Novel mididystrophin

Abstract

The present invention relates to mididystrophin comprising at least the rod domains R1, R8, R9, R10, R11, R12, R16, R17, and R24 of dystrophin, and to the use thereof for treating muscular dystrophy, particularly Duchenne muscular dystrophy (DMD).

Description

[Technical Field]

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

[0002] This application reports that the use of a dual AAV vector system enables the mass production of active cleaved dystrophin exhibiting important functional domains. [Background technology]

[0003] Duchenne muscular dystrophy (DMD) is the most common progressive muscle degenerative disease, affecting approximately 1 in 3,500 to 5,000 male births. DMD is caused by a deletion or mutation in the gene encoding dystrophin, located on the X chromosome. Dystrophin is required for the assembly of the dystrophin-glycoprotein complex, providing mechanical and functional connections between the cytoskeleton and extracellular matrix of muscle fibers. The absence of functional dystrophin leads to fibrosis, inflammation, necrosis, and muscle replacement by scarring and adipose tissue, resulting in progressive muscle weakness and premature death from respiratory and heart failure between the ages of 20 and 40 [Moser, H., Hum Genet, 1984. 66(1): 17-40].

[0004] A milder form of the disease, called Becker muscular dystrophy (BMD), is distinguished from DMD by its later onset, later reliance on wheelchair assistance, and longer lifespan. BMD typically corresponds to mutations that maintain the leading frame [Muntoni F et al, Lancet Neurol, 2003. 2(12): 731-40].

[0005] There are no available treatments or effective interventions for DMD [Rodino-Klapac, LR et al., Curr Neurol Neurosci Rep, 2013. 13(3): 332] or BMD. Conventional treatments are limited to supportive care, partially alleviating signs and symptoms but not directly targeting the disease mechanism or reversing the phenotype.

[0006] Currently, several treatment schemes are being developed for DMD, including in vivo gene therapy, cell transplantation therapy, pharmacological rescue and exon skipping of DMD nonsense mutations, or gene editing schemes to repair the dystrophin gene reading frame. All of these schemes have challenges to overcome, including efficiency, targeting different muscle groups, optimizing delivery, long-term expression of transgenes, and potential immune responses [Jarmin et al., Expert Opin Biol Ther, 2014. 14(2): 209-30].

[0007] Various gene transfer methods for DMD aim to compensate for dystrophin loss of function and offer the potential to treat all patients with a single drug.

[0008] The dystrophin gene is the largest known gene in the human genome, exceeding 2.5 Mb or approximately 2% of the entire human X chromosome. The dystrophin gene consists of 79 exons (full-length cDNA: 11.1 kb) and encodes the dystrophin protein, which has 3685 amino acids and a density of 427 kD. The dystrophin protein is defined by four structural regions (Figure 1A): the NH2-terminal actin-binding domain (exons 1-8), the intermediate rod domain (24 spectrin-like repeat sequences R1-24 and 4 hinge regions H1-4; exons 9-62), the cysteine-rich (CR) domain (exons 63-69), and the carboxy-terminal (CT) domain (exons 70-79).

[0009] Due to its excessively large cDNA size, it cannot be accommodated in known gene therapy vector systems, and in particular, in adeno-associated virus (AAV) vectors, which are one of the promising candidates for efficient gene transfer to various muscle groups depending on the AAV serotype orientation. The AAV vector has the potential to exhibit long-term genetic transduction in both dividing muscle cells (muscle fibers and cardiomyocytes) and non-dividing muscle cells (mature myotubes).

[0010] In fact, the main limitation of AAV is its cargo capacity, which is thought to be limited to approximately 5kb, the genome size of the parent virus [Wu Z. et al., Mol Ther., 2010, 18(1): 80-86; Lai Y. et al., Mol Ther., 2010, 18(1): 75-79; Wang Y. et al., Hum Gene Ther Methods, 2012, 23(4): 225-33]. Larger vector genomes resulted in fragmentation of the packaged genome, a heterogeneous genome population with a wide size distribution, and lower expression efficiency [Wu Z. et al., Mol Ther., 2010, 18(1): 80-86].

