muscle stem cell dysfunction, which impairs muscle regeneration and leads to muscle weakness, in addition to muscle fiber fragility. The impact of satellite cell dysfunction in DMD-associated muscle weakness is a relatively recent discovery, which could provide a tremendous improvement over current strategies based on AAV-dystrophin delivery by preserving the main binding sites and key molecular functions of dystrophin.

[0023] Par1b, also known as Marks, is a serine-threonine kinase that associates with dystrophin and ensures asymmetric division by regulating satellite cell polarity (Dumont et al., Nat Med, 2015, 21(12):1455-63). Lack of dystrophin leads to downregulation of Par1b and loss of asymmetric division, which is required for proper generation of myogenic precursors. Therefore, maintenance of Par1b binding sites (R8-R9) in quasi-dystrophin is important to ensure satellite cell polarity and asymmetric division for generation of myogenic precursors and efficient muscle regeneration.

[0024] It also appears important to preserve the binding site for nNOS (neuronal nitric oxide synthase), since dystrophin-mediated assembly of nNOS, a cytosolic transmembrane protein, is known to be required for NO production in the sarcolemma, a key event for controlling blood flow regulation in skeletal muscle (Ervasti, Biochim Biophys Acta, 2007, 1772(2):108-17).

[0025] definition The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0026] "About" or "approximately," as used herein when referring to a measurable value, e.g., an amount, a period over time, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the particular value, as variations are appropriate in performing the disclosed methods.

[0027] Ranges: Throughout this disclosure, various aspects of the invention can be described in the form of a range. It will be understood that the description in the form of a range is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to include all possible subranges specifically disclosed as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to include the specifically disclosed subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0028] "Isolated" means removed or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0029] In the context of the present invention, the following commonly occurring abbreviations for nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine and "U" refers to uridine.

[0030] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA or cDNA may also include introns to the extent that nucleotide sequences encoding proteins may contain introns in some forms.

[0031] "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers or macromolecules in biological processes, either with a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the resulting biological properties. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of a gene or cDNA, can be referred to as encoding a protein or other product of that gene or cDNA.

[0032] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes any nucleic acid sequence obtained by any means available in the art, including but not limited to recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques, PCR, etc., as well as by synthetic means.

[0033] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit as to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids joined together by peptide bonds. The term, as used herein, refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and longer chains, generally referred to in the art as proteins, of which there are many types. For example, "polypeptides" include biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide can be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0034] "Identical" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, then the molecules are homologous or identical at that position. The percentage of homology / identity between two sequences is a function of the number of matching positions common to the two sequences divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences are identical, then the two sequences are 60% identical. Generally, the comparison is made when the two sequences are aligned to obtain maximum homology / identity.

[0035] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid to cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0036] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0037] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, necessary to initiate the specific transcription of a polynucleotide sequence.

[0038] The term "promoter / regulatory sequence" as used herein means a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be the core promoter sequence, and in other instances, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0039] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of the gene product in a cell under most or all physiological conditions of the cell.

[0040] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the production of the gene product in a cell only if an inducer corresponding to the promoter is present in the cell.

[0041] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes production of a gene product in a cell preferentially if the cell is a cell of the tissue type corresponding to the promoter.

[0042] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal, or cells thereof, suitable for the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0043] A "disease" is a state of health in an animal where the animal is unable to maintain homeostasis and where the animal's health continues to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis but where the animal's health is less favorable than would be expected in the absence of the disorder. A disorder, if left untreated, does not necessarily cause a further deterioration of the animal's health.

[0044] A disease or disorder is "alleviated" or "ameliorated" if the severity of the symptoms of the disease or disorder, the frequency with which the patient experiences such symptoms, or both, are reduced. This also includes halting the progression of the disease or disorder. A disease or disorder is "cured" if the severity of the symptoms of the disease or disorder, the frequency with which the patient experiences such symptoms, or both, are eliminated.

[0045] A "therapeutic" treatment is a treatment administered to a subject who exhibits symptoms of a pathology, with the intent of reducing or eliminating such symptoms.

[0046] "Treating a disease or disorder," as used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject. Disease and disorder are used interchangeably herein in the context of treatment.

[0047] An "effective amount" of a compound is an amount of the compound sufficient to provide a beneficial effect to the subject to which the compound is administered. The phrase "therapeutically effective amount" as used herein refers to an amount sufficient or effective to prevent or treat (delay or prevent the onset, prevent, inhibit, reduce, or reverse) a disease or condition, as well as to alleviate the symptoms of such disease. An "effective amount" of a delivery vehicle is an amount sufficient to effectively bind to or deliver a compound. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] According to a first aspect, the present invention relates to quasi-dystrophin (QD), preferably functional quasi-dystrophin, more preferably of human origin.

[0049] In the scope of this application, quasi-dystrophin refers to a peptide or protein that is shorter than native or wild-type dystrophin. In the context of the present invention, the terms "micro-dystrophin", "mini-dystrophin" and "quasi-dystrophin" have the same meaning. Since the protein according to the present invention is larger than previously described micro-dystrophins, such as those described by Yue et al. (MD1), the term "quasi-dystrophin" will be used elsewhere in this application.

[0050] According to a specific embodiment, the quasi-dystrophin according to the present invention has a size of more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, or even more than 60% of the size of full-length dystrophin (for example, 3685 amino acids in the case of human type). According to a preferred embodiment, the quasi-dystrophin according to the present invention has a size of more than 61.1%, more than 62%, more than 63%, more than 64%, or even more than 65% of the size of full-length dystrophin. For human type, this means that according to the present invention, the quasi-dystrophin advantageously contains more than 2361 amino acids, and still advantageously contains more than 2400 amino acids, such as 2406 aa (WL2) or 2427 aa (WL1).

[0051] The structure of dystrophin is well documented (see FIG. 1A), and active fragments thereof have been disclosed. As would be understood in the art, an active fragment is one or more portions of the full-length sequence that retain at least some of the biological function of the full-length sequence.

[0052] "Functional" truncated dystrophin or quasi-dystrophin means that the corresponding peptide or protein is capable of exerting at least some of the functions of the wild-type dystrophin protein and further capable of at least partially ameliorating one or more of the symptoms associated with the absence of native dystrophin, in particular fibrous degeneration, inflammation, necrosis, replacement of muscle with scarring and fatty tissue, muscle weakness, respiratory and cardiac failure, as well as premature death.

[0053] The quasi-dystrophin according to the invention preferably exhibits (to a greater or lesser extent) at least one of the properties disclosed for micro-dystrophins in the prior art, in particular those disclosed by Yue et al. (Mol Ther, 2006. 14(1): 79-87) or Kodippili et al. (Human Gene Therapy, 2018, 29 (3), 299-311).

[0054] Preferred properties are, among others: - binding to at least one DAP ("dystrophin associated protein"), in particular to syntrophin, dystrobrevin, nNOS and / or PAR-1b proteins; - Recruitment of the DAP complex in the sarcolemma; - Rescue of the microtubule network; - Protects muscles from damage; - Conservation of the overall structure of the protein and the organization of the spectrin repeat (R) domains; - restoring muscle structure and function, most importantly skeletal muscles, but also cardiac muscles and the diaphragm; - More generally, improvement in muscle function, ambulation, cardiac function, respiratory function, survival, quality of life and / or expectation.

