Novel mididystrophins

Abstract

The present invention concerns mididystrophins comprising at least the rod domains R1, R8, R9, R10, R11, R12, R16, R17 and R24 of the dystrophin and their use for treating a muscular dystrophy, in particular a Duchenne muscular dystrophy (DMD).

Description

[0001] NOVEL MIDIDYSTROPHINS

[0002] The present invention relates to gene therapy vectors which are useful in the treatment or prevention of dystrophic diseases, especially Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).

[0003] The present application reports that the use of dual AAV vector systems allows the production of large amount of active truncated dystrophins displaying important functional domains.

[0004] BACKGROUND OF THE INVENTION

[0005] Duchenne muscular dystrophy (DMD) is the most frequent progressive muscle degenerative disease, affecting approximately one in 3,500 to 5000 male births. 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, and provides a mechanical and functional link between the cytoskeleton of the muscle fiber and the extracellular matrix. The absence of functional dystrophin causes fiber degeneration, inflammation, necrosis and replacement of muscle with scar and fat tissue, resulting in progressive muscle weakness and premature death due to respiratory and cardiac failure between the second and fourth decade of life (Moser, H., Hum Genet, 1984. 66(1): 17-40).

[0006] A milder form of the disease called Becker muscular dystrophy (BMD) is distinguished from DMD by delayed onset, later dependence on wheelchair support, and longer life span. BMD usually corresponds to mutations maintaining the reading frame (Muntoni F et al, Lancet Neurol, 2003. 2(12): 731-40).

[0007] There is no cure nor effective treatment available for DMD (Rodino-Klapac, L.R. etal., Curr Neurol Neurosci Rep, 2013. 13(3): 332) or BMD. Conventional therapies are limited to supportive care, which partially alleviates signs and symptoms, but does not directly target the disease mechanism nor reverse the phenotype. There are currently several therapeutic strategies being developed for DMD including in vivo gene therapy, cell transplantation therapy, pharmacologic rescue of DMD nonsense mutations and exon skipping or gene editing strategies to repair the dystrophin gene reading frame. All of these strategies have problems to overcome, including efficiency, targeting different muscle groups, optimization of delivery, longterm expression of the transgene, and potential immune response (J armin et al., Expert Opin Biol Ther, 2014. 14(2): 209-30).

[0008] Different gene transfer approaches for DMD aim to compensate for dystrophin loss- of-function and offer the potential to treat all patients using a single medication.

[0009] The dystrophin gene is the largest known gene in the human genome, spanning over 2.5Mb or some 2% of the entire X chromosome in humans. It consists of 79 exons (full- length cDNA: 11 , Ikb), which encodes for a 3685 amino acids, 427kD dystrophin protein. The dystrophin protein is defined by four structural regions (Figure 1 A). These are the actin binding domain at the NH2 terminus (exons 1 to 8), central rod domain (24 spectrin-like repeats Rl-24 and 4 Hinge regions Hl -4; exons 9 to 62), cysteine-rich (CR) domain (exons 63 to 69), and carboxy-terminal (CT) domain (exons 70 to 79).

[0010] The cDNA size is too large to fit inside known gene therapy vector systems, especially in Adeno-Associated Virus (AAV) vector which is one of the promising candidates with efficient gene transfer into various muscle groups depending on tropism of AAV serotypes. AAV vector has a potential to show long term gene transduction in both dividing (myofibres and cardiomyocytes) and non-dividing (mature myotubes) muscle cells.

[0011] Indeed, a major limitation of AAV is its cargo capacity which is thought to be limited to around 5 kb, the size of parental viral genome (Wu Z. et al., Mol Ther., 2010, 18(1): SO- 86; Lai Y. etal., Mol Ther., 2010, 18(1): 75-79; Wang Y. etal., Hum Gene Ther Methods, 2012, 23(4): 225-33). Larger vector genomes resulted in truncated packaged genomes, heterogeneous population of genome with broad size distribution, and lower expression efficiency (Wu Z. et al., Mol Ther., 2010, 18(1): 80-86). To overcome the DNA packaging limitation of AAV (<5 kb), several research groups have attempted to engineer synthetic truncated but functional dystrophins (MD, also known as “microdystrophin” or “minidystrophin”). A series of microdystrophins has been designed to encode truncated dystrophins optimized to contain the more clinically important regions of the protein. Such regions have generally been thought to lie within dystrophin’s N-terminal and cysteine-rich domains.

[0012] A microdystrophin, which contains the first 3 and the last of the 24 spectrin-like repeats without the C-terminal domain (AR4-R23 / ACT), named MD1, displayed highly functional activity to restore dystrophin and co-localise with syntrophin and dystrobrevin, but it failed to recruit nNOS at the sarcolemma in mdx mouse model (Yue etal., Mol Ther, 2006. 14(1): 79-87). Its therapeutic value has been reported in document WO2015 / 197869.

[0013] However, the relevance of the deleted regions, e.g. of the R16-R17 nNOS binding site and / or the R8-R9 Parlb binding site and / or the RIO to R17 binding sites with F-actin, in muscle function remains questioned.

[0014] As a further strategy, it has been proposed to produce larger microdystrophins, i.e. quasidystrophins, relying on a recombination event and using a dual AAV vector system. As known in the art, the two vectors of the dual AAV system can be overlapping vectors, trans-splicing AAV vectors or hybrid trans-splicing AAV vectors (see e.g. Pryadkina et al., Molecular Therapy, 2015, 2, 15009).

[0015] Based thereon, Kodippili et al. (Human Gene Therapy, 2018, 29 (3), 299-311) reported expression of a canine AH2-R15 mini dystrophin using a pair of dual AAV9 vectors in a canine model of Duchenne Muscular Dystrophy (DMD).

[0016] Besides, two further quasidystrophins (WL1 : AR4-R7AR10-R15AR18-R19 and WL2: AR4-R7AR10-R13AR18-R21) have been reported in documents WO2020 / 193636 (US2022 / 204574) and Albini S. et al. (International Journal of Molecular Sciences, 2023, 24(14), p. 11421).

[0017] Moreover, in documents W02023 / 004125 and Tasfaout H. et al. (Research Square, 2023, doi.org / 1.21203 / rs.3-2924001 / vl), it has been reported the use of split inteins for producing large versions of truncated polypeptides, such as a AR5-R15 quasidystrophin. Anyway, there is still a need in the art for producing high levels of active truncated dystrophins.

[0018] BRIEF SUMMARY OF THE INVENTION

[0019] The present invention aims at alleviating or curing the devastating Duchenne muscular dystrophy (DMD) as well as Becker muscular dystrophy (BMD) by expressing a shorter but functional dystrophin polypeptide, called mididystrophin, using dual AAV vector systems.

[0020] Over the last few years it has indeed been revealed that DMD pathology is caused by myofiber fragility as well as muscle stem cell dysfunction that impairs muscle regeneration and lead to muscle wasting. The impact of satellite cell dysfunction in DMD associated muscle wasting is a relatively recent finding and could ameliorate by far the current strategies based on AAV-dystrophin delivery by preserving key binding sites and key molecular function of dystrophin.

[0021] In particular, the dystrophin constructs according to the present invention differ for the inclusion of key domains important to boost muscle membrane (sarcolemma) binding, cytoskeleton (Actin) binding or microtubule binding activity. They offer new therapeutic tools useful for membrane stability of myofibers and consequently, muscle force generation.

[0022] Definitions

[0023] The articles “a” and “an” are used herein to refer to one or to more than one (z.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.

[0024] “About” or “approximately” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0025] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0026] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0027] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0028] 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 that encodes a protein or a RNA or a cDNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0029] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (ie., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0030] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR and the like, and by synthetic means.

[0031] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0032] “Identical” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or identical at that position. The percent of homology / identity between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched then the two sequences are 60% identical. Generally, a comparison is made when two sequences are aligned to give maximum homology / identity.

[0033] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior 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 an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno- associated virus vectors, retroviral vectors, and the like.

[0034] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can 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 the recombinant polynucleotide.

[0035] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0036] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0037] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0038] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell. A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell preferentially if the cell is a cell of the tissue type corresponding to the promoter.

[0039] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0040] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0041] A disease or disorder is “alleviated” or “ameliorated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced. This also includes halting progression of the disease or disorder. A disease or disorder is “cured” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is eliminated.

[0042] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

[0043] As used herein, “treating a disease or disorder” means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. Disease and disorder are used interchangeably herein in the context of treatment.

[0044] An “effective amount” of a compound is that amount of compound which is 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 that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or condition, including alleviating symptoms of such diseases. An “effective amount” of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound. DETAILED DESCRIPTION OF THE INVENTION

[0045] According to a first aspect, the present invention concerns a mididystrophin, advantageously a functional midi dystrophin, more advantageously of human origin.

[0046] In the frame of the present application, “mididystrophin” means a peptide or protein which is shorter than the native or wild type dystrophin. In the context of the invention, the terms “mididystrophin”, “microdystrophin”, “minidystrophin” and “quasidystrophin” have the same meaning.

[0047] According to a particular embodiment, a mididystrophin according to the invention has a size of less than 2400 amino acids (aa), the typical size of a so-called quasidystrophin, or even less than 2350 or 2300 aa. According to a further embodiment, a mididystrophin according to the invention has a size of more than 1200 aa, the size of the so-called MD1 microdystrophin, or even more than 1300 aa, 1400 aa, 1500 aa, 1600 aa, 1700 aa, 1800 aa, 1900 aa, 2000 aa or even 2100 aa.