[0011] To overcome the DNA packaging limitations of AAV (less than 5kb), several research groups are attempting to create cleaved but functional synthetic dystrophins [MD, also known as "microdystrophins" or "minidystrophins"]. A series of microdystrophins are designed to encode cleaved dystrophins optimized to contain more clinically important regions of the protein. Such regions are generally thought to be located within the N-terminal domain and cysteine-rich domain of dystrophin.

[0012] A microdystrophin named MD1, containing the first three and last of 24 spectrin-like repeat sequences and lacking a C-terminal domain (ΔR4~R23 / ΔCT), exhibited highly functional activity, restoring dystrophin and co-localizing with syntrophin and dystroblevin, but failed to mobilize nNOS at the muscle cell membrane in the mdx mouse model [Yue et al., Mol Ther, 2006. 14(1): 79-87]. Its therapeutic value has been reported in the literature WO2015 / 197869.

[0013] However, the relationship between deleted regions, such as the R16-R17 nNOS binding sites and / or the R8-R9 Par1b binding sites and / or the R10-R17 binding sites and F-actin, and their role in muscle function remains unclear.

[0014] Further plans have proposed the production of larger microdystrophins, i.e., quasi-dystrophins, which depend on recombinant events and utilize a dual AAV vector system. As is well known in the art, the two vectors in the dual AAV system may be duplicate vectors, trans-splicing AAV vectors, or hybrid trans-splicing AAV vectors (see, e.g., Pryadkina et al., Molecular Therapy, 2015, 2, 15009).

[0015] Based on this, Kodippili et al. (Human Gene Therapy, 2018, 29 (3), 299-311) reported the expression of canine ΔH2~R15 mini-dystrophin using a pair of dual AAV9 vectors in a canine model of Duchenne muscular dystrophy (DMD).

[0016] In addition, two further pseudo-dystrophins (WL1: ΔR4~R7ΔR10~R15ΔR18~R19, and WL2: ΔR4~R7ΔR10~R13ΔR18~R21) have been reported in reference WO2020 / 193636 (USA 2022 / 204574) and Albini S. et al. (International Journal of Molecular Sciences, 2023, 24(14), p. 11421).

[0017] Furthermore, the use of split inteins to produce larger versions of cleaved polypeptides, such as ΔR5~R15 pseudodystrophin, has been reported in references WO2023 / 004125 and Tasfaout H. et al. (Research Square, 2023, doi.org / 1.21203 / rs.3-2924001 / v1).

[0018] In any case, there is still a need in this field for producing high levels of active cleavage-type dystrophin. [Overview of the project] [Problems that the invention aims to solve]

[0019] The present invention aims to alleviate or cure destructive Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) by expressing a shorter but functional dystrophin polypeptide called mididystrophin using a dual AAV vector system.

[0020] In recent years, it has actually been revealed that DMD pathology is caused by myofiber fragility and dysfunction of muscle stem cells that impairs muscle regeneration and leads to muscle wasting. The impact of satellite cell dysfunction in DMD-related muscle wasting is a relatively recent discovery and could significantly improve current AAV-dystrophin delivery-based programs by preserving important binding sites and important molecular functions of dystrophin.

Means for Solving the Problems

[0021] In particular, dystrophin constructs according to the present invention differ in the inclusion of domains that are key to enhancing sarcolemma (muscle cell membrane) binding, cytoskeleton (actin) binding or microtubule binding activity. They provide new therapeutic tools useful for membrane stability of muscle fibers and, as a result, muscle force generation.

[0022] Definitions The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0023] "About" or "approximately" when used herein in reference to a measurable value such as an amount, a temporal duration, etc., means an inclusion of a variation of ±20%, ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0. The variation of 1% from a particular value and such variation is appropriate for performing the disclosed method.