[0055] As known in the art, the properties may be tested in vitro on various cells expressing dystrophin, for example on iPS-derived human DMD myogenic cells, ex vivo on muscle fibers isolated from various animal models, or in vivo on animal model-based or patients suffering from DMD or BMD. Animal models include, for example, the mdx mouse (Foster H. et al., Mol Ther, 2008. 16(11):p.1825-32), the mdx 4cv Mouse (Decrouy et al., Gen Ther, 1997. 4(5): 401-8), D2.B10-mdx / J mouse (Coley et al., Human Molecular genetics, 2016. 25(1): 130-45), CXMDj dog (Koo et al., J Gene Med, 2011. 13(9): 497-506) or GRMD dog (Le Guiner et al., Mol Ther., 2014. 22(11): 1923-35). Mouse models are commonly used to test new constructs encoding microdystrophin. However, this model has drawbacks, since mice present a less severe form of the disease without an immune response. Another animal model is the dog, which is considered more reliable for predicting the therapeutic potential of gene therapy products in humans. Rat models such as that disclosed by Larcher et al. (Plos One, 2014, 9(10), e110371) are also of great interest as they present a cardiomyopathy.

[0056] As mentioned above, full-length dystrophin (Figure 1A) is characterized by distinct domains: - N-terminal domain that binds to actin (CH1CH2); - 4 hinge domains (H1-H4); - 24 spectrin-like repeats or rod domains (R1-R24); - Cysteine-rich (CR) domain; - C-terminal (CT) domain.

[0057] According to one embodiment, the quasi-dystrophin according to the invention has at least one domain which, compared to full-length dystrophin, advantageously lacks at least one spectrin-like repeat (R).

[0058] According to one aspect, the quasi-dystrophin of the present invention contains at least one major protein binding site, in particular for F-actin, nNOS protein and PAR-1b protein. Advantageously, the quasi-dystrophin of interest contains binding sites for nNOS and / or PAR1b. The binding site for nNOS has been shown to be present in repeats 16 and 17 (R16, R17) of the rod domain (Lai et al., J. Clin. Invest., 2009. 119:624-635), while the binding of dystrophin repeats 8 and 9 (R8, R9) to PAR1b has been demonstrated in vitro (Yamashita et al., Biochem. Biophys. Res. Commun., 2010. 391: 812-817). According to a preferred embodiment, the quasi-dystrophin of interest contains R16-17 and / or R8-9 rod domain.

[0059] According to this aspect, the invention relates to a quasidystrophin comprising the R8, R9, R16 and R17 rod domains.

[0060] According to another embodiment, the quasidystrophin of the invention is characterized by an N-terminal portion lacking or having the R4, R5, R6 and R7 rod domains removed (ΔR4-R7).

[0061] In other words, the quasi-dystrophin of the present invention has an N-terminal portion defined as follows, in the following order from its N-terminus: - N-terminal domain that binds to actin (CH1CH2); - H1 hinge domain; - the R1, R2 and R3 rod domains; - H2 hinge; - R8 and R9 rod domains.

[0062] Such quasi-dystrophin presents in its N-terminal part the structure CH1CH2H1R1R2R3H2R8R9.

[0063] According to a preferred embodiment, such a quasidystrophin further contains the R16 and R17 rod domains.

[0064] According to a particular embodiment, the quasi-dystrophin of the present invention contains at least the spectrin-like repeats of the prior art MD1 micro-dystrophin (FIG. 1B), namely R1-R2-R3 and R24.

[0065] Advantageously, the quasi-dystrophin according to the invention contains additional rod (R) domains compared to MD1 micro-dystrophin, advantageously at least those selected from the group consisting of R14, R15, R20, R21, R22 and R23. According to a preferred embodiment, the quasi-dystrophin further contains R22-R23, and optionally R14-R15 or R20-R21.

[0066] According to a specific embodiment, the quasidystrophin according to the invention lacks the following rod domain: - R4-R7, R10-R15 and R18-R19. In other words, such a quasidystrophin contains R1-R3, R8-R9, R16-R17 and R20-R24; or - R4-R7, R10-R13 and R18-R21. In other words, such a quasi-dystrophin contains R1-R3, R8-R9, R14-R17 and R22-R24.

[0067] Advantageously, the quasi-dystrophin according to the invention further comprises: - the complete N-terminal domain corresponding to amino acids 1 to 252 of SEQ ID NO: 2 or 3; - a complete cysteine-rich (CR) domain, corresponding to amino acids 1822 to 2102 of SEQ ID NO: 2 or SEQ ID NO: 20, respectively, or amino acids 1801 to 2081 of SEQ ID NO: 3; - a partial or full-length C-terminal domain, advantageously a full-length C-terminal domain corresponding to amino acids 2103 to 2427 of SEQ ID NO: 2 or SEQ ID NO: 20, respectively, or amino acids 2082 to 2406 of SEQ ID NO: 3. Possible partial C-terminal domains are truncated C-terminal domains of MD1, MD2, MD3 or MD4, such as those disclosed in WO2016 / 177911; - at least one hinge (H) domain, optionally selected from the group consisting of H1, H2, H3 and H4, advantageously from at least H1, H2 and H4, optionally selected from the group consisting of H1, H2, H3 and H4.

[0068] According to a specific embodiment, the quasi-dystrophin according to the invention comprises: - ΔR4-R7ΔR10-R15ΔR18-R19 quasidystrophin (further named WL1), advantageously the quasidystrophin of SEQ ID NO: 2; or - ΔR4-R7ΔR10-R13ΔR18-R21 quasidystrophin (further named WL2), advantageously the quasidystrophin of SEQ ID NO: 3 It is.

[0069] According to one preferred embodiment, the quasi-dystrophin according to the invention is ΔR4-R7ΔR10-R15ΔR18-R19 or ΔR4-R7ΔR10-R13ΔR18-R21 quasi-dystrophin. According to another preferred embodiment, the quasi-dystrophin according to the invention consists of or comprises the sequence of SEQ ID NO:2 or SEQ ID NO:3.

[0070] According to one embodiment, the quasidystrophin is "substantially identical" to the quasidystrophin disclosed therein, in particular to the quasidystrophin of the sequence of SEQ ID NO: 2 or SEQ ID NO: 3, i.e. about 60% identical thereto, preferably about 70% identical, more preferably about 80% identical, even more preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even more preferably about 99% identical thereto.

[0071] According to a specific embodiment, the ΔR4-R7ΔR10-R15ΔR18-R19 quasi-dystrophin according to the invention comprises the two following mutations in its coding sequence (compared to SEQ ID NO: 2): - serine instead of arginine at position 49 (R → S); and / or - Serine instead of phenylalanine at position 748 (F → S) It contains at least one of the following:

[0072] According to a specific embodiment, the ΔR4-R7ΔR10-R15ΔR18-R19 quasidystrophin of the present invention consists of or comprises SEQ ID NO:20.

[0073] According to a further aspect, the present invention relates to a nucleic acid sequence encoding quasi-dystrophin as defined above.

[0074] According to one embodiment, the nucleic acid sequence encoding quasisdystrophin according to the invention comprises or consists of SEQ ID NO:4.

[0075] According to another embodiment, the nucleic acid sequence encoding quasidystrophin according to the present invention is "substantially identical" to the sequence of SEQ ID NO: 4, i.e., about 60% identical thereto, preferably about 70% identical, more preferably about 80% identical, even more preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even more preferably about 99% identical thereto.

[0076] According to a preferred embodiment, the nucleic acid sequence according to the invention comprises or consists of SEQ ID NO:5 or SEQ ID NO:22.

[0077] According to another embodiment, the nucleic acid sequence encoding quasidystrophin according to the invention comprises or consists of SEQ ID NO:21.