[0048] According to a specific embodiment, a mididystrophin according to the invention has a size of more than 35%, 40%, 45%, 50%, 55% or even more than 57% of the size of the full- length dystrophin (e.g. 3685 amino acids for the human version). According to a preferred embodiment, it has a size of more than 58%, 59%, 60%, 61% or even 62% of the size of the full-length dystrophin.

[0049] In relation to the human version, this means that according to the invention, the mi di dystrophin advantageously contains more than 2137 amino acids, still advantageously more than 2200 amino acids, e.g. 2139 aa (LP1 in the examples), 2294 aa (LP2 in the examples), and 2206 aa (LP3 in the examples).

[0050] The structure of dystrophin is well documented (see Figure 1A) and active fragments thereof have been disclosed. As it would be understood in the art, an active fragment is a portion or portions of a full-length sequence that retain at least some of the biological functions of the full-length sequence.

[0051] A “functional” truncated dystrophin or mididystrophin means that the corresponding peptide or protein is able to perform at least some of the functions of the wild-type dystrophin protein and is able to alleviate, at least partially, one or more of the symptoms associated with the absence of a native dystrophin, especially fiber degeneration, inflammation, necrosis, replacement of muscle with scar and fat tissue, muscle weakness, respiratory and cardiac failure, as well as premature death.

[0052] It is preferred that the mididystrophin according to the invention displays (to a greater or lesser extent) at least one of the properties disclosed in relation to the microdystrophins of the prior art, especially 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).

[0053] Among others, preferred properties are:

[0054] - Binding with at least one DAP (“dystrophin associated proteins”), especially with actin (F-actin), synemin, syntrophin, dystrobrevin, nNOS and / or PAR-lb proteins;

[0055] - Recruitment of the DAP complex at the sarcolemma through lipid binding;

[0056] - Rescue of the microtubule network;

[0057] - Muscle protection from damage;

[0058] - Preservation of the global structure of the protein and organization of spectrin repeat (R) domains;

[0059] - Restoration of muscle structure and function. Of particular interest are the skeletal muscles, but also the cardiac muscle and the diaphragm;

[0060] - More generally, amelioration of muscular function, gait, cardiac function, respiratory function, survival, quality and / or expectancy of life.

[0061] As known in the art, said properties can be tested in vitro on various cells expressing dystrophin, e.g. using muscle organoids derived from DMD patients’s induced pluripotent stem cells, ex vivo on muscle fibres isolated from various animal models, or in vivo based on animal models or even on patients suffering from DMD or BMD. Animal models are e.g. the mdx mouse (Foster H. etal., Mol Ther, 2008. 16(11): p. 1825-32), the mdx^'' mouse (Decrouy et al., Gen Ther, 1997. 4(5): 401-8), the D2.B10-mdx / J mouse (Coley etal., Human Molecular genetics, 2016. 25(1): 130-45), the CXMDj dog (Koo et al., J Gene Med, 2011. 13(9): 497-506) or the GRMD dog (Le Guiner et al., Mol Ther., 2014. 22(11): 1923-35). The mouse model is commonly used to test new constructs encoding microdystrophins. However, this model has drawbacks because the mouse displays a less severe form of the disease, without immune reactions. The other animal model is the dog which is considered more reliable to predict the therapeutic potential of a gene therapy product in humans. The rat model as disclosed by Larcher et al. (Pios One, 2014, 9(10), el 10371) is also very interesting since it displays cardiomyopathy. As mentioned above, the full-length human dystrophin (Fig. 1A) is characterized by different domains: a N-terminal domain (CH1CH2)

[0062] 4 hinge domains (Hl to H4)

[0063] 24 spectrin-like repeats or rod domains (R1 to R24) a cysteine-rich (CR) domain a C-terminal (CT) domain.

[0064] According to one embodiment, a mi di dystrophin according to the invention has at least one domain lacking, advantageously at least one spectrin-like-repeat (R), compared to the full- length dystrophin.

[0065] According to one aspect, mididystrophins of the invention contain at least one key protein binding site, especially for F-actin, the nNOS protein and the PAR-lb protein. Advantageously, mididystrophins of interest contain the binding site of nNOS and / or PARlb. The binding site of nNOS was shown to lie in repeats 16 and 17 (R16, R17) of the rod domain (Lai et al., J. Clin. Invest., 2009. 119:624-635) while a binding of dystrophin repeats 8 and 9 (R8, R9) to PARlb was demonstrated in vitro (Yamashita et al., Biochem. Biophys. Res. Commun., 2010. 391 : 812-817). According to a preferred embodiment, mi di dystrophins of interest contain the R16-17 and / or R8-9 rod domains.

[0066] According to this aspect, the invention concerns a mididystrophin comprising at least one or even two, three or even four rod domains selected in the group consisting from: R8, R9, R16 and R17. According to a specific embodiment, a midi dystrophin according to the invention contains the R8, R9, R16 and R17 rod domains.

[0067] Further functions of rod domains (R) in the human dystrophin have been reported:

[0068] - Rl, R2, R3, R10, Rl l and R12 (besides the cysteine-rich domain) bind to lipids and therefore contribute to the recruitment of the DAP complex at the sarcolemma;

[0069] - Rl l, R12, R13, R14, R15 (besides CHI and CH2) bind to F-actin;

[0070] - R4, R5, R6, R7, R8, R9, R10, Rl l, R12, R13, R14, R15, R20, R21, R22, R23 and R24 bind to microtubules. According to this aspect, the invention concerns a mididystrophin comprising: at least one or even several rod domains selected in the group consisting from: Rl, R2, R3, RIO, Rl l, R12. According to a specific embodiment, a mididystrophin according to the invention comprises at least Rl, RIO, Rl 1 and R12. According to another embodiment, a mididystrophin according to the invention comprises at least Rl, R2 and R3; and / or at least one or even several rod domains selected in the group consisting from: RIO, Rl l, R12, R13, R14, R15, R16 and R17, or Rl l, R12 and possibly R13. According to a specific embodiment, a mididystrophin according to the invention comprises at least Rl l and R12. According to another embodiment, a mididystrophin according to the invention comprises at least Rl l, R12 and R13; and / or at least one or even several rod domains selected in the group consisting from: RIO, Rl l, R12, R13, R20, R21, R22, R23 and R24. According to a specific embodiment, a mididystrophin according to the invention comprises at least R24. According to another embodiment, a mididystrophin according to the invention comprises R20, R21, R22, R23 and R24.

[0071] According to another aspect, the mididystrophins of the invention are characterized by a N-terminal part lacking or deprived of at least the R4, R5, R6 and R7 rod domains (AR4- R7).

[0072] In other words, a mididystrophin according to the invention has a N-terminal part defined as follows, from its N terminal end and in the following order: a N-terminal domain which binds to actin (CH1CH2); the Hl hinge domain; the Rl rod domain or the Rl, R2 and R3 rod domains; the H2 hinge;

[0073] - the R8 and R9 rod domains.

[0074] Such a mi di dystrophin displays the structure CH1CH2H1R1R2R3H2R8R9 or CHlCH2HlRlH2R8R9in its N terminal part.

[0075] According to a preferred embodiment, such a mi di dystrophin further contains the R16 and R17 rod domains. According to a particular embodiment, the mididystrophins of the invention contains at least the spectrin-like-repeats of the MD1 microdystrophin of the prior art, i.e. at least R1 and R24 or even Rl, R2, R3 and R24.

[0076] Advantageously, mididystrophins according to the invention contain further rod (R) domains compared to the MD1 microdystrophin, advantageously at least one chosen in the group consisting of RIO, Rl l, R12, R13, R20, R21, R22 and R23.

[0077] According to specific embodiments, mididystrophins according to the invention lack the following rod domains:

[0078] R4 to R7, R14 to R15 and R18 to R23. In other words, such a midi dystrophin (LP1 below) contains Rl to R3, R8 to R13, R16 to R17 and R24; or

[0079] R2 to R7, R13 to R15 and R18 to R19. In other words, such a midi dystrophin (LP2 below) contains Rl, R8 to R12, R16 to R17 and R20 to R24; or

[0080] R4 to R7, RIO to R15 and R18 to R19. In other words, such a midi dystrophin (LP3 below) contains Rl to R3, R8 to R9, R16 to R17 and R20 to R24.

[0081] Advantageously, it further contains: a complete N-terminal domain, corresponding to amino acids 1 to 246 of e.g. SEQ ID NO: 1 a complete cysteine-rich (CR) domain, corresponding to amino acids 3080 to 3360 of the full-length dystrophin a partial or full-length C-terminal domain, advantageously the full-length C- terminal domain corresponding to amino acids 3361 to 3685 of the full-length dystrophin. Possible partial C-terminal domains are the truncated C-terminal domains of MD1, MD2, MD3 or MD4 as disclosed in WO2016 / 177911

[0082] - Possibly at least one hinge (H) domain, chosen in the group consisting of Hl, H2, H3 and H4, advantageously at least Hl, H2 and H4, possibly Hl, H2, H3 and H4.