[0024] Scope: Throughout this disclosure, various aspects of the invention may be presented in range form. It should be understood that descriptions in range form are merely for convenience and brevity and should not be interpreted as inflexible limitations on the scope of the invention. Therefore, a range description should be considered to include all possible subranges and the individual numerical values ​​within those ranges, as specifically disclosed. For example, a range description such as 1-6 should be considered to include the specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numerical values ​​within those ranges, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0025] "Isolated" means that it has been modified or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from its naturally occurring coexisting material is "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-native environment, such as a host cell.

[0026] In the context of this invention, the following abbreviations for commonly existing nucleic acid bases are used: "A" refers to adenosine, "C" to cytosine, "G" to guanosine, "T" to thymidine, and "U" to uridine.

[0027] Unless otherwise specified, “nucleotide sequences encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Furthermore, the phrase “nucleotide sequences encoding a protein, RNA, or cDNA” may include introns to the extent that a nucleotide sequence encoding a protein contains introns in some versions.

[0028] "Code" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to act as a template for the synthesis of other polymers and macromolecules having either a specific nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a specific amino acid sequence in biological processes, as well as the resulting biological properties. Therefore, if the transcription and translation of mRNA corresponding to a gene produce a protein in a cell or other biological system, that gene codes for a protein. Both the coding and non-coding strands, whose nucleotide sequences are identical to the mRNA sequence and are typically shown in sequence listings, and which are used as templates for the transcription of a gene or cDNA, can be said to code for a protein, or other products of that gene or cDNA.

[0029] 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 are interchangeable as used herein. Those skilled in the art have general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed to monomeric “nucleotides.” Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., as well as by synthetic means.

[0030] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds containing amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, this term refers to both short chains, also commonly called, for example, peptides, oligopeptides, and oligomers in the art, and longer chains, of which there are many types, commonly called proteins in the art. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among many others. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0031] "Identical" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. Molecules are homologous or identical at a given position if the positions in both sequences being compared are occupied by the same base or amino acid monomer subunit—for example, if the positions in each of two DNA molecules are occupied by adenine. The percentage 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 being compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences match, the two sequences are 60% identical. Generally, a comparison is made when the two sequences are aligned and give maximum homology / identity.

[0032] A “vector” is a composition of substances containing isolated nucleic acids that can be used to deliver isolated nucleic acids into the interior of a cell. Many vectors are known in the art and include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors.

[0033] An "expression vector" refers to a vector containing recombinant polynucleotides that include an expression control sequence operably ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be supplied by host cells or in an in vitro expression system. Expression vectors include all vectors known in the art that incorporate recombinant polynucleotides, such as cosmids, plasmids (e.g., naked or liposome-containing), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0034] As used herein, the term "promoter" is defined as a DNA sequence that is recognized by a cellular synthetic mechanism, or introduced synthetic mechanism, and is required to initiate a specific transcription of a polynucleotide sequence.

[0035] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence required for the expression of a gene product operably ligated to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, it may also include an enhancer sequence and other regulatory elements required for the expression of the gene product. The promoter / regulatory sequence may, for example, be a promoter / regulatory sequence that expresses a gene product in a tissue-specific manner.

[0036] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or identifying a gene product, results in the gene product being produced within the cell under most or all physiological conditions.

[0037] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or identifying a gene product, results in the gene product being produced in the cell only if a corresponding inducer is present within the cell.

[0038] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that codes for or is identified by a gene, results in the preferential production of a gene product within the cell if the cell is a tissue type corresponding to the promoter.

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

[0040] "Disease" is a health condition in which an animal is unable to maintain homeostasis, and if the disease is not treated, the animal's health continues to deteriorate. In contrast, "disorder" in animals is a health condition in which the animal is able to maintain homeostasis, but the animal's health is less desirable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily lead to a further decline in the animal's health.