[0078] According to another embodiment, the nucleic acid sequence encoding quasidystrophin according to the present invention is "substantially identical" to the sequence of SEQ ID NO: 21, i.e., about 60% identical thereto, preferably about 70% identical, more preferably about 80% identical, even more preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even more preferably about 99% identical thereto.

[0079] According to a preferred embodiment, the quasi-dystrophin of the present invention is produced using the so-called double AAV vector system.

[0080] The expression system according to the present invention is typically composed of two AAV vectors. After recombination based on the overlapping region common to the two AAV vectors in vivo, the resulting quasi-dystrophin is produced in large quantities. Therefore, the nucleic acid sequence encoding quasi-dystrophin is divided into two parts, namely, the 5' sequence encoding its N-terminal part and the 3' sequence encoding its C-terminal part. The 3' end of the 5' sequence and the 5' end of the 3' sequence are homologous or even identical so that homologous recombination can occur.

[0081] According to a further aspect, the present invention provides a dual AAV vector system comprising two AAV vectors, the first AAV vector comprises, between the 5' and 3' AAV ITRs, a first nucleic acid sequence encoding an N-terminal portion of quasi-dystrophin; the second AAV vector comprises, between the 5' and 3' AAV ITRs, a second nucleic acid sequence encoding a C-terminal portion of quasi-dystrophin; The first and second nucleic acid sequences are related to an AAV vector system, which comprises an overlapping region that allows for the production of the quasi-dystrophin of the present invention by recombination.

[0082] Within the scope of this application, as explained below, the terms "overlap region" (or "overlap sequence") and "region of homology" (or "region of sequence homology") have the same meaning and are used interchangeably.

[0083] In other words, the present invention relates to a composition comprising a recombinant adeno-associated virus (AAV) vector, preferably a total of two AAV vectors, carrying a complementary construct that allows expression of a functional quasi-dystrophin of the present invention.

[0084] In the scope of this application, the term "composition" can be replaced by "association", "combination" or "expression system". This means that the two AAV vectors must function together and be in contact so that homologous recombination can occur and then produce an active protein. However, they can be found in a single composition or in two separate compositions that are optionally mixed before use.

[0085] According to a specific embodiment, the first adeno-associated viral (AAV) vector comprises: i) the 5'ITR (inverted terminal repeat) sequence of AAV; ii) the portion of the gene controlled by the promoter; iii) AAV 3'ITR sequence Includes.

[0086] In addition, the second adeno-associated virus (AAV) vector iv) the 5'ITR (inverted terminal repeat) sequence of AAV; v) a gene portion, advantageously followed by a polyadenylation signal; vi) AAV 3'ITR sequence Includes.

[0087] These two AAV vectors have complementary sequences that will form a functional unit upon recombination. As known to those skilled in the art, recombination occurs through recognition of homologous sequences present in each of the AAV vectors by the cellular DNA repair pathways.

[0088] Therefore, the genetic parts of the two AAV vectors meet the following requirements: - the genetic portions of the first and second AAV vectors together comprise an open reading frame encoding a quasi-dystrophin of the invention, preferably a quasi-dystrophin of human origin; - both gene portions of the first and second AAV vectors contain regions of homology that allow the reconstitution of said open reading frame after homologous recombination; must be met.

[0089] "Dual AAV system" refers to a vector system composed of two AAV vectors, in which each vector carries a part of the sequence encoding the quasi-dystrophin of the present invention to be delivered to a cell, and the open reading frame (ORF) encoding said quasi-dystrophin is reconstituted in the cell by the interaction between the first and second nucleic acid sequences. According to the present invention, the dual vector system of the present invention implements vectors that contain sequences that allow (homologous) recombination, i.e. overlapping vectors. Therefore, the quasi-dystrophin protein is reconstituted by implementing homologous recombination by adding appropriate overlapping regions to each of the parts of the dystrophin gene introduced in each AAV vector.

[0090] According to the invention, each adeno-associated virus (AAV) vector of the system comprises an expression construct, also called an "expression cassette" or "insert". In the context of this application, said "insert" is advantageously defined as a nucleic acid sequence located between the 5' and 3' ITR ("inverted terminal repeat") sequences of the AAV genome.

[0091] According to the general knowledge in the art, the size of the insert should not be significantly larger than the length of the wild-type AAV genome.For example, AAV2 contains two ITR sequences of 145bp each, and has a genome (including ITR sequences) of 4682pb.

[0092] In certain embodiments, the nucleic acid sequence encoding a portion of quasidystrophin introduced into each AAV vector has a length of less than 5 kb, e.g., less than 4.9 kb, less than 4.8 kb, less than 4.7 kb, less than 4.6 kb, or less than 4.5 kb.

[0093] Advantageously, in order to limit the size constraints of AAV packaging, said nucleic acid sequences correspond to exons, in other words, they are preferably cDNA fragments.

[0094] Advantageously, the reading frame formed by combining the two AAVs encodes quasi-dystrophin (MD), advantageously functional quasi-dystrophin, more advantageously of human origin, as disclosed above.

[0095] According to a preferred embodiment, the quasi-dystrophin produced using the claimed dual AAV vector system contains at least 2000 amino acids (aa), advantageously at least 2100 aa, 2200 aa, 2300 aa, 2400 aa or 2500 aa. According to another embodiment, the micro-dystrophin produced using the claimed AAV vector contains no more than 3000 amino acids (aa), advantageously no more than 2900 aa, 2800 aa, 2700 aa, 2600 aa, 2550 aa or 2500 aa. In a preferred embodiment, the quasi-dystrophin produced using the claimed dual AAV vector system contains between 2400 and 2500 aa, i.e. has a size corresponding to about 70% of the size of full-length dystrophin.

[0096] In the dual vector system according to the invention, the first and second nucleic acid sequences present regions of sequence homology to promote homologous recombination between the molecules, thus generating a large quasi-dystrophin transgene by recombining the two vectors of the dual AAV system, i.e. the nucleic acid sequences encoding the N-terminal and C-terminal parts of quasi-dystrophin, respectively. In a particular embodiment implementing this overlap system, the length of the region of sequence homology can be varied widely, as long as the size of the resulting insert (including the 5'- and 3'-ITR sequences, and any expression control sequences) is compatible with the size constraints for encapsidation in the AAV vector. Those skilled in the art are well aware of these size constraints and can adapt the size of both the first and second nucleic acid sequences, and therefore the region of sequence homology, according to this knowledge. Thus, in a particular embodiment, the region of sequence homology is a polynucleotide sequence of the dystrophin gene having a length of less than 4599 nucleotides, such as less than 4500, 4000, 3500, 3000, 2500, 2000, 1500 or 1000 nucleotides. In another particular embodiment, the region of sequence homology is a polynucleotide sequence of the quasi-dystrophin sequence having a length of at least 100 nucleotides, such as at least 100, 200, 300, 400, 500, 600, 700 or 800 nucleotides. In a further particular embodiment, the region of sequence homology is a polynucleotide sequence of the dystrophin gene having a length of between 100 and 1000 nucleotides, in particular between 500 and 1000 nucleotides, such as between 700 and 900 nucleotides, advantageously between 750 and 850 nucleotides.

[0097] According to another embodiment, the region of homology contains at least one complete spectrin-like domain (R), preferably two complete spectrin-like domains (R), and optionally further comprises two shortened hinge domains (H), optionally one hinge domain (H). They correspond to the consecutive domains in native dystrophin, preferably. They are located in the middle part of native dystrophin, for example between R14 and R21.