[0083] According to specific embodiments, the mididystrophin according to the invention is: a AR4-R7AR14-R15AR18-R23 mididystrophin, advantageously of structure CH1CH2H1R1R2R3H2R8R9R10R11R12R13R16R17R24H4CRCT (further named LP1), more advantageously of sequence SEQ ID NO: 1; or a AR2-R7AR13-R15AR18-R19 mididystrophin, advantageously of structure CH1CH2H1R1H2R8R9R10R11R12R16R17H3R20R21R22R23R24H4CRCT (further named LP2), more advantageously of sequence SEQ ID NO: 2; or a AR4-R7AR10-R15AR18-R19 midi dystrophin, advantageously of structure CHI CH2H1R1R2R3H2R8R9R16R17H3R20R21R22R23R24H4CRCT (further named LP3), more advantageously of sequence SEQ ID NO: 3.

[0084] According to one embodiment, the mididystrophin is “substantially identical”, that is, is about 60% identical, 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 to the mididystrophins disclosed therein, especially those of sequence SEQ ID NO: 1 to SEQ ID NO: 3, advantageously SEQ ID NO: 1 to SEQ ID NO: 2. According to an embodiment, such a mididystrophin is functional, optionally has the same function / activity as the mididystrophin encoded by SEQ ID NO: 1 to SEQ ID NO: 3, advantageously SEQ ID NO: 1 to SEQ ID NO: 2.

[0085] According to a further aspect, the invention relates to a nucleic acid sequence encoding a mi di dystrophin as defined above.

[0086] According to one embodiment, the nucleic acid sequence encoding a mi di dystrophin according to the invention comprises or consists of a sequence selected in the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, advantageously SEQ ID NO: 4 to SEQ ID NO: 6.

[0087] According to another embodiment, the nucleic acid sequence encoding a mididystrophin according to the invention is “substantially identical”, that is, is about 60% identical, 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 to any of the sequences SEQ ID NO: 4 to SEQ ID NO: 8, advantageously SEQ ID NO: 4 to SEQ ID NO: 6.

[0088] The expression system according to the invention is typically composed of 2 AAV vectors, i.e. a so-called dual AAV vector system.

[0089] According to a preferred embodiment, the mididystrophins of the invention are produced using such a dual AAV vector system.

[0090] By “dual AAV system”, it is meant a vector system composed of two AAV vectors, in which system each vector carries a part of a sequence encoding the mididystrophin of the invention to be delivered to a cell and an open reading frame (ORF) encoding said mi di dystrophin is reconstituted by interaction between the first and the second nucleic acid sequences into the cell.

[0091] According to another aspect, the present invention also concerns an AAV vector corresponding to the first AAV vector or to the second AAV vector of the dual AAV vector system according to the invention.

[0092] In other words, the invention concerns a composition comprising recombinant adeno- associated viral (AAV) vectors, preferably two in number, carrying complementary constructs allowing the functional mididystrophin of the invention to be expressed.

[0093] In the frame of the invention, the term “composition” can be replaced by “association” “combination” or “expression system”. It means that the two AAV vectors work together and have to be in contact so that the active protein of interest can be produced. However, they can be found in a single composition, or in two distinct compositions possibly mixed before use.

[0094] According to the invention, each adeno-associated viral (AAV) vector of the system comprises an expression construct, also named “expression cassette” or “insert”. In the frame of the present application, said “insert” is advantageously defined as the nucleic acid sequence located between the 5’ and 3’ ITR (’’Inverted Terminal Repeat”) sequences of the AAV genome.

[0095] According to common knowledge in the art, the size of the insert should not largely exceed the wild-type AAV genome length. For example, AAV2 contains 2 ITR sequences of 145 bp each and has a genome of 4682 pb (including the ITR sequences).

[0096] In a particular embodiment, the nucleic acid sequences encoding a part of the mi di dystrophin introduced in each AAV vector have a length of less than 5 kb, such as less than 4.9, 4.8, 4.7, 4.6 or 4.5 kb.

[0097] To advantage, and to limit the constraint of the size of the AAV packaging, said nucleic acid sequences correspond to exons. In other words, they are preferably cDNA fragments.

[0098] To advantage, the reading frame formed from combining the two AAVs encodes a mi di dystrophin (MD), advantageously a functional mididystrophin, more advantageously of human origin, as disclosed above. According to a preferred embodiment, the mididystrophin to be produced with the claimed dual AAV vector system contains at least 2000 amino acids (aa), advantageously at least 2100 aa, 2200 aa or 2300 aa. According to another embodiment, the microdystrophin to be produced with the claimed AAV vector contains no more than 3000 amino acids (aa), advantageously no more than 2900 aa, 2800 aa, 2700 aa, 2600aa 2550 aa or 2500 aa. In a preferred embodiment, a mididystrophin to be produced with the claimed dual AAV vector system contains between 2100 and 2300 aa, i.e. has a size corresponding to about 60% of the size of the full-length dystrophin.

[0099] Each AAV vector comprises the nucleic acid sequences encoding the relevant part of the dystrophin gene (N-terminal and C-terminal part, respectively), advantageously as defined above, but also all the sequences required for a proper expression of said mididystrophin, after reconstitution of the whole gene or protein.

[0100] According to one embodiment, especially for the overlap approach, the first and second nucleic acid sequences of the first and second AAV vectors are placed under the control of regulatory sequence(s). Advantageously, the first nucleic acid sequence is preceded by a promoter optionally followed by an intron, and the second nucleic acid sequence is followed by a polyadenylation signal. Alternatively, especially for the intein approach, the first and second nucleic acid sequences are preceded by a promoter optionally followed by an intron and followed by a polyadenylation signal.

[0101] According to a specific embodiment, a first adeno-associated viral (AAV) vector comprises: i) an AAV 5’ITR (Inverted Terminal Repeat) sequence; ii) a gene portion controlled by a promoter; iii) an AAV 3TTR sequence.

[0102] In addition, the second adeno-associated viral (AAV) vector comprises: iv) an AAV 5’ITR (Inverted Terminal Repeat) sequence; v) a gene portion advantageously followed by a polyadenylation signal; vi) an AAV 3’ITR sequence.

[0103] According to another specific embodiment, both the first and second adeno-associated viral (AAV) vectors comprise:

[0104] -an AAV 5’ITR (Inverted Terminal Repeat) sequence; - a gene portion controlled by a promoter and advantageously followed by a polyadenylation signal;

[0105] - an AAV 3’ITR sequence.

[0106] Such promoters can be natural or synthetic (artificial) promoters, inducible or constitutive.

[0107] In one embodiment, the promoter is a ubiquitous promoter or has a low tissue-specificity. As an example, the expression vector can harbor the phosphoglycerate kinase 1 (PGK), EFl, ACTA1, P-actin, Desmin, all MCK variants, cardiac Troponin and CMV promoter.

[0108] In a preferred embodiment, the promoter sequence is chosen in order to adequately govern the expression of the nucleic acid sequence placed under its control, in terms of expression level, but also of tissue specificity.

[0109] In one embodiment, the expression vector comprises a muscle specific promoter. Such a promoter allows a robust expression in the skeletal muscles, in the diaphragm and possibly in the cardiac muscle, as well as in satellite cells. Examples of suitable promoters known by the skilled person are e.g. the desmin promoter, the muscle creatine kinase (MCK) promoter, truncated creatine kinase promoters such as e.g. CK6, CK7 or CK8 promoter, the Syn promoter, MyoD, Myf5, Vcam, Pax3 and Pax7 satellite cell promoter. Another promoter is the synthetic promoter C5-12 (spC5-12). It is also possible to use a hybrid promoter comprising sequences from two or more transcriptional regulatory elements (see e.g. PCT / EP2019 / 053061).

[0110] Advantageously, the first nucleic acid sequence is placed under the control of a musclespecific promoter. In other words, the first AAV vector further comprises a muscle-specific promoter which is operably linked to the nucleic acid sequence encoding the N-terminal part of a dystrophin.

[0111] As known in the art, a non-exhaustive list of other possible regulatory sequences to be introduced in one or in the other AAV vector is:

[0112] - a polyadenylation signal, advantageously in 3’ of the sequence encoding the functional microdystrophin;

[0113] - sequences for transcript stabilization, e.g. intron;

[0114] - enhancer sequences ; - miRNA target sequences, which can inhibit the expression of the sequence encoding the functional dystrophin in non target tissues, in which said expression is not desired, for example where it can be toxic.

[0115] According to one aspect, the intron is selected in the group consisting of a human beta globin b2 (or HBB2) intron, a FIX intron and a chicken beta-globin intron, wherein said intron is optionally a modified intron such as a modified HBB2 intron, a modified FIX intron, or a modified chicken beta-globin intron.

[0116] According to another embodiment, the polyadenylation signal is selected among 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.

[0117] According to a specific embodiment, the dual AAV vector system according to the invention contains at least one of the following elements, advantageously all of them: a CK8 promoter, advantageously of sequence SEQ ID NO: 23; and a SV40 polyA signal, advantageously of sequence SEQ ID NO: 24. a Chimeric Intron, advantageously of sequence SEQ ID NO: 25.

[0118] According to a first aspect, such a dual AAV vector system is dedicated to the so- called overlap approach:

[0119] After in vivo recombination based on the overlapping region shared by the 2 AAV vectors, high amounts of the resulting mididistrophin are produced. Therefore, the nucleic acid sequence encoding the mididystrophin is split into 2 parts, i.e. a 5’ sequence encoding its N-terminal part and a 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 take place.