[0041] A disease or disorder is “relieved” or “improved” if the severity of its symptoms, the frequency with which the patient experiences such symptoms, or both are reduced. This also includes halting the progression of the disease or disorder. A disease or disorder is “cured” if the severity of its symptoms, the frequency with which the patient experiences such symptoms, or both are eliminated.

[0042] "Therapeutic" treatments are procedures administered to subjects exhibiting pathological signs with the aim of reducing 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 the subject. Diseases and disorders are used interchangeably herein in the context of treatment.

[0044] The “effective dose” of a compound is the amount of the compound sufficient to provide a beneficial effect to the target to which it is administered. The phrase “therapeutic effective dose,” as used herein, means a sufficient or effective amount to prevent or treat a disease or condition (delaying or preventing its onset, preventing, inhibiting, reducing, or reversing its progression), including alleviating the symptoms of the disease. The “effective dose” of a delivery medium is the amount sufficient to effectively bind to or deliver the compound. [Modes for carrying out the invention]

[0045] According to a first aspect, the present invention relates to mididystrophin, preferably functional mididystrophin, and more preferably mididystrophin of human origin.

[0046] Within the framework of this application, “mididystrophin” means a peptide or protein shorter than native or wild-type dystrophin. In the context of the present invention, the terms “mididystrophin,” “microdystrophin,” “minidystrophin,” and “pseudodystrophin” have the same meaning.

[0047] According to certain embodiments, the mididystrophin according to the present invention has a size of less than 2400 amino acids (aa), which is the typical size of a so-called pseudodystrophin, or even less than 2350 or 2300 aa. According to further embodiments, the mididystrophin according to the present invention has a size of more than 1200 aa, which is the size of a 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 more than 2100 aa.

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

[0049] With respect to the human version, this means that, in accordance with the present invention, mididystrophin contains more than 2137 amino acids, more preferably more than 2200 amino acids, for example, 2139aa (LP1 in example), 2294aa (LP2 in example), and 2206aa (LP3 in example).

[0050] The structure of dystrophin has been well-established (see Figure 1A), and its active fragment has been disclosed. As can be understood in the art, the active fragment is a part or portion of the full-length sequence that retains at least some of the biological functions of the full-length sequence.

[0051] "Functional" cleaved 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 can at least partially alleviate one or more of the symptoms associated with the absence of native dystrophin, in particular fibrosis, inflammation, necrosis, muscle replacement by scarring and adipose tissue, muscle weakness, respiratory failure and heart failure, and premature death.

[0052] The mididystrophin according to the present invention preferably exhibits (to a greater or lesser extent) at least one of the properties disclosed in relation to prior art microdystrophins, particularly 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 the many characteristics, the desirable ones are: - Binding to at least one DAP ("dystrophin-related protein"), particularly actin (F-actin), synemin, syntrophin, dystroblevin, nNOS and / or PAR-1b protein, - Recruitment of the DAP complex in the muscle cell membrane via lipid binding, - Rescue of microtubule networks, - Muscle protection from injury, - Conservation of the overall protein structure and organization of spectrin repeat (R) domains. - Restoration of muscle structure and function (skeletal muscle, and also cardiac muscle and diaphragm, which are of particular interest), - More generally, improvements in muscle function, walking ability, cardiac function, respiratory function, survival, quality of life, and / or life expectancy. That is the case.

[0054] As is well known in the art, the properties can be tested in vitro in various cells expressing dystrophin, ex vivo in muscle fibers isolated from various animal models, for example using muscle organoids derived from induced pluripotent stem cells of DMD patients, or in vivo based on animal models or even patients suffering from DMD or BMD. Animal models include, for example, the mdx mouse [Foster H. et al., Mol Ther, 2008. 16(11): p. 1825-32], mdx 4cv These include mice [Decrouy et al., Gen Ther, 1997. 4(5): 401-8], D2.B10-mdx / J mice [Coley et al., Human Molecular Genetics, 2016. 25(1): 130-45], CXMDj dogs [Koo et al., J Gene Med, 2011. 13(9): 497-506], or GRMD dogs [Le Guiner et al., Mol Ther., 2014. 22(11): 1923-35]. Mouse models are commonly used to test novel constructs encoding microdystrophins. However, this model has drawbacks because mice exhibit less severe forms of the disease that do not involve an immune response. Another animal model is the dog, which is considered more reliable for predicting the therapeutic potential of gene therapy products in humans. Furthermore, the rat model disclosed by Larcher et al. (PLOS One, 2014, 9(10),e110371) is very interesting because it exhibits cardiomyopathy.