[0098] According to a specific embodiment, the overlap region comprises: - abbreviated R16, R17, H3 and abbreviated R20, or - shortened R14, R15, R16 and shortened R17; or - Shortened H3, R20 and R21 corresponds to the sequence encoding

[0099] In one particular embodiment, the first nucleic acid sequence may encode the N-terminal domains of dystrophin, H1, R1-R3, H2, R8, R9, R16, R17, H3 and a truncated R20, and the second nucleic acid sequence encodes the truncated R16, R17, H3, R20-R24, H4, CR domains and the C-terminal domain of dystrophin (full length or truncated as disclosed above, advantageously full length).

[0100] According to a specific embodiment, the first nucleic acid sequence comprises or consists of SEQ ID NO: 6 or SEQ ID NO: 23, and the second nucleic acid sequence comprises or consists of SEQ ID NO: 7. The 800 bp overlap region of the sequence of SEQ ID NO: 12 corresponds to nucleotides 3779 to 4578 of SEQ ID NO: 6 (or SEQ ID NO: 23) and nucleotides 173 to 972 of SEQ ID NO: 7.

[0101] According to another embodiment, the first nucleic acid sequence comprises nucleotides 646 to 4578 of SEQ ID NO:6 (or SEQ ID NO:23) and the second nucleic acid sequence comprises nucleotides 173 to 4323 of SEQ ID NO:7.

[0102] According to a specific embodiment, the first nucleic acid sequence comprises or consists of SEQ ID NO: 8 and the second nucleic acid sequence comprises or consists of SEQ ID NO: 9. The 800 bp overlap region corresponds to nucleotides 3798 to 4597 of SEQ ID NO: 8 and nucleotides 193 to 992 of SEQ ID NO: 9. According to a further embodiment, the 800 bp overlap region has the sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0103] According to another embodiment, the first nucleic acid sequence comprises nucleotides 665 to 4597 of SEQ ID NO:8 and the second nucleic acid sequence comprises nucleotides 193 to 4343 of SEQ ID NO:9.

[0104] In other particular embodiments, the first nucleic acid sequence may encode the N-terminal domains of dystrophin, H1, R1-R3, H2, R8, R9, R14, R15, R16 and a truncated R17, and the second nucleic acid sequence encodes the truncated R14, R15, R16, R17, R22-R24, H4, CR domains and the C-terminal domain of dystrophin (full length or truncated as disclosed above, advantageously full length).

[0105] According to a specific embodiment, the first nucleic acid sequence comprises or consists of SEQ ID NO: 13 and the second nucleic acid sequence comprises or consists of SEQ ID NO: 14. The 800 bp overlap region corresponds to nucleotides 3701 to 4500 of SEQ ID NO: 13 and nucleotides 182 to 981 of SEQ ID NO: 14. According to a further embodiment, the 800 bp overlap region has the sequence of SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.

[0106] According to another embodiment, the first nucleic acid sequence comprises nucleotides 646 to 4500 of SEQ ID NO:13 and the second nucleic acid sequence comprises nucleotides 182 to 4347 of SEQ ID NO:14.

[0107] In a further particular embodiment, the first nucleic acid sequence may encode the N-terminal domains of dystrophin, H1, R1-R3, R16, R17, R18, R19, H3, R20 and R21, and the second nucleic acid sequence encodes the truncated H3, R20-R24, H4, CR domains and the C-terminal domain of dystrophin (full length or truncated as disclosed above, advantageously full length).

[0108] According to a specific embodiment, the first nucleic acid sequence comprises or consists of SEQ ID NO: 18 and the second nucleic acid sequence comprises or consists of SEQ ID NO: 19. The 375 bp overlap region corresponds to nucleotides 4091 to 4465 of SEQ ID NO: 18 and nucleotides 193 to 567 of SEQ ID NO: 19.

[0109] The nucleic acid sequence encoding quasidystrophin is preferably of human origin, but may also be a dog, rat, mouse or non-human primate sequence. In one embodiment, the nucleic acid sequence originates from the organism to which it will be administered, and is preferably a human sequence for administration to humans.

[0110] According to the present invention, the regions having sequence homology, i.e. overlapping regions, may be sequences optimized for recombination, such as the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, which are optimized from the corresponding wild-type sequence (nucleotides 193 to 992 of SEQ ID NO: 9), or the sequences shown in SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, which are optimized from the corresponding wild-type sequence (nucleotides 182 to 981 of SEQ ID NO: 14).

[0111] In a known manner, there are various methods to optimize protein-encoding sequences to increase mRNA levels (recombination and transcription) and / or protein levels (translation). Within the scope of the present application, sequences in the vectors of the dual AAV vector system of the present invention are advantageously optimized to increase recombination and, in some cases, to increase expression of the quasi-dystrophin polypeptide in vivo.

[0112] In implementation, the following sequence: - a first nucleic acid sequence encoding an N-terminal portion of dystrophin; - a second nucleic acid sequence encoding a C-terminal portion of dystrophin; and / or - a region of overlap between the first and second nucleic acid sequences may be optimized.

[0113] In certain embodiments, both the non-overlapping sequences and recombination regions of dystrophin are optimized. Sequence optimization can include several changes in the nucleic acid sequence, including codon optimization, increasing GC content, reducing the number of CpG islands, reducing the number of alternative open reading frames (ARFs), and / or reducing the number of splice donor and splice acceptor sites. Due to the degeneracy of the genetic code, different nucleic acid molecules may code for the same protein. It is also well known that the genetic codes of different organisms are often biased towards using one of several codons that code for the same amino acid over others. Through codon optimization, changes are introduced into the nucleotide sequence that take advantage of the codon bias present in a given cellular environment, so that the resulting codon-optimized nucleotide sequence is more likely to be expressed in such a given cellular environment at a relatively high level compared to a non-codon-optimized sequence. In a preferred embodiment of the present invention, such a sequence-optimized nucleotide sequence encoding a functional quasi-dystrophin is codon-optimized, for example by taking advantage of human-specific codon frequency bias, so that its expression and stability in human cells is improved compared to a non-codon-optimized nucleotide sequence encoding the same protein. In certain embodiments, the entire sequence of quasi-dystrophin, i.e., the non-overlapping and overlapping regions, is optimized to improve its production by the target or host cell, preferably in humans.

[0114] According to another aspect, the present invention also relates to an AAV vector corresponding to the first AAV vector or the second AAV vector of the dual AAV vector system according to the present invention.

[0115] Each AAV vector advantageously contains not only nucleic acid sequences encoding the relevant parts of the dystrophin gene (respectively the N-terminal and C-terminal parts) as defined above, but also all sequences necessary for the proper expression of said quasi-dystrophin after recombination and reconstruction of the entire gene.

[0116] According to one embodiment, the first and second nucleic acid sequences of the first and second AAV vectors are under the control of regulatory sequences. Advantageously, the first nucleic acid sequence is preceded by a promoter, optionally followed by an intron, and the second nucleic acid sequence is preceded by a polyadenylation signal.

[0117] Such promoters may be natural or synthetic (artificial) promoters, and may be inducible or constitutive.

[0118] In one embodiment, the promoter is a ubiquitous promoter or has low tissue specificity. By way of example, the expression vector may contain the promoters of phosphoglycerate kinase 1 (PGK), EF1, ACTA1, β-actin, desmin, all MCK variants, cardiac troponin and CMV.

[0119] In a preferred embodiment, the promoter sequence is selected to appropriately govern the expression of the nucleic acid sequence placed under its control, both in terms of expression level and also in terms of tissue specificity.