[0120] According to a further aspect, the present invention concerns a dual AAV vector system comprising two AAV vectors, wherein a first AAV vector comprises, between 5’ and 3’ AAV ITRs, a first nucleic acid sequence that encodes a N-terminal part of the mididystrophin, and a second AAV vector comprises, between 5’ and 3’ AAV ITRs, a second nucleic acid sequence that encodes a C-terminal part of the mididystrophin, wherein the first and second nucleic acid sequences comprise an overlapping region that permits the production of the mididystrophin of the invention by recombination. In the frame of the invention and as explained below, the terms “overlapping region” (or “overlapping sequences”) and “region of homology” (or “region of sequence homology”) have the same meaning and are used interchangeably.

[0121] These two AAV vectors have complementary sequences which will form a functional unit at the time of recombination. As known by the skilled person, recombination occurs by the recognition of homologous sequences present on each of the AAV vectors, thanks to the cellular DNA repair pathway.

[0122] Therefore, the gene portions of the two AAV vectors have to fulfil the following requirements:

[0123] - the gene portions of the first and second AAV vectors together comprise an open reading frame which codes for the mididystrophin of the invention, preferably of human origin;

[0124] - the gene portions of the first and second AAV vectors both comprise a region of homology which allows, after homologous recombination, the reconstitution of said open reading frame.

[0125] According to this aspect, the dual vector system of the invention implements vectors comprising sequences allowing (homologous) recombination, i.e. overlapping vectors. Therefore, a mididystrophin protein is reconstituted by implementation of homologous recombination by adding an appropriate overlapping region to each of part of the dystrophin gene introduced in each AAV vector.

[0126] In the dual vector system according to this first embodiment, the first and second nucleic acid sequences display a region of sequence homology to promote intermolecular homologous recombination, thus generating the large mididystrophin transgene by recombining the two vectors of the dual AAV system, i.e. the nucleic acid sequences encoding the N-terminal part and the C-terminal part of the mididystrophin, respectively. In a particular embodiment implementing this overlapping system, the length of the region of sequence homology may vary to a large extent as long as the size of the resulting inserts (including the 5'- and 3'-ITR sequences, and any expression control sequence) is compatible with the size limit for encapsidation within an AAV vector. One skilled in the art is well aware of this size limit and is able to adapt the size of both the first and second nucleic acid sequences, and therefore of the region of sequence homology, according to this knowledge. Therefore, 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, for example of 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 midi dystrophin sequence having a length of at least 100 nucleotides, such as of 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 comprised between 500 and 1500 nucleotides, in particular between 700 and 1500 nucleotides, advantageously between 800 and 1450 nucleotides.

[0127] According to a specific embodiment, the first nucleic acid sequence comprises or consists of SEQ ID NO: 11 or SEQ ID NO: 15 or SEQ ID NO: 19 and the second nucleic acid sequence comprises or consists of SEQ ID NO: 12 or SEQ ID NO: 16 or SEQ ID NO: 20.

[0128] According to one embodiment, the first nucleic acid sequence may encode the N-terminal domain of dystrophin, Hl, Rl, R2, R3, H2, R8, R9, R10, R11, R12 and a truncated R13, wherein the second nucleic acid sequence encodes R9, R10, Rl l, R12, R13, R16, R17, R24, H4, the CR domain and the C-terminal domain of dystrophin (full-length or truncated as disclosed above, advantageously full-length).

[0129] According to a specific embodiment, the first nucleic acid sequence comprises or consists of SEQ ID NO: 11 and the second nucleic acid sequence comprises or consists of SEQ ID NO: 12.

[0130] According to another embodiment, the first nucleic acid sequence may encode the N- terminal domain of dystrophin, Hl, Rl, H2, R8, R9, R10, Rl l, R12, R16, R17 and a truncated R20, wherein the second nucleic acid sequence encodes R6, R17, H3, R20, R21, R22, R23, R24, H4, the CR domain and the C-terminal domain of dystrophin (full-length or truncated as disclosed above, advantageously full-length).

[0131] According to a further specific embodiment, the first nucleic acid sequence comprises or consists of SEQ ID NO: 15 and the second nucleic acid sequence comprises or consists of SEQ ID NO: 16.

[0132] According to a further embodiment, the first nucleic acid sequence may encode the N- terminal domain of dystrophin, Hl, Rl, R2, R3, H2, R8, R9, R16, R17, H3 and R20, wherein the second nucleic acid sequence encodes a truncated R16, R17, H3, R20, R21, R22, R23, R24, H4, the CR domain and the C-terminal domain of dystrophin (full-length or truncated as disclosed above, advantageously full-length). According to a specific embodiment, the first nucleic acid sequence comprises or consists of SEQ ID NO: 19 and the second nucleic acid sequence comprises or consists of SEQ ID NO: 20.

[0133] According to a second aspect, such a dual AAV vector system is dedicated to the so- called intein approach:

[0134] As known in the art, protein splicing is an intramolecular reaction of a particular protein in which an internal protein segment (called a split intein or an intein) is removed from a precursor protein with a ligation of C-terminal and N-terminal external proteins (called exteins) on both sides. This precursor protein contains three segments: an N-extein followed by the intein followed by a C-extein. After splicing has taken place, the resulting protein contains the N-extein linked to the C-extein.

[0135] In case of a dual AAV vector, the first AAV allows the production of the N-extein followed by a first part of intein (first fusion protein) and the second AAV allows the production of the second part of intein followed by the C-extein (said second fusion protein). Said first and second fusion polypeptides are produced in the cell and the first and second parts of the intein promote joining of the first and second parts of the extein, thereby delivering the extein to the cell.

[0136] Therefore, the nucleic acid sequence encoding the mididystrophin is split into 2 parts, i.e. a 5’ sequence encoding its N-terminal part and a 3’sequence encoding its C- terminal part. The 3 ’end of the 5’sequence is fused to a first part of the split intein, advantageously its N-terminal part, and the 5 ’end of the 3’sequence is fused to the second part of the split intein, advantageously its C-terminal part.

[0137] Examples of split inteins are e.g. disclosed in WO2013 / 045632. Preferred split inteins to be used in the frame of the invention are GP41-1 and IMPDH1, advantageously GP41-1. A sequence of the N-terminal part of GP41-1 is shown in sequence SEQ ID NO: 21.

[0138] A sequence of the C-terminal part of GP41-1 is shown in sequence SEQ ID NO: 22.

[0139] According to a further aspect, the present invention concerns a dual AAV vector system comprising two AAV vectors, wherein a first AAV vector comprises, between 5’ and 3’ AAV ITRs, a first nucleic acid sequence that encodes the N-terminal part of a mididystrophin fused to the N- terminal part of a split intein, and a second AAV vector comprises, between 5’ and 3’ AAV ITRs, a second nucleic acid sequence that encodes the C-terminal part of the split intein fused to the C- terminal part of the mididystrophin, wherein the first and second parts of the split inteins promote joining of the first and second parts of the mididystrophin, thereby permitting the production of the mididystrophin according to the invention.

[0140] According to a specific embodiment, the first nucleic acid sequence comprises or consists of SEQ ID NO: 9 or SEQ ID NO: 13 or SEQ ID NO: 17 and the second nucleic acid sequence comprises or consists of SEQ ID NO: 10 or SEQ ID NO: 14 or SEQ ID NO: 18.

[0141] According to one embodiment, the first nucleic acid sequence may encode the N-terminal domain of dystrophin, Hl, Rl, R2, R3, H2, R8, R9, RIO, a truncated R11, fused to the N- terminal part of the GP41-1 gene, wherein the second nucleic acid sequence encodes the C-terminal part of the GP41-1 gene fused to the rest of the truncated Rl 1, R12, R13, R16, R17, R24, H4, the CR domain and the C-terminal domain of dystrophin (full-length or truncated as disclosed above, advantageously full-length).

[0142] According to a specific embodiment, the first nucleic acid sequence comprises or consists of SEQ ID NO: 9 and the second nucleic acid sequence comprises or consists of SEQ ID NO: 10.

[0143] According to another embodiment, the first nucleic acid sequence may encode the N- terminal domain of dystrophin, Hl, Rl, H2, R8, R9, RIO, Rl 1, R12, R16, a truncated R17, fused to the N-terminal part of the GP41-1 gene, wherein the second nucleic acid sequence encodes the C-terminal part of the GP41-1 gene fused to the rest of the truncated R17, H3, R20, R21, R22, R23, R24, H4, the CR domain and the C-terminal domain of dystrophin (full-length or truncated as disclosed above, advantageously full-length).

[0144] According to a further 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.

[0145] According to a further embodiment, the first nucleic acid sequence may encode the N- terminal domain of dystrophin, Hl, Rl, R2, R3, H2, R8, R9, R16, R17, H3 fused to the N- terminal part of the GP41-1 gene, wherein the second nucleic acid sequence encodes the C-terminal part of the GP41-1 gene fused toR20, R21, R22, R23, R24, H4, the CR domain and the C-terminal domain of dystrophin (full-length or truncated as disclosed above, advantageously full-length). According to a specific embodiment, the first nucleic acid sequence comprises or consists of SEQ ID NO: 17 and the second nucleic acid sequence comprises or consists of SEQ ID NO: 18.

[0146] The nucleic acid sequence encoding the midi dystrophin is advantageously of human origin but can also be a canine, a rat, a murine or a non-human primate sequence. In one embodiment, the nucleic acid sequence originates from the organism it will be administered to, advantageously a human sequence for administration in humans.