[0055] As mentioned above, full-length human dystrophin (Figure 1A) has various domains, - N-terminal domain (CH1CH2), - 4 hinged domains (H1~H4), - 24 spectrin-like repeat sequences or rod domains (R1~R24), - Cysteine-rich (CR) domain, - C-terminal (CT) domain It is characterized by:

[0056] According to one embodiment, the mididystrophin according to the present invention has at least one domain which is advantageously lacking at least one spectrin-like repeat sequence (R) compared to full-length dystrophin.

[0057] According to one embodiment, the mididystrophin of the present invention contains at least one important protein binding site, particularly for F-actin, the nNOS protein, and the PAR-1b protein. Advantageously, the mididystrophin of interest contains binding sites for nNOS and / or PAR1b. The nNOS binding site has been shown to be located in the repeat sequences 16 and 17 (R16, R17) of the rod domain (Lai et al., J. Clin. Invest., 2009. 119:624-635), while the binding of dystrophin repeat sequences 8 and 9 (R8, R9) to PAR1b has been demonstrated in vitro (Yamashita et al., Biochem. Biophys. Res. Commun., 2010. 391: 812-817). According to a preferred embodiment, the mididystrophin of interest contains the R16-17 and / or R8-9 rod domains.

[0058] In accordance with this embodiment, the present invention relates to a mididystrophin comprising at least one, and even more than two, three, or even four, rod domains selected from the group consisting of R8, R9, R16, and R17. According to a particular embodiment, the mididystrophin according to the present invention contains the R8, R9, R16, and R17 rod domains.

[0059] Further functions of the rod domain (R) in human dystrophin have been reported. - R1, R2, R3, R10, R11, and R12 (in addition to the cysteine-rich domain) bind to lipids and therefore contribute to the recruitment of the DAP complex in the muscle cell membrane. - R11, R12, R13, R14, and R15 (in addition to CH1 and CH2) bind to F-actin. - R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R20, R21, R22, R23 and R24 bind to microtubules.

[0060] In accordance with this embodiment, the present invention is - At least one or more rod domains selected from the group consisting of R1, R2, R3, R10, R11, and R12 (according to a particular embodiment, the mididystrophin according to the present invention includes at least R1, R10, R11, and R12; according to another embodiment, the mididystrophin according to the present invention includes at least R1, R2, and R3), and / or - At least one or more rod domains selected from the group consisting of R10, R11, R12, R13, R14, R15, R16 and R17, or R11, R12 and possibly R13 (according to a particular embodiment, the mididystrophin according to the present invention includes at least R11 and R12; according to another embodiment, the mididystrophin according to the present invention includes at least R11, R12 and R13), and / or - At least one or more rod domains selected from the group consisting of R10, R11, R12, R13, R20, R21, R22, R23, and R24 (according to a particular embodiment, the mididystrophin according to the present invention includes at least R24; according to another embodiment, the mididystrophin according to the present invention includes R20, R21, R22, R23, and R24) Regarding mididystrophin, including.

[0061] In another embodiment, the mididystrophin of the present invention is characterized by an N-terminal portion lacking or having at least R4, R5, R6, and R7 rod domains (ΔR4~R7).