[0120] In one embodiment, the expression vector comprises a muscle-specific promoter. Such a promoter allows robust expression in skeletal muscle, diaphragm, possibly cardiac muscle, as well as satellite cells. Examples of suitable promoters known to those skilled in the art are, for example, the desmin promoter, the muscle creatine kinase (MCK) promoter, truncated creatine kinase promoters, such as the CK6, CK7 or CK8 promoters, the Syn promoter, the MyoD, Myf5, Vcam, Pax3 and Pax7 satellite cell promoters. Another promoter is the synthetic promoter C5-12 (spC5-12). It is also possible to use hybrid promoters that contain sequences from two or more transcriptional regulatory elements (see, for example, PCT / EP2019 / 053061).

[0121] Advantageously, the first nucleic acid sequence is under the control of a muscle-specific promoter, in other words, the first AAV vector further comprises a muscle-specific promoter operably linked to the nucleic acid sequence encoding the N-terminal portion of dystrophin.

[0122] As known in the art, a non-limiting list of other possible regulatory sequences that can be introduced into one or the other AAV vector includes: - a polyadenylation signal, preferably 3' to the sequence coding for the functional microdystrophin; - sequences for transcription stabilization, e.g. introns; - enhancer sequences; - miRNA target sequences capable of inhibiting expression of a functional dystrophin coding sequence in non-target tissues where expression of said sequence is not desired, for example where this may be toxic It is.

[0123] According to one embodiment, the intron is selected from the group consisting of a human beta globin b2 (or HBB2) intron, a FIX intron and a chicken beta-globin intron, said intron being optionally a modified intron, such as a modified HBB2 intron, a modified FIX intron, or a modified chicken beta-globin intron.

[0124] According to another embodiment, the polyadenylation signal is selected from the human beta globin polyadenylation signal, the bovine growth hormone polyadenylation signal, the SV40 polyadenylation (pA) signal, or another naturally occurring or artificial polyadenylation signal.

[0125] According to a specific embodiment, the dual AAV vector system according to the invention comprises the following elements: - the spC5-12 promoter, advantageously corresponding to nucleotides 173 to 506 of SEQ ID NO: 6 (or SEQ ID NO: 23); a chimeric intron, advantageously corresponding to nucleotides 507 to 639 of SEQ ID NO: 6 (or SEQ ID NO: 23), and - the SV40pA signal, advantageously corresponding to nucleotides 4324 to 4545 of SEQ ID NO: 7 The compound preferably contains at least one of, and preferably all of, the following:

[0126] For cloning purposes and for production of viral particles, the expression construct can be inserted into a plasmid suitable for selection, replication and production of quasi-dystrophin.

[0127] According to the present invention, the viral vector containing the expression construct is an adeno-associated viral (AAV) vector.

[0128] Adeno-associated virus (AAV) vectors are becoming a powerful gene delivery tool for the treatment of various disorders. AAV vectors have many characteristics that make them ideally suited for gene therapy, such as lack of pathogenicity, mild immunogenicity, and the ability to transduce postmitotic cells and tissues in a stable and efficient manner. The expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by selecting the appropriate combination of AAV serotype, promoter, and delivery method.

[0129] In one embodiment, the coding sequence is contained in an AAV vector. More than 100 naturally occurring AAV serotypes are known. Many natural variants in AAV capsid exist, allowing the identification and use of AAV with properties that are particularly suitable for dystrophic pathology. AAV viruses can be engineered using conventional molecular biology techniques, allowing these particles to be optimized for cell-specific delivery of nucleic acid sequences, minimizing immunogenicity, adjusting stability and particle lifespan, efficient degradation, and precise delivery to the nucleus.

[0130] As mentioned above, the use of AAV is a common mode of exogenous delivery of DNA because AAV is relatively non-toxic, provides efficient gene transfer, and can be easily optimized for specific purposes. Among the well-characterized serotypes of AAV isolated from humans or non-human primates (NHPs), human serotype 2 is the first AAV developed as a gene transfer vector. Other currently used AAV serotypes include AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In addition, non-natural engineered variants and chimeric or hybrid AAVs can also be useful.

[0131] Desired AAV fragments for assembly into vectors include the cap proteins, including vp1, vp2, vp3 and hypervariable regions, the rep proteins, including rep78, rep68, rep52, and rep40, and the sequences encoding these proteins. These fragments can be readily utilized in a variety of vector systems and host cells.

[0132] Such fragments can be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, but are not limited to, AAVs with non-naturally occurring capsid proteins. Such artificial capsids can be generated by any suitable technique using selected AAV sequences (e.g., fragments of vp1 capsid protein) in combination with heterologous sequences that can be obtained from different selected AAV serotypes, non-contiguous parts of the same AAV serotype, non-AAV viral sources, or non-viral sources. Artificial AAV serotypes can be, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Thus, exemplary AAVs, or engineered AAVs, include AAV2 / 8 (US 7,282,199), AAV2 / 5 (available from the National Institutes of Health), AAV2 / 9 (WO 2005 / 033321), AAV2 / 6 (US 6,156,303), AAVrh8 (WO 2003 / 042397), and rh74-AAV9 (EP 18305399.0), among others. In one embodiment, vectors useful in the compositions and methods described herein contain, at a minimum, sequences encoding a selected AAV serotype capsid, e.g., AAV8 capsid, or a fragment thereof. In another embodiment, useful vectors contain, at a minimum, sequences encoding a selected AAV serotype rep protein, e.g., AAV8 rep protein, or a fragment thereof. Optionally, such vectors may contain both AAV cap and rep proteins. In vectors in which both AAV rep and cap are provided, the AAV rep and AAV cap sequences may both be of one serotype origin, for example all of AAV8 origin. Alternatively, vectors may be used in which the rep sequence is from a different AAV serotype than that providing the cap sequence.In one embodiment, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or a host cell and a vector). In another embodiment, the rep sequences are fused in frame to the cap sequence of a different AAV serotype to form a chimeric AAV vector, e.g., AAV2 / 8 (US 7,282,199).

[0133] According to one embodiment, each of the first and second AAV vectors comprises a capsid derived from an AAV, such as AAV1, AAV2, variant AAV2, AAV3, variant AAV3, AAV3B, variant AAV3B, AAV4, AAV5, AAV6, variant AAV6, AAV7, AAV8, AAV9, AAV2G9, AAV10, such as AAVcy10 and AAVrh10, AAVrh74, AAVdj, AAV-Anc 80, AAV-LK03, AAV2i8, and porcine AAV, such as AAVpo4 and AAVpo6 capsids, or AAV vectors having chimeric capsids, particularly AAV8, AAV9, AAVrh74, AAV2i8 capsids or AAV9-rh74 chimeric capsids, particularly AAV8, AAV9 or AAVrh74 capsids, more particularly AAV8 or AAV9 capsids.

[0134] In the AAV vector used in the present invention, the AAV genome is advantageously a single-stranded (ss) nucleic acid.

[0135] As known in the art, recombinant viral particles can be obtained, for example, by triple transfection of 293HEK cells, the herpes simplex virus system and the baculovirus system, or using specific cell lines. Advantageously, the viral particles are obtained by triple transfection of 293HEK cells.

[0136] Vector titers are usually expressed as viral genomes / ml (vg / ml).

[0137] The present invention also relates to cells, in particular muscle cells, transduced with the above disclosed dual AAV vector system.

[0138] According to another aspect, the present invention relates to a composition, advantageously a therapeutic composition or a medicament, comprising the above disclosed dual AAV vector system and optionally other active molecules (other gene therapy products, chemical molecules, peptides, proteins, etc.) intended for the treatment of the same or another disease.