[0147] In a known manner, there are different ways to optimize a sequence encoding a protein, so as to increase the mRNA level (recombination and transcription) and / or the protein level (translation). In the frame of the invention, the sequences in the vectors of the dual AAV vector system of the invention are advantageously optimized for increasing recombination, and / or for increasing the expression of the mididystrophin polypeptide in vivo.

[0148] In a particular embodiment, the sequences encoding the mididystrophin of the invention are optimized. Sequence optimization may include a number of changes in a nucleic acid sequence, including: introduction of a consensus Kozak sequence (GCCACC) before AUG start codon within mRNA, to improve initiation of translation codon optimization, preferably adaptation to the codon bias of Homo sapiens genes increase of GC content and decrease of the number of CpG islands: in general, regions of very high (>80%) or very low (<30%) GC content are avoided decrease or suppression of internal TATA-boxes, chi-sites, ribosomal entry sites, AT -rich or GC-rich sequence stretches, RNA instability motifs, repeat sequences and RNA secondary structures decrease of the number of alternative open reading frames (ARFs) decrease of the number of (cryptic) splice donor and splice acceptor sites.

[0149] Because of the degeneracy of the genetic code, different nucleic acid molecules may encode the same protein. It is also well known that the genetic codes of different organisms are often biased towards using one of the several codons that encode the same amino acid over the others. Through codon optimization, changes are introduced in a nucleotide sequence that take advantage of the codon bias existing in a given cellular context so that the resulting codon optimized nucleotide sequence is more likely to be expressed in such given cellular context at a relatively high level compared to the non-codon optimised sequence. In a preferred embodiment of the invention, such sequence optimized nucleotide sequence encoding a functional mi di dystrophin is codon-optimized to improve its expression and stability in human cells compared to non-codon optimized nucleotide sequences coding for the same protein, for example by taking advantage of the human specific codon usage bias. In a particular embodiment, the whole sequence of the mi di dystrophin is optimized for improving its production by the target or host cell, advantageously in humans.

[0150] For cloning purposes and production of viral particles, the expression construct can be inserted in a plasmid suitable for selection, replication and production of the mididystrophin.

[0151] According to a specific embodiment of the invention, the viral vector is an adeno- associated viral (AAV) vector.

[0152] Adeno-associated viral (AAV) vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, moderate immunogenicity, and the ability to transduce post-mitotic cells and tissues in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.

[0153] In one embodiment, the encoding sequence is contained within an AAV vector. More than 100 naturally occurring serotypes of AAV are known. Many natural variants in the AAV capsid exist, allowing identification and use of an AAV with properties specifically suited for dystrophic pathologies. AAV viruses may be engineered using conventional molecular biology techniques, making it possible to optimize these particles for cell specific delivery of nucleic acid sequences, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivery to the nucleus.

[0154] As mentioned above, the use of AAVs is a common mode of exogenous delivery of DNA as it is relatively non-toxic, provides efficient gene transfer, and can be easily optimized for specific purposes. Among the serotypes of AAVs isolated from human or non-human primates (NHP) and well characterized, human serotype 2 is the first AAV that was 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 AAV can also be useful. Desirable AAV fragments for assembly into vectors include the cap proteins, including the vpl, vp2, vp3 and hypervariable regions, the rep proteins, including rep 78, rep 68, rep 52, and rep 40, and the sequences encoding these proteins. These fragments may be readily utilized in a variety of vector systems and host cells.

[0155] Such fragments may 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, without limitation, AAV with a non-naturally occurring capsid protein. Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vpl capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV serotype, non-contiguous portions of the same AAV serotype, from a non- AAV viral source, or from a non-viral source (i.e. its capsid comprises VP capsid proteins derived from at least two different AAV serotypes, or comprises at least one chimeric VP protein combining VP protein regions or domains derived from at least two AAV serotypes). An artificial AAV serotype may be, without limitation, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid. Moreover, a peptide (P) can be introduced in said capsids, for example into a variable region of the cap gene, possibly to modify the AAV tropism.

[0156] In one embodiment, the vectors useful in the compositions and methods described herein contain, at a minimum, sequences encoding a selected AAV serotype capsid, e.g., an AAV9 capsid, or a fragment thereof. In another embodiment, useful vectors contain, at a minimum, sequences encoding a selected AAV serotype rep protein, e.g., AAV9 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 can both be of one serotype origin, e.g., all AAV9 origin. Alternatively, vectors may be used in which the rep sequences are from an AAV serotype, which differs from that which is providing the cap sequences. 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, these rep sequences are fused in frame to cap sequences of a different AAV serotype to form a chimeric AAV vector.

[0157] In some embodiments the AAV vector comprises a genome and a capsid derived from AAVs of different serotypes. Thus exemplary AAVs, or artificial AAVs, include AAV2 / 8 (US 7,282,199), AAV2 / 5 (available from the National Institutes of Health), AAV2 / 9 (W02005 / 033321), AAV2 / 6 (US 6,156,303), AAVrhlO (W02003 / 042397), AAVrh74 (W02003 / 123503), AAV9-rh74 hybrid or AAV9-rh74-Pl hybrid (WO2019 / 193119; W02020 / 200499; EP20306005.8).

[0158] According to one embodiment, the AAV is of serotype 2, 5, 8 or 9, or an AAVrh74. Advantageously, the claimed vector comprises a capsid selected from the group consisting of AAV8 capsid, AAV9 capsid, AAV9-rh74 capsid and AAV9-rh74-Pl capsid.

[0159] According to a particular embodiment, the AAV is an AAV of serotype 9 (AAV9).

[0160] In the AAV vectors used in the present invention, the AAV genome may be either a single stranded (ss) nucleic acid or a double stranded (ds) / self complementary (sc) nucleic acid molecule.

[0161] Advantageously and as mentioned above, the nucleic acid sequence of interest is inserted between the ITR (« Inverted Terminal Repeat ») sequences of the AAV vector. Typically, ITR sequences originate from AAV2 or AAV9, advantageously AAV2.

[0162] As known in the art, recombinant viral particles can be obtained, e.g., by tri-transfection of 293 HEK cells, by the herpes simplex virus system and by the baculovirus system, or using specific cell lines. The vector titers are usually expressed as viral genomes per ml (vg / ml).

[0163] The invention also concerns cells transduced with the dual AAV vector system as disclosed above, especially muscle cells.

[0164] According to another aspect, the present invention concerns a composition, advantageously a therapeutic composition or medicament, comprising the dual AAV vector system as disclosed above and possibly other active molecules (other gene therapy products, chemical molecules, peptides, proteins, . . .), dedicated to the treatment of the same disease or another disease.

[0165] The present invention then provides pharmaceutical compositions comprising the dual AAV vector system or the first or second AAV vector of said system. Such compositions comprise a therapeutically effective amount of the therapeutic (the nucleic acid or vector of the invention), and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. or European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic 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. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions 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.

[0166] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. 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 E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0167] In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to release pain at the site of the injection.

[0168] In one embodiment, the composition according to the invention is suitable for administration in humans. The composition is preferably in a liquid form, advantageously a saline composition, more advantageously a phosphate buffered saline (PBS) composition or a Ringer-Lactate solution.

[0169] The amount of the therapeutic (i.e. an expression system or a vector or a cell) of the invention which will be effective in the treatment of the target diseases can be determined by standard clinical techniques. In addition, in vivo and / or in vitro assays may optionally be employed to help predict optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, the physical characteristics of the individual under consideration such as sex, age and weight, concurrent medication, other factors and the seriousness of the disease, and should be decided according to the judgment of the practitioner and each patient’s circumstances.

[0170] Suitable administration should allow the delivery of a therapeutically effective amount of the gene therapy product to the target tissues, especially skeletal muscles and possibly heart and diaphragm. In the context of the invention, when the gene therapy product is a viral vector, the therapeutic dose is defined as the quantity of viral particles (vg for viral genomes) containing the transgene administered per kilogram (kg) of the subject.

[0171] In case of a treatment comprising administering a viral vector, such as an AAV vector, to the subject, typical doses of the vector are of at least IxlO8vector genomes per kilogram body weight (vg / kg), such as at least IxlO9vg / kg, at least IxlO10vg / kg, at least IxlO11vg / kg, at least IxlO12vg / kg at least IxlO13vg / kg, at least IxlO14vg / kg, at least 1015vg / kg. Specifically, the dose can be between 5.1011vg / kg and 1014vg / kg, e.g. 1, 2, 3, 4, 5, 6, 7, 8 or 9.1013vg / kg. A lower dose of e.g. 1, 2, 3, 4, 5, 6, 7, 8 or 9.1012vg / kg can also be contemplated in order to avoid potential toxicity and / or immune reactions. As known by the skilled person, a dose as low as possible giving a satisfying result in term of efficiency is preferred.

[0172] Available routes of administration are topical (local), enteral (system-wide effect, but delivered through the gastrointestinal (GI) tract), or parenteral (systemic action, but delivered by routes other than the GI tract). The preferred route of administration of the compositions disclosed herein is parenteral which includes intramuscular administration (i.e. into the muscle) and systemic administration (i.e. into the circulating system). In this context, the term “injection” (or “perfusion” or “infusion”) encompasses intravascular, in particular intravenous (IV), intramuscular (IM), intraocular, intrathecal or intracerebral administration. Injections are usually performed using syringes or catheters.