[0062] In other words, the mididystrophin according to the present invention has the following defined N-terminal portion, starting from its N-terminus in the following order: - N-terminal domain (CH1CH2) that binds to actin, - H1 hinge domain, - R1 rod domain or R1, R2 and R3 rod domains - H2 hinge, - R8 and R9 rod domains Such mididystrophines exhibit the structure CH1CH2H1R1R2R3H2R8R9 or CH1CH2H1R1H2R8R9 in their N-terminal region.

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

[0064] According to a particular embodiment, the mididystrophin of the present invention contains at least a spectrin-like repeat sequence of prior art MD1 microdystrophin, i.e., at least R1 and R24 or even R1, R2, R3 and R24.

[0065] Advantageously, the mididystrophin according to the present invention contains additional rod (R) domains compared to MD1 microdystrophin, preferably at least one domain selected from the group consisting of R10, R11, R12, R13, R20, R21, R22, and R23.

[0066] According to a particular embodiment, the mididistrophin according to the present invention has the following rod domains, - R4~R7, R14~R15 and R18~R23 [in other words, such mididystrophin (LP1 below) contains R1~R3, R8~R13, R16~R17 and R24], or - R2~R7, R13~R15 and R18~R19 [in other words, such mididystrophin (LP2 below) contains R1, R8~R12, R16~R17 and R20~R24], or - R4~R7, R10~R15 and R18~R19 [In other words, such mididystrophin (LP3 below) contains R1~R3, R8~R9, R16~R17 and R20~R24] It lacks.

[0067] To its advantage, that is, - For example, the complete N-terminal domain corresponding to amino acids 1-246 of Sequence ID No. 1, - The complete cysteine-rich (CR) domain corresponding to amino acids 3080-3360 of full-length dystrophin. - Partial or full-length C-terminal domain, preferably a full-length C-terminal domain corresponding to amino acids 3361-3685 of full-length dystrophin (possibly a partial C-terminal domain is a cleaved C-terminal domain of MD1, MD2, MD3, or MD4 as disclosed in WO2016 / 177911), - Selected from the group consisting of H1, H2, H3, and H4, possibly at least one hinge (H) domain, favorably at least H1, H2, and H4, possibly H1, H2, H3, and H4 It further contains

[0068] According to a particular embodiment, the mididystrophin according to the present invention is - ΔR4~R7ΔR14~R15ΔR18~R23 mididystrophin, preferably the mididystrophin of the structure CH1CH2H1R1R2R3H2R8R9R10R11R12R13R16R17R24H4CRCT (further designated as LP1), more preferably the mididystrophin of the sequence of sequence number 1, or - ΔR2~R7ΔR13~R15ΔR18~R19 mididystrophin, favorably mididystrophin of structure CH1CH2H1R1H2R8R9R10R11R12R16R17H3R20R21R22R23R24H4CRCT (further named LP2), more favorably mididystrophin of sequence number 2, or - ΔR4~R7ΔR10~R15ΔR18~R19 mididystrophin, favorably structured CH1CH2H1R1R2R3H2R8R9R16R17H3R20R21R22R23R24H4CRCT mididystrophin (further named LP3), more favorably sequenced mididystrophin of sequence number 3 That is the case.

[0069] According to one embodiment, a mididystrophin is "substantially identical" to the mididystrophins disclosed herein, particularly the sequences of SEQ ID NOs: 1 to 3, favorably SEQ ID NOs: 1 to 2, i.e., 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. According to one embodiment, such a mididystrophin is functional and may have the same function / activity as the mididystrophins encoded by SEQ ID NOs: 1 to 3, favorably SEQ ID NOs: 1 to 2.

[0070] In a further embodiment, the present invention relates to a nucleic acid sequence encoding a mididystrophin as defined above.

[0071] According to one embodiment, the nucleic acid sequence encoding mididystrophin according to the present invention comprises or comprises sequences selected from the group consisting of SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8, and preferably SEQ ID NOs. 4 to SEQ ID NOs. 6.