[0139] The invention then provides pharmaceutical compositions comprising a dual AAV vector system or the first or second AAV vector of said system. Such compositions comprise a therapeutically effective amount of a therapeutic agent (nucleic acid or vector of the invention) and a pharmaceutically acceptable carrier. In specific embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States or European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals or humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.

[0140] The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if necessary. These compositions can take the form of solutions, suspensions, emulsions, sustained-release formulations, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions will contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the subject.

[0141] In a preferred embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition adapted for intravenous administration to humans.Typically, the composition for intravenous administration is a solution in sterile isotonic aqueous buffer.If necessary, the composition may also contain a solubilizing agent and a local anesthetic, such as lidocaine, to relieve pain at the injection site.

[0142] In one embodiment, the composition according to the invention is suitable for administration in humans. The composition is preferably in the form of a liquid, advantageously a saline composition, more advantageously a phosphate buffered saline (PBS) composition or lactated Ringer's solution.

[0143] The amount of the therapeutic agent (i.e., nucleic acid or vector) of the present invention that is expected to be effective in treating dystrophic disease can be determined by standard clinical techniques.In addition, in vivo and / or in vitro assays can be optionally employed to help predict optimal dosage ranges.The exact dose to be employed in the formulation will also depend on the route of administration, body weight and severity of disease, and should be determined according to the professional judgment and circumstances of each patient.

[0144] The AAV vector or composition disclosed above can be used as a medicament, in particular as a gene therapy product, to be administered to a subject in need thereof. In another aspect, the present invention relates to the use of the AAV vector or composition disclosed above for the preparation of a medicament.

[0145] Suitable administration should allow delivery of a therapeutically effective amount of the gene therapy product to target tissues, particularly skeletal muscle, and possibly the diaphragm and heart. In the context of the present invention, when the gene therapy product is a viral vector containing a nucleic acid sequence encoding quasi-dystrophin, the therapeutic dose is defined as the amount of viral particles (vg relative to the viral genome) containing the quasi-dystrophin sequence administered per kilogram (kg) of subject.

[0146] Available routes of administration are topical (local), enteral (systemic action, but delivered via the gastrointestinal (GI) tract), or parenteral (systemic action, but delivered by a route other than the gastrointestinal tract). A preferred route of administration of the compositions disclosed herein is parenteral, examples of which include intramuscular administration (i.e., into the muscle) and systemic administration (i.e., into the circulatory system). In this context, the term "injection" (or "perfusion" or "infusion") includes intravascular, specifically intravenous (IV), and intramuscular (IM) administration. Injections are usually performed using a syringe or catheter.

[0147] In one embodiment, systemic delivery of the composition involves administering the composition near the local treatment site, i.e., into a vein or artery in the immediate vicinity of the weakened muscle. In certain embodiments, the invention involves local delivery of the composition to produce a systemic effect. This route of administration is commonly referred to as "local (locoregional) infusion," "administration by isolated limb perfusion," or "high pressure intravenous limb perfusion," and has been used successfully as a method of gene delivery in muscular dystrophies (Zheng Fan et al. (2012, Molecular Therapy 20(2), 456-461).

[0148] The preferred method of administration according to the present invention is systemic administration. Systemic injection paves the way for injection throughout the body of the subject to reach all of the muscles of the subject's body, including the heart and diaphragm, and then for the actual treatment of these systemic, yet untreatable, diseases. In certain embodiments, systemic delivery includes delivering the composition to the subject so that the composition is available throughout the subject's entire body.

[0149] According to a preferred embodiment, systemic administration is via injection of the composition into a blood vessel, i.e., intravascular (intravenous or intraarterial) administration, including intrauterine administration. According to one embodiment, the composition is administered via a peripheral vein by intravenous injection. Alternatively, systemic administration is via intramuscular injection.

[0150] When delivered systemically, compositions containing the dual AAV vector system of the invention are preferably administered in doses of 10 15 at a dose of less than or equal to 10 vg / kg, or even 14 vg / kg, advantageously 10 12 vg / kg~10 14 vg / kg, more preferably 5×10 12 vg / kg~10 14 vg / kg, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 x 10 13 vg / kg, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 × 10 12 Doses of 1000 mg / kg or even lower may be envisaged to avoid possible toxicity and / or immune reactions, and as known to those skilled in the art, the lowest possible dose is preferred, taking into account satisfactory results in terms of efficacy.

[0151] In a specific embodiment, the treatment comprises a single administration of the composition.

[0152] In one embodiment, the presence of the AAV vector and / or expression of quasidystrophin, as well as the associated therapeutic benefits, are observed for up to one month, or up to three or six months, or even one, two, five, ten or even longer, over the course of the subject's lifetime.

[0153] In accordance with the present invention, the subject is preferably a human, but may also be a mouse, rat, non-human primate, or dog.

[0154] "Dystrophic disease" in the context of the present invention means a disease associated with a defect in the dystrophin gene. This defect can be a deletion or mutation that causes low or no expression, the introduction of a premature stop codon in the open reading frame, or the production of an inactive protein. A preferred dystrophic disease is Duchenne and Becker muscular dystrophy (DMD / BMD), which is caused by a mutation in the dystrophin gene. The mutation can result in the absence or low level of dystrophin expression, or the production of a partially or completely inactive, possibly truncated, protein.

[0155] The subjects who may benefit from the composition of the present invention include all patients who have been diagnosed with muscular dystrophy or are at risk of developing such muscular dystrophy.Subsequently, the subjects to be treated can be selected based on the identification of mutations or deletions in the dystrophin gene by any method known to those skilled in the art, such as sequencing the dystrophin gene, and / or through the evaluation of the expression or activity level of dystrophin by any method known to those skilled in the art.Therefore, said subjects include both subjects who already exhibit symptoms of dystrophic disease and subjects who are at risk of developing said disease.In one embodiment, said subjects include subjects who already exhibit symptoms of dystrophic disease.In another embodiment, said subjects are outpatients and early non-ambulatory patients.

[0156] According to one embodiment, the present invention relates to a dual AAV vector system as disclosed above or a composition comprising said AAV vector for use in the treatment of a dystrophic disease. According to another embodiment, the present invention relates to the use of an AAV vector as disclosed above or a composition comprising said AAV vector for the preparation of a medicament for the treatment of a dystrophic disease.

[0157] In other words, the present invention provides a method for treating a dystrophic disease in a subject, comprising administering to the subject the above disclosed dual AAV vector system or a composition comprising said system.

[0158] Such a dual AAV vector system and compositions comprising said system are intended in particular for gene therapy, in particular for the treatment of subjects suffering from Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). A first goal is to provide a safe (non-toxic) treatment. A further objective is to provide an efficient treatment that makes it possible to postpone, slow down or prevent the onset of the disease, possibly improving the phenotype of the patient, which can be easily monitored at a clinical level. In a subject, the AAV vectors and compositions according to the invention are - can be used to improve the function of muscles, most importantly the skeletal muscles, but also the cardiac muscles and the diaphragm; - Can be used to improve gait; - Can be used to improve heart function; - Can be used to improve respiratory function; - It can be used to extend survival and, more generally, to improve quality and expectations of life.

[0159] The practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the understanding of those skilled in the art, unless otherwise specified. Such techniques are fully described in the literature, such as, for example, "Molecular Cloning: A Laboratory Manual", 4th Edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting", (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and therefore may be considered in making and practicing the invention. Techniques that are particularly useful for certain embodiments will be discussed in the sections that follow.

[0160] The disclosures of any and all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entireties.