[0173] In one embodiment, systemic delivery of the composition comprises administering the composition near a local treatment site, i.e. in a vein or artery nearby a weakened muscle. In certain embodiments, the invention comprises the local delivery of the composition, which produces systemic effects. This route of administration, usually called “regional (loco-regional) infusion”, “administration by isolated limb perfusion” or “high-pressure transvenous limb perfusion” has been successfully used as a gene delivery method in muscular dystrophy.

[0174] According to one aspect, the composition is administered to an isolated limb (loco- regional) by infusion or perfusion. In other words, the invention comprises the regional delivery of the composition in a leg and / or arm by an intravascular route of administration, i.e. a vein (transvenous) or an artery, under pressure. This is usually achieved by using a tourniquet to temporarily arrest blood circulation while allowing a regional diffusion of the infused product, as e.g. disclosed by Toromanoff et al. (2008).

[0175] In one embodiment, the composition is injected in a limb of the subject. When the subject is a human, the limb can be the arm or the leg. According to one embodiment, the composition is administered in the lower part of the body of the subject, e.g. below the knee, or in the upper part of the body of the subject, e.g., below the elbow.

[0176] A preferred method of administration according to the invention is systemic administration. Systemic injection opens the way to an injection of the whole body, in order to reach the entire muscles of the body of the subject including the heart and the diaphragm and then a real treatment of these systemic and still incurable diseases. In certain embodiments, systemic delivery comprises delivery of the composition to the subject such that composition is accessible throughout the body of the subject.

[0177] According to a preferred embodiment, systemic administration occurs via injection of the composition in a blood vessel, i.e. intravascular (intravenous or intra-arterial) administration. According to one embodiment, the composition is administered by intravenous injection, through a peripheral vein.

[0178] In a specific embodiment, the treatment comprises a single administration of the composition.

[0179] In one embodiment, the presence of the gene therapy product and / or the expression of the functional micro-utrophin, as well as the associated therapeutic benefits, are observed for up to 1 month, or 3 months or 6 months or even 1 year, 2 years, 5 years, 10 years, or even more the whole life of the subject.

[0180] According to the invention, the subject is preferably a human, but can also be a mouse, a rat, a non-human primate, or a dog. Subjects that could benefit from the compositions of the invention include all patients diagnosed with a muscular dystrophy or at risk of developing such a muscular dystrophy. A subject to be treated can then be selected based on the identification of mutations or deletions in the dystrophin gene by any method known to the one skilled in the art, including for example sequencing of the dystrophin gene, and / or through the evaluation of the dystrophin level of expression or activity by any method known to the one skilled in the art. Therefore, said subjects include both subjects already exhibiting symptoms of a dystrophic disease and subjects at risk of developing said disease. In one embodiment, said subjects include subjects already exhibiting symptoms of a dystrophic disease. In another embodiment, said subjects are ambulatory patients and early non-ambulant patients.

[0181] Such compositions are notably intended for gene therapy in a subject, particularly for the treatment of diseases due the deficiency of the above-identified proteins, especially dystrophin.

[0182] Such compositions are notably intended for the treatment of a neuromuscular disease, especially a muscular dystrophy involving a defective dystrophin, i.e. a dystrophic disease.

[0183] “Dystrophic disease” in the context of the invention means a disease linked to a defect in the dystrophin gene. This defect can be deletions or mutations leading to low level of expression or absence of expression, introduction of a premature stop codon in the open reading frame, or the production of an inactive protein. Preferred dystrophic diseases are Duchenne and Becker muscular dystrophy (DMD / BMD) caused by mutations of the dystrophin gene. Said mutations can result in the absence or a low level of dystrophin expression, or in the production of a partially or fully inactive, possibly truncated, protein.

[0184] According to one embodiment, the invention concerns 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 invention concerns 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.

[0185] In other words, the present invention provides a method for treating a dystrophic disease especially DMD and BMD, in a subject, comprising administrating to the subject a dual AAV vector system as disclosed above or a composition comprising said system. A first target of the invention is to provide a safe (not toxic) and non-immunogenic treatment. A further aim is to provide an efficient treatment which allows to postpone, slow down or prevent the development of the disease, and possibly to ameliorate the phenotype of the patient which can be easily monitored at the clinical level. In a subject, AAV vectors and compositions according to the invention can be used: for ameliorating muscular function. Of particular interest are the skeletal muscles, but also the cardiac muscle and the diaphragm; for ameliorating gait; for ameliorating cardiac function; for ameliorating respiratory function; for prolonging survival, more generally to ameliorate the quality and the expectancy of life.

[0186] The amelioration of said functions can be assessed by the methods described in the examples below or known from the skilled person.

[0187] More generally and according to further embodiments, the invention is useful for: increasing muscular force, muscular endurance and / or muscle mass in a subject; reducing fibrosis in a subject; reducing contraction-induced injury in a subject;

[0188] - treating muscular dystrophy in a subject; reducing degenerating fibers or necrotic fibers in a subject suffering from muscular dystrophy; reducing inflammation in a subject suffering from muscular dystrophy; reducing levels of creatine kinase (or any other dystrophic marker) in a subject suffering from muscular dystrophy;

[0189] - treating myofiber atrophy and hypertrophy in a subject suffering from muscular dystrophy; decreasing dystrophic calcification in a subject suffering from muscular dystrophy; decreasing fatty infiltration in a subject; decreasing central nucleation in a subject.

[0190] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, fourth 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, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0191] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

[0192] 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.

[0193] EXPERIMENTAL EXAMPLES

[0194] The invention is further described in detail by reference to the following experimental examples and the attached figures. These examples are provided for purposes of illustration only, and are not intended to be limiting.

[0195] FIGURES

[0196] Figure 1: Scheme of the different forms of dystrophins:

[0197] A / Scheme of the full-length dystrophin

[0198] B / Scheme of the LP1 mididystrophin (AR4-R7AR14-R15AR18-R23) according to the invention

[0199] C / Scheme of the LP2 mididystrophin (AR2-R7AR13-R15AR18-R19) according to the invention.

[0200] D / Scheme of the LP3 midi dystrophin (AR4-R7AR10-R15AR18-R19) according to the invention.

[0201] Figure 2: production of the LP1 midi dystrophin using the overlap approach:

[0202] A / Scheme of the general strategy

[0203] B / Scheme of the first AAV vector of the dual AAV vector system C / Scheme of the second AAV vector of the dual AAV vector system

[0204] D / Scheme of the mididystrophin so obtained

[0205] Figure 3: production of the LP1 midi dystrophin using the intein approach:

[0206] A / Scheme of the general strategy

[0207] B / Scheme of the first AAV vector of the dual AAV vector system

[0208] C / Scheme of the second AAV vector of the dual AAV vector system

[0209] D / Scheme of the mididystrophin so obtained

[0210] Figure 4: production of the LP2 mididystrophin using the overlap approach:

[0211] A / Scheme of the general strategy

[0212] B / Scheme of the first AAV vector of the dual AAV vector system C / Scheme of the second

[0213] AAV vector of the dual AAV vector system

[0214] D / Scheme of the mididystrophin so obtained

[0215] Figure 5: production of the LP2 mididystrophin using the intein approach:

[0216] A / Scheme of the general strategy

[0217] B / Scheme of the first AAV vector of the dual AAV vector system

[0218] C / Scheme of the second AAV vector of the dual AAV vector system

[0219] D / Scheme of the mididystrophin so obtained

[0220] Figure 6: production of the LP3 mididystrophin using the overlap approach:

[0221] A / Scheme of the general strategy

[0222] B / Scheme of the first AAV vector of the dual AAV vector system

[0223] C / Scheme of the second AAV vector of the dual AAV vector system

[0224] D / Scheme of the mididystrophin so obtained

[0225] Figure 7: production of the LP3 mididystrophin using the intein approach:

[0226] A / Scheme of the general strategy

[0227] B / Scheme of the first AAV vector of the dual AAV vector system

[0228] C / Scheme of the second AAV vector of the dual AAV vector system

[0229] D / Scheme of the mididystrophin so obtained

[0230] Figure 8: Quantification of dystrophin production

[0231] Western blot analysis using the JESS system of Gastrocnecmius muscles from

[0232] - wild-type mice injected with saline buffer (WT) mdx mice injected with saline buffer (Saline) mdx mice injected with the dual AAV vector system of the invention ensuring the production of LP1 by the intein approach (Mi di -Dy s 1) mdx mice injected with the dual AAV vector system of the invention ensuring the production of LP2 by the intein approach (Mi di -Dy s 2) mdx mice injected with an AAV vector ensuring the production of MD1 as disclosed in the prior art (Micro-dys) Figure 9: Quantification of dystrophin positive fibers

[0233] The percentage (%) of positive dystrophin myofibers is shown in the Gastrocnecmius muscle (A) and in the Tibialis Anterior (TA) muscle (B) of wild-type mice injected with saline buffer (WT) (n=5) mdx mice injected with saline buffer (Saline) (n=5) mdx mice injected with the dual AAV vector system of the invention ensuring the production of LP1 (Mi di -Dy s 1) (n=5) mdx mice injected with the dual AAV vector system of the invention ensuring the production of LP2 (Mi di -Dy s 2) (n=5) mdx mice injected with an AAV vector ensuring the production of MD1 as disclosed in the prior art (Micro-dys) (n=5)