[0072] According to another embodiment, the nucleic acid sequence encoding mididystrophin according to the present invention is "substantially identical" to the sequences of SEQ ID NOs: 4 to 8, preferably any of SEQ ID NOs: 4 to 6, i.e., 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.

[0073] The expression system according to the present invention typically consists of two AAV vectors, i.e., a so-called dual AAV vector system.

[0074] According to a preferred embodiment, the mididystrophin of the present invention is produced using such a dual AAV vector system.

[0075] A "dual AAV system" means a vector system consisting of two AAV vectors, each of which has a portion of the sequence encoding the mididystrophin of the present invention, which is delivered to the cell, and the open reading frame (ORF) encoding the mididystrophin is reassembled into the cell by interaction between the first and second nucleic acid sequences.

[0076] In another embodiment, the present invention also relates to an AAV vector corresponding to a first AAV vector or a second AAV vector in a dual AAV vector system according to the present invention.

[0077] In other words, the present invention relates to a composition comprising a recombinant adeno-associated virus (AAV) vector, preferably two in number, having a complementary construct that enables the expression of the functional mididystrophin of the present invention.

[0078] Within the framework of this invention, the term “composition” can be replaced by “association,” “combination,” or “expression system.” This means that two AAV vectors must work together and be in contact so that the desired active protein can be produced. However, they may be found in a single composition or in two separate compositions that may be mixed before use.

[0079] In accordance with the present invention, each adeno-associated virus (AAV) vector in the system includes an expression construct, also named “expression cassette” or “insert.” Within the framework of this application, the “insert” is defined as a nucleic acid sequence advantageously located between the 5' and 3' ITR (“terminal inversion” sequences) of the AAV genome.

[0080] According to common knowledge in the relevant technical field, the size of the insert should not significantly exceed the length of the wild-type AAV genome. For example, AAV2 contains two ITR sequences, each 145 bp long, and has a genome (including the ITR sequences) of 4682 pb.

[0081] In certain embodiments, the nucleic acid sequence encoding a portion of mididystrophin, introduced into each AAV vector, has a length of less than 5 kb, for example, less than 4.9, 4.8, 4.7, 4.6, or 4.5 kb.

[0082] Advantageously, to limit the size constraints of AAV packaging, the nucleic acid sequences correspond to exons. In other words, they are preferably cDNA fragments.

[0083] Advantageously, the leading frame formed by combining two AAVs encodes the mididystrophin (MD) disclosed above, advantageously functional mididystrophin, and more advantageously human-derived mididystrophin.

[0084] According to a preferred embodiment, the mididystrophin produced by the claimed dual AAV vector system contains at least 2000 amino acids (aa), preferably at least 2100 aa, 2200 aa, or 2300 aa. According to another embodiment, the microdystrophin produced by the claimed AAV vector contains 3000 or fewer amino acids (aa), preferably 2900 or fewer aa, 2800 or fewer aa, 2700 or fewer aa, 2600 or fewer aa, 2550 or fewer aa, or 2500 or fewer aa. In a preferred embodiment, the mididystrophin produced by the claimed dual AAV vector system contains 2100 to 2300 aa, i.e., has a size corresponding to about 60% of the size of full-length dystrophin.

[0085] Each AAV vector contains nucleic acid sequences encoding the relevant portions of the dystrophin gene (the N-terminal and C-terminal portions, respectively), preferably the nucleic acid sequences defined above, but also all sequences necessary for the proper expression of mididystrophin after the reconstruction of the entire gene or protein.

[0086] In one embodiment, particularly in the duplication method, the first and second nucleic acid sequences of the first and second AAV vectors are controlled by one or more regulatory sequences. Advantageously, the first nucleic acid sequence may be preceded by a promoter followed by an intron, and the second nucleic acid sequence may be followed by a polyadenylation signal. Alternatively, particularly in the intein method, the first and second nucleic acid sequences may be preceded by a promoter followed by an intron, and then followed by a polyadenylation signal.

[0087] According to a particular embodiment, the first