[0161] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. EXAMPLES

[0162] Experimental Example The present invention will now be described in more detail by reference to the following experimental examples and the accompanying drawings, which are provided merely for illustrative purposes and are not intended to be limiting. [Brief description of the drawings]

[0163] [Figure 1] 1 shows schemes of different forms of dystrophin: A / scheme of full-length dystrophin; B / scheme of MD1 microdystrophin disclosed by Yue et al.; C / scheme (DD) of ΔH2-R15 quasi-dystrophin (SEQ ID NO: 1) disclosed by Kodippili et al.; D / scheme (WL1) of ΔR4-R7ΔR10-R15ΔR18-R19 quasi-dystrophin (SEQ ID NO: 2) according to the present invention; E / scheme (WL2) of ΔR4-R7ΔR10-R13ΔR18-R21 quasi-dystrophin (SEQ ID NO: 3) according to the present invention. [Figure 2A] Figure 2 shows the analysis of the production levels of different forms of dystrophin. A / Western blotting with anti-dystrophin antibody. TA muscles of 3-month-old mdx mice were injected with 1x1010vg of AAV. TA muscles were harvested 30 days after injection and processed for Western blotting with an antibody against dystrophin (DysB). Panels 6-8 (Opt) show the levels of protein WL1 (280 kDa) obtained with the dual AAV vector system according to the invention compared with those obtained with AAV2 / 9-hMD1 (panel 9: 138 kDa), human control samples (FL: panel 2: 427 kDa) and mdx (KO: panels 3 and 4), respectively. Control muscles from age-matched mdx and C57BL / 10 were injected with saline only. [Figure 2B]Figure 1 shows the analysis of the production levels of different forms of dystrophin. B and C / 1-2 month old WT mice were injected with 1-3 x 1010 vg of AAV into the TA muscle. TA muscles were harvested 30 days after injection and processed for Western blotting with antibodies against dystrophin (DysB and Dys2) and α-actinin levels (α-actinin levels were used as normalization group). Comparison of protein levels expressed from different constructs: - WL1wt (expressed from SEQ ID NO:8+9): panels 1-3 in the upper blot; - WL2wt (expressed from SEQ ID NO:13+14: panels 4-6 in the upper blot; - WL1Opt (expressed from SEQ ID NO:6+7): panels 7-9 in the upper blot; - PBS: panel 10 in the upper blot; - DD (expressed from SEQ ID NO:18+19): panels 1-3 in the lower blot; - WL1Opt (expressed from SEQ ID NO:6+7): panels 4-6 in the lower blot; - PBS: panel 7 in the lower blot. [Figure 2C] Figure 1 shows the analysis of the production levels of different forms of dystrophin. B and C / 1-2 month old WT mice were injected with 1-3 x 1010 vg of AAV into the TA muscle. TA muscles were harvested 30 days after injection and processed for Western blotting with antibodies against dystrophin (DysB and Dys2) and α-actinin levels (α-actinin levels were used as normalization group). [Diagram 3]Figure 1: Evaluation of dystrophin expression and distribution in TA muscle. One-month-old Dba2_Mdx mice were intravenously injected with either: - 5'AAV vector harboring SEQ ID NO:6 and 3'AAV vector harboring SEQ ID NO:7 (7 mice were injected; n=3 representative mice for WB); - 5'AAV vector harboring only SEQ ID NO:6 (2 mice were injected; n=1 representative mice for WB); - 3'AAV vector harboring only SEQ ID NO:7 (2 mice were injected; n=1 representative mice for WB); - no vector, i.e. PBS (5 mice were injected; n=2 representative mice for WB). TA muscles were harvested 70 days after injection and processed for Western blotting or cryosectioning. A / Western blotting (WB) with anti-dystrophin antibodies (Dys N-ter and Dys C-ter) and α-actinin antibodies. Expected size = 280 kDa; full length dystrophin = 427 kDa. Immunolabeling in frozen sections with B / Dys N-ter antibody. The percentage of positive fibers is given in each figure. [Figure 4A] Figure 3: Evaluation of the therapeutic effect of dystrophin in TA muscles treated as in Figure 3. Staining of sections with Hematoxylin, Phloxine, Saffron (HPS). [Figure 4B-1] Figure 4: Evaluation of the therapeutic effect of dystrophin in TA muscles treated as in Figure 3. Expression levels of TMEM8C (top left). [Figure 4B-2] Figure 4 shows the evaluation of the therapeutic effect of dystrophin in TA muscles treated as in Figure 3. Expression levels of CD11b (top right). [Figure 4B-3] Evaluation of the therapeutic effect of dystrophin in TA muscles treated as in Figure 3. Fibronectin (bottom) expression levels. [Diagram 5] FIG. 4 shows the evaluation of dystrophin expression in the hearts of mice treated as in FIG. 3 by Western blotting with anti-dystrophin antibodies (Dys N-ter and Dys C-ter) and α-actinin antibodies. [Figure 6] Figure 1: Evaluation of dystrophin expression and distribution in the TA (tibialis anterior) and DIA (diaphragm). Dba2_Mdx mice were intravenously injected with either: - 5'AAV vector harboring SEQ ID NO: 6 and 3'AAV vector harboring SEQ ID NO: 7 (5'mut+3' or double Dys mut); 5 mice were injected; - 5'AAV vector harboring SEQ ID NO: 23 and 3'AAV vector harboring SEQ ID NO: 7 (5'cor+3' or double Dys cor); 5 mice were injected; For comparison, non-injected (NI) Dba2_Mdx mice (n=3) and Dba2_WT mice were included in the experiment. TA and DA muscles were harvested 3 weeks after injection and processed for Western blotting or cryosectioning. A / Western blotting (WB) with anti-dystrophin antibody (Dys2) and α-actinin antibody (used to normalize samples). Expected size = 280 kDa; full length dystrophin = 427 kDa; L = ladder; HL = high molecular weight ladder, T+ = positive control. B / Immunolabeling in frozen sections with Dys2 antibody. The percentage of positive fibers is given in each figure. [Figure 7] FIG. 7 shows evaluation of the therapeutic effect of dystrophin in muscles treated as in FIG. 6: expression levels of TMEM8C (top left), CD11b (top right) and fibronectin (bottom).

[0164] Materials and Methods In vivo gene transfer. Different mouse models were used in this study: wild type C57BL / 10 and C57BL / 6J; mdx and Dba_2Mdx (D2.B10-mdx / J). All mouse procedures were performed under appropriate biological containment according to protocols approved by the ethical committee at the CERFE of Evry animal facility. A protocol of three plasmid constructs was used to produce adeno-associated virus vectors. After sacrifice, tissues (TA muscle and heart) were collected, flash frozen in liquid nitrogen-cooled isopentane and stored at -80°C.

[0165] Western blot. Total protein was extracted from tissue samples. Protein extracts were separated by NuPAGE™ gel and then transferred to nitrocellulose membrane. Membranes were then blocked with Odyssey blocking buffer and PBS, and then hybridized with sufficient antibodies (anti-dystrophin DysB or Dys2 antibodies, rabbit alpha-actinin (Life Technologies)) and secondary anti-mouse or anti-rabbit-conjugated (680 or 800) antibodies.

[0166] Immunohistochemistry TA muscle cryosections were stained using the Mouse on Mouse (MOM) kit (Vector Labs). Primary antibodies were incubated overnight at 4°C, followed by three washes with PBS-0.1% tween and incubation with goat anti-mouse or goat anti-rabbit secondary antibodies Alexa 594 (Life Technologies). An antibody against dystrophin (Dys2, 1:100, mouse monoclonal, Novocastra) was used.

[0167] HPS staining. TA muscle cryosections were stained using hematoxylin (nuclear stain), phloxine (intracytoplasmic stain) and saffron (collagen).