[0234] Figure 10: Fibrosis and calcium deposit quantification in the Gastrocnecmius muscle The percentage (%) of collagen deposit area (A) and of calcium deposit area (B) is shown for: wild-type mice injected with saline buffer (WT) (n=5) mdx mice injected with saline buffer (Saline) (n=5) mdx mice injected with the dual AAV vector system of the invention ensuring the production of LP1 (Mi di -Dy s 1) (n=5) mdx mice injected with the dual AAV vector system of the invention ensuring the production of LP2 (Mi di -Dy s 2) (n=5) mdx mice injected with an AAV vector ensuring the production of MD1 as disclosed in the prior art (Micro-dys) (n=5)

[0235] MATERIAL AND METHODS

[0236] 1 / Test of AAV9-encoding for LPl, LP2, LP3 and uDys in vitro using muscle organoids derived from DMD patients’ s induced pluripotent stem cells

[0237] AAV production and organoids infection

[0238] Recombinant AAV are produced at Genethon using AAV9 serotype. Purification is performed using affinity chromatography and titration is done by ddPCR using ITRs primers. For optimization of infection, an AAV9-CK8-GFP construct is used. The microdystrophin transgene used in the study, under the control of CK8 promoter, is an optimized version of construct used for GENETHON’ s preclinical investigation and clinical trial, with deletion from spectrin-like repeats 4 to 23 and full C-terminal truncation, here referred as pDys (see WO2015 / 197869). Infection in organoids is performed delivering the AAV9 particles into the differentiation media at day 7, at two different doses: 1E+10 vg / organoids (low dose) and 5E+10 vg / organoids (high dose). Media are replaced after 24 hr from the infection and changed daily until day 14.

[0239] Generation of MYOrganoids

[0240] MYOrganoids from iPSC are generated adapting the protocol described for engineered heart tissue (Tiburcy et al. 2020). In particular, 1.25 x 106hIPSC-committed (24 hours after induction with Doxycycline) are resuspended in 77 pl of growth media supplemented with hES cell Recovery (Stemgent) and molded in hydrogel composed by 40 pl of Bovine Collagen solution 6mg / ml (Sigma-Aldrich), 17.8 pl of Matrigel Growth Factor reduced (Coming) 10% v / v 3), 40pl 2X DMEM (Gibco) 4) and 5.2 pl of NaOH 1.5N 5). For the generation of MYOrganoids including Fibroblasts, 1.25 x 105(1 : 10) immortalized fibroblasts are included in the iP SC -committed mix before hydrogel inclusion. The hydrogels are casted into 48-well plate TM5 MyrPlate (Myriamed), containing in each well a pair of flexible poles (static stretchers) that supports the growth of the engineered tissue in a ring shape. After 1 hour of polymerization at 37°C, media is added for 24 hours. At day 2 of the 3D, growth media (SKM02) is replaced by differentiation media (SKM03plus) and changed every day until dayl4.

[0241] Muscle force analysis

[0242] Functional analyses are carried out at day 14 after 3D casting. Contraction experiments are performed using the MyoDynamics Muscle Strip System 840 MD (DMT Technologies) and CS4 stimulator (DMT Technologies). All functional analysis are performed at 37°C, 5% CO2 / 95% 02, in Tyrode’s solution supplemented with 25 mM NaHCO3. Optimal muscle length is determined by gradually stretching the muscle until there is no further increase in the twitch tension. Functional tests are performed under isometric and eccentric conditions. Organoids are electrically stimulated with 250 pulses of 30V, 4ms width at the 125Hz of frequency for both isometric and eccentric contractions. For eccentric analysis, organoids are 1 mm stretched at the 6.5 cm / s during the muscular contraction. Each organoid is subjected to 1 isometric contraction, 10 eccentric contractions and 1 isometric contraction. Data collection and analysis are done by Powerlab device and LabChart software (AD instruments) respectively. Fatigue is represented as percentage drop force between the first and the last isometric contraction. Force is normalized by CSA (Cross Section Area) and expressed as mN / mm2.

[0243] Protein expression analysis by WB and IHC Western blot analysis The reconstituted MIDI-dystrophin is monitored by Western Blot by size selection (range 260-280KDa). Organoids proteins are extracted in RIPA buffer supplemented with Protease Inhibitor Cocktail EDTA-free (Roche) and Benzonase by homogenization. Total proteins are then quantified by BCA method, thanks to the Pierce BCA protein assay kit (Invitrogen) accordingly to manufacturer’s instructions. Protein detection has been performed by capillary western blot, thanks to the JESS protein simple (Bio-techne), accordingly to manufacturer’s directions. Micro-dystrophin detection is performed by the antibody DysB and Dys 2, recognizing N-t and C-t respectively, and its expression has been quantified by total protein normalization.

[0244] Immunocytochemistry

[0245] MYOrganoids are fixed if 4% methanol-free PFA overnight at day 14. For whole mount staining, fixed MYOrganoids are permeabilized, stained and dehydrated with the MACS clearing kit (Miltenyi) accordingly to manufacturer’s instructions. Whole mount-stained organoids are then imaged with confocal microscope (LEICA STED SP8) at 10X magnification.

[0246] For staining on transversal or longitudinal sections, fixed MYOrganoids are dehydrated with a gradient of sucrose (7.5%-30%) over-day and embedded in OCT matrix in plastic mold. After 24 hr, embedded organoids are processed with the cryostat (LEICA) with 15 pm thick sections. Slices are then dried and fixed again with 4% methanol-free PFA (Invitrogen). Fixed sections are then blocked with serum cocktail (5% Goat serum and 5% Fetal bovine serum), before being stained overnight at +4°C with dystrophin primary antibodies. After that, slices are washed 3 times in PBS and hybridized with Al exaFluor secondary antibody accordingly to the host species of the first antibody. Stained slides are then covered with Fluoromont + Dapi (Ref) and glass slide 1.5H. For imaging, sections are scanned with AxioScan microscope and confocal Leica SP8.

[0247] 2 / Evaluation of therapeutic efficiency of constructs pre-selected in vitro in the aggressive DMD mouse model DBA2-mdx

[0248] Midi-Dystrophins design and viral particle preparation

[0249] Mi di -Dystrophin (LP1 and LP2; see below) protein sequence was derived from dystrophin muscular isoform (Dp427m; RefSeq: NM 004006.3) and optimized to remove the rare human codons, CpG and possible alternative ORFs by GeneArt service (Life Technologies).

[0250] Gene fragments were produced by GeneArt gene synthesis (Life Technologies) and cloned into a plasmid containing CK8 promoter, and poly A SV40. Plasmids were amplified by endotoxin-free methods. For recombinant AAV production, HEK293-T cells (obtained from Stanford University School of Medicine), cultured in suspension, were transfected with the three plasmids coding for the adenovirus helper proteins, the AAV Rep and Cap proteins, and the ITR- flanked transgene expression cassette.

[0251] Three days after transfection, cells were harvested, chemically lysed and treated with benzonase (Merck-Millipore, Darmstadt, Germany). After filtration, viral capsids were purified by affinity chromatography, formulated in sterile PBS, and the vector stocks were stored at -80 °C. Titers of AAV vector were determined by using digital droplet polymerase chain reaction (ddPCR). Viral particles were treated with DNAse I for 30 minutes at 37°C (Invitrogen, MA, USA) and then viral DNA was amplified by using poly A SV40 specific primers with ddPCR Supermix for probes (Biorad, CA, USA).

[0252] Capillary western blot

[0253] Capillary western blot of Mi di -Dy s (LP1 and LP2) and Micro-Dys (MD1) was performed on a Simple Western™ Jess system (ProteinSimple, Bio-Techne) according to the manufacturer’s instructions, using a 60-440 kDa separation module (ProteinSimple SMW004) and the anti-mouse detection module (Protein simple DM-002). In brief, protein samples consisting of midi-dystrophin or micro-dystrophin proteins were obtained from muscle lysates. Proteins were extracted in RIPA buffer and 2.5 ng were used for Dystrophin detection in mdx mice injected with vehicle, AAV-Mi di -Dystrophins or AAV- Micro-Dystrophin. DysB primary antibody was used for recognizing the N-term part of dystrophin (Leica, IL, US) and Mancho-11 primary antibody (DSHB, IA, US) was used for recognition of the C-term part of dystrophin.

[0254] Animal care and use

[0255] DBA2 (B6; 129S4-DBA2tmlCpr / J, strain #000671) and DBA2-mdx (D2.B10- Dmdmdx / J) mice were supplied by the Jackson Laboratory. All animal procedures were approved by the National Ethical Committee, C2EA 51 (Evry, France), and the French Ministry of Research (MESRI) and received a national agreement number (APAFIS #38006). Four-week-old Dba2_mdx and Dba2_WT male mice were injected by retro- orbital injection with dual Midi-dystrophins (LP1 and LP2) AAV9 vectors at the dose for each vector of 2E+13 vg / Kg (total dose of 4E+13 vg / Kg) and with Micro-Dystrophin AAV9 vector at the dose of 2 x 1013vg / kg. AAV9 injected mice were compared to Dba2_mdx mice injected with PBS (negative control) as well as to the Dba2 WT mice (positive control). Seven weeks post-injection, muscles and serum collected for molecular and histological analysis. 5 mice were tested per experimental condition. Histological staining

[0256] Skeletal muscles were sampled and frozen in isopentane cooled in liquid nitrogen. Transverse cryosections (8-10 pm) were prepared from frozen muscles, air dried, and stored at -80°C. Muscle sections were processed for Sirius red and red Alizarin histological staining. Sections were visualized on an Axioscan Z1 automated slide scanner (Zeiss, Germany), using a Plan APO 10X / 0.45 NA objective.

[0257] Sections were immunostained overnight at 4°C with primary antibodies specific for dystrophin (N term-, DysB, LEICA) and laminin (ThermoFisher, MA, USA). Following three washes with PBS, the muscle sections were incubated for 1 hour at room temperature with a goat secondary antibody conjugated with Alexa Fluor 488 or 594 dye (Molecular Probe, dilution 1 : 1000). The sections were then mounted using DAPI-Fluoromount-G (Southern Biotech) and visualized either on a LEICA TCS-SP8 confocal microscope (Leica, IL, USA) with a 63X APO CS2 1.4 NA objective or on an Axioscan Z1 automated slide scanner (Zeiss, Germany) with a Plan APO 10X / 0.45 NA objective.

[0258] Fibrosis and calcium deposit quantification

[0259] Sirius Red and Alizarin stained transverse sections were used for fibrosis and calcification assessment using QuPath (version 0.3.2). For each muscle scan, a small artificial neural network was trained to classify positive and negative pixels, and subsequently used to quantify fibrotic and calcified areas.

[0260] Dystrophin positive fibers quantification

[0261] Muscle sections were stained with laminin to label cell membranes. The Cellpose2 cyto2 model 10 was fine-tuned using manually labeled images of myofibers based on laminin staining. This fine-tuning was performed with hyperparameters set to 200 epochs, a learning rate of 0.05, and a weight decay of 0.0001. The labeled dataset was carefully prepared to allow the model to simultaneously segment myofibers while ignoring areas with low-quality staining.

[0262] Once fine-tuned, these models were utilized to extract myofiber masks. The reconstruction of myofiber masks from whole-scan images was performed using the Cellpose package 10. These reconstructed masks were then converted into Regions of Interest (ROIs), with each ROI corresponding to an individual myofiber, using the Labels To Rois.py FIJI plugin 11.

[0263] Muscle sections were co-labeled with laminin for membrane labeling and dystrophin for further analysis. As with the initial labeling, ROIs were generated based on membrane labeling and used for the subsequent quantification of dystrophin signals using a FIJI macro. Serum biomarkers quantification

[0264] For serum MY0M3 quantification, a custom sandwich ELISA assay was employed. Initially, a polyclonal MY0M3 antibody (Proteintech, 17692-1-AP) diluted 1 : 10 was coated onto a 96-well plate and incubated overnight at 4°C. After this incubation, the plate was washed three times with PBST and then blocked with a saturation solution of 3% BSA in PBS. Dilutions of the serum samples were then added to the plate and incubated for 2 hours at room temperature. For detection, a monoclonal antibody (Proteogenix, REF: 51- H1-B4) coupled with SULFO-TAG (MSD) was used, and the plate was incubated for an additional 2 hours at room temperature. After incubation, the plate was washed three times with wash buffer (0.05% Tween-20 in PBS). The absorbance of the SULFO-TAG was measured using the MESO Quickplex SQ 120 (MSD). To quantify the MY0M3 concentration in the serum samples, a set of concentration-defined MY0M3 peptides (His- tagged, Proteogenix) was included in the same experiment and served as the standard for calculation.

[0265] Creatine kinase (CK) measurement

[0266] CK quantifications are performed starting with 10 pl of mouse serum by colorimetric assay with the FUJI DRI-CHEM nx500 system (DMV Imaging), used to measure creatine phosphokinase concentration.

[0267] Muscle force evaluation

[0268] For the escape test, mice are placed inside 30 cm-long tube and attached to a horizontal tension transducer by their tail. In response to gentle pinching of the tail, the mice tried to escape within the tube. A short peak of force is recorded by the force transducer and, typically, 15 pinches are made, and the top 5 pulling tensions are averaged and divided by the weight of the mouse. The test can be done only once in a lifetime because of a memory effect. Data are reported as maximum peak and the mean of the five peaks normalized to body weight. In the 2-limb grip strength test, the grip force is measured using the grip strength meter (Bioseb https: / / www.bioseblab.com / accessed on 1 December 2021; France Grip Test 25N). Three independent measurements are performed, and the mean value of grip strength normalized to weight is calculated.

[0269] Statistical analysis

[0270] All data are analyzed by GraphPad Prism 9.5.1 software. Parametric tests such as t-tests and ANOVA were used for statistical comparison. To compare two groups, initially, the F-test was used to compare variances. If there was no difference in variances, a statistical comparison was performed using an unpaired t-test. To compare multiple groups, we used one-way ANOVA with Tukey’s correction for multiple comparison tests. Results were considered significantly different at p < 0.05. Graphs were generated using Graphpad Prism v9 or R version 3.6.2, The figures display the mean ± standard deviation.

[0271] RESULTS:

[0272] Construction of AAV vectors

[0273] Different recombinant AAV2 / 9 vectors were constructed using either the “INTEIN” approach or the “OVERLAP” approach of dual AAV vectors.

[0274] Both approaches are well known in the art. The “INTEIN” approach is e.g. disclosed in WOOl / 29243 and the “OVERLAP” approach is used in WO2020 / 193636 for the production of quasidystrophins. Table 1 : List of tested dystrophins

[0275] Production and activity of the mini / mididystrophins

[0276] Figure 8 shows that at the protein level, the microdystrophin according to the prior art is expressed at a level from 2.5 to 4 times superior to the midi dystrophins of the invention (LP1 and LP2) produced using the intein approach.

[0277] In terms of distribution and activity, figures 9 and 10 show that both midi dystrophins of the invention (LP1 and LP2) and the microdystrophin (MD1) of the prior art show a similar profile, especially an equal therapeutic efficacy.

[0278] Taking into account the different amounts of proteins present in the muscles, it can be concluded that the midi dystrophins according to the invention, when expressed at the same protein levels than the microdystrophin of the prior art, are more performant.

Claims

CLAIMS1. A mididystrophin comprising at least the rod domains Rl, R8, R9, RIO, R11, R12, R16, R17 and R24 of the dystrophin.

2. The mididystrophin according to claim 1 deprived of the R4, R5, R6, R7 rod domains (AR4-R7), ofthe R14, R15 rod domains (AR14-R15), and ofthe R18, R19, R20, R21, R22, R23 rod domains (AR18-R23).

3. The mididystrophin according to claim 2 having the structure CH1CH2H1R1R2R3H2R8R9R10R11R12R13R16R17R24H4CRCT.

4. The midi dystrophin according to claim 2 or 3 having the sequence SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

5. The mididystrophin according to claim 1 deprived of the R2, R3 R4, R5, R6, R7 rod domains (AR2-R7), of the R13, R14, R15 rod domains (AR13-R15), and of the R18, R19 rod domains (AR18-R19).

6. The mididystrophin according to claim 5 having the structure CH1CH2H1R1H2R8R9R10R11R12R16R17H3R20R21R22R23R24H4CRCT.

7. The midi dystrophin according to claim 5 or 6 having the sequence SEQ ID NO: 2 or a sequence having at least 90% identity thereto.

8. A nucleic acid sequence encoding the midi dystrophin according to claims 1 to 7, advantageously having a sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and a sequence having at least 70% identity thereto.

9. A dual AAV vector system comprising two AAV vectors, wherein a first AAV vector comprises, between 5’ and 3’ AAV ITRs, a first nucleic acid sequence that encodes a N-terminal part of a mididystrophin, and a second AAV vector comprises, between 5’ and 3’ AAV ITRs, a second nucleic acid sequence that encodes a C-terminal part of a mididystrophin, wherein the first and second nucleic acid sequences comprise an overlapping region that permits the production by recombination of the mididystrophin according to any of claims10. The dual AAV vector system according to claim 9, wherein the first nucleic acid sequence has the sequence SEQ ID NO: 11 or SEQ ID NO: 15 and the second nucleic acid sequence has the sequence SEQ ID NO: 12 or SEQ ID NO: 16.

11. A dual AAV vector system comprising two AAV vectors, wherein a first AAV vector comprises, between 5’ and 3’ AAV ITRs, a first nucleic acid sequence that encodes the N-terminal part of a mididystrophin fused to the N- terminal part of a split intein, advantageously from the GP41.1 gene, and a second AAV vector comprises, between 5’ and 3’ AAV ITRs, a second nucleic acid sequence that encodes the C-terminal part of the split intein, advantageously from the GP41.1 gene, fused to the C-terminal part of the midi dystrophin, wherein the first and second parts of the split inteins promote joining of the first and second parts of the mididystrophin, thereby permitting the production of the mididystrophin according to any of claims 1 to 7.

12. The dual AAV vector system according to claim 11, wherein the first nucleic acid sequence has the sequence SEQ ID NO: 9 or SEQ ID NO: 13 and the second nucleic acid sequence has the sequence SEQ ID NO: 10 or SEQ ID NO: 14.

13. A cell transduced with the dual AAV vector system according to any one of claims 9 to 12, advantageously a muscle cell.

14. A composition comprising, in a pharmaceutically acceptable carrier, the dual AAV vector system according to any of claims 9 to 12 or the cell according to claim 13.

15. The dual AAV vector system according to any of claims 9 to 12, the cell according to claim 13 or the composition according to claim 14 for use in a method for treating a muscular dystrophy, in particular a Duchenne muscular dystrophy (DMD).

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