[0168] PCR quantification of dystrophic profiles. Expression of TMEM8C (to measure fibrosis / TGE Mm00481256_m1, ThermoFisher), CD11b (to measure inflammation / TGE Mm00434455_m1) and fibronectin (to measure fibrosis / TGE Mm01256744_m1) was quantified in cDNA collected from frozen sections by RT-quantitative PCR (RTqPCR).

[0169] result: 1) Construction of AAV vectors Various recombinant AAV2 / 8 or AAV2 / 9 vectors were constructed to assess their relative efficiency for dystrophin production:

[0170] [Table 1]

[0171] 2) Analysis of dystrophin profiles following intramuscular (IM) delivery To evaluate the level of transduction and expression in muscle of proteins produced from the dual vector system according to the invention, an in vivo analysis of the vectors was performed. One-month-old dystrophin-deficient (mdx) mice were intramuscularly injected with 1e10vg of AAV vector. Tibialis anterior (TA) muscles were harvested 30 days after injection and processed with an antibody against dystrophin (DysB) for Western blotting (Figure 2A): Panels 6-8 (Opt) show the levels of protein WL1OPT (280 kDa) obtained with the dual AAV vector system according to the invention compared to those obtained with AAV2 / 9-hMD1 (Panel 9: 138 kDa), human dystrophin (FL: Panel 2: 427 kDa) and mdx (KO: Panels 3 and 4), respectively. Control muscles from age-matched mdx and C57BL / 10 were injected with saline only. As shown, these data confirm that truncated dystrophin according to the invention can be efficiently produced based on a dual AAV vector system.

[0172] Further experiments were performed to evaluate different dual AAV vector systems. One-month-old wt mice were intramuscularly injected with 1-3e10vg of AAV vectors. Tibialis anterior (TA) muscles were harvested 30 days after injection and processed for Western blotting with antibodies against dystrophin (Dys2 and DysB) and α-actinin. Figure 2B and C show the resulting quantification of protein levels after normalization with α-actinin using StudioLight software.

[0173] FIG. 2B shows results demonstrating that quasi-dystrophin according to the invention (WL1Opt) is produced at significantly higher levels than WL1wt and WL2wt, and at levels superior to those obtained with the prior art (DD) construct.

[0174] Therefore, further experiments were carried out using the constructs identified as most promising for functional evaluation.

[0175] 3) Analysis of dystrophin profiles after intravenous (IV) injection Using A / WL1Opt After IM analysis, WL1Opt was selected for intravascular (IV) injection. Four-week-old Dba_2Mdx mice were given the preparation by intravascular injection (5e11vg / mouse) (5'+3', n=7; 5' alone, n=2; 3' alone, n=2; PBS, n=5). Endpoint analysis was performed 70 days after gene transfer.

[0176] 3-1 In muscles After intravenous injection, quasi-dystrophin production based on the dual AAV vector system according to the present invention was confirmed (FIG. 3A).

[0177] Immunohistochemical analysis of TA muscle sections (FIG. 3B) showed labeling of dystrophin in the membrane, confirming suitable expression of quasi-dystrophin according to the invention, although there was some heterogeneity in expression levels between mice.

[0178] To evaluate the therapeutic effect of the quasi-dystrophin according to the present invention administered by intravenous injection, TA sections were stained with HPS, revealing the overall state of the tissue, especially inflammation, fibrosis and fibrosis.Compared to wt and mdx mice, the quasi-dystrophin-expressing mice of the present invention show an intermediate profile (Figure 4A).This observation was confirmed by measuring related markers (see Figure 4B).Improvements can be observed in the levels in treated animals between the levels observed in wt and mdx mice for each criterion.

[0179] 3-2 In the heart Besides its beneficial effects in muscle as reported above, the transgenic quasi-dystrophin according to the invention is also expressed in the hearts of injected mice (see FIG. 5).

[0180] Use of B / WL1Cor It was found that codon optimization of WL1 resulted in two mutations in the WL1 ORF (R49S and F748S). Therefore, the corresponding sequence in the 5' AAV vector (SEQ ID NO: 6) was modified (AGC at position 790 was changed to AGG, and TCT at position 2887 was changed to TTC).

[0181] The experiment was then repeated using both constructs: the new (cor) double Dys vector system (mutation corrected; 5'cor+3') corresponding to WL1Cor (sequence number 22) and the old (mut) double Dys vector system (5'mut+3', n=5) corresponding to WL1Opt (sequence number 5).

[0182] As shown in Figures 6A / 6B, adequate quasi-dystrophin production was observed with both dual AAV vector systems in the tibialis anterior and diaphragm muscles even at early stages (3 weeks) after intravenous injection.

[0183] Regarding the level of expression, the new (cor) double Dys vector system (mutation corrected; 5'cor+3'), an optimized version encoding the native quasi-dystrophin, appears to give promising results.

[0184] Regarding the therapeutic action of the quasi-dystrophin so produced, FIG. 7 confirms that both constructs result in a similar profile of treated mice, i.e. an improvement in relevant markers of regeneration, inflammation and fibrosis.

Claims

1. Quasidystrophin (QD), having the sequence of SEQ ID NO:2 or a sequence having at least 90% identity thereto.

2. The quasidystrophin of claim 1 having the sequence of SEQ ID NO:

20.

3. A quasi-dystrophin having the sequence of SEQ ID NO:3 or a sequence having at least 90% identity thereto.

4. A nucleic acid encoding the quasi-dystrophin of any one of claims 1 to 3.

5. 5. The nucleic acid according to claim 4, comprising or consisting of the sequence SEQ ID NO: 4 or a sequence having at least 70% identity thereto.

6. 6. The nucleic acid according to claim 5, comprising or consisting of the sequence of SEQ ID NO: 5 or SEQ ID NO:

22.

7. A dual AAV vector system comprising two AAV vectors, - the first AAV vector comprises, between the 5' and 3' AAV ITRs, a first nucleic acid sequence encoding an N-terminal portion of quasi-dystrophin; - the second AAV vector comprises, between the 5' and 3' AAV ITRs, a second nucleic acid sequence encoding a C-terminal portion of quasi-dystrophin; A dual AAV vector system, wherein the first and second nucleic acid sequences comprise an overlapping region that allows for recombinant production of the quasi-dystrophin of any one of claims 1 to 3.

8. The dual AAV vector system of claim 7, wherein the first nucleic acid sequence has the sequence of SEQ ID NO: 6 or SEQ ID NO: 23, and the second nucleic acid sequence has the sequence of SEQ ID NO:

7.

9. The dual AAV vector system of claim 7, wherein the first nucleic acid sequence has the sequence of SEQ ID NO:8 and the second nucleic acid sequence has the sequence of SEQ ID NO:

9.

10. A cell transduced with the dual AAV vector system of any one of claims 7 to 9.

11. The cell of claim 10, which is a muscle cell.

12. 12. A composition comprising a dual AAV vector system according to any one of claims 7 to 9 or a cell according to claim 10 or 11 in a pharma- ceutically acceptable carrier.

13. A medicament comprising the dual AAV vector system of any one of claims 7 to 9, a cell of claim 10 or 11, or a composition of claim 12.

14. A medicament for treating muscular dystrophy comprising a dual AAV vector system according to any one of claims 7 to 9, a cell according to claim 10 or 11 or a composition according to claim 12.

15. A medicament for treating Duchenne muscular dystrophy (DMD), comprising a dual AAV vector system according to any one of claims 7 to 9, a cell according to claim 10 or 11, or a composition according to claim 12.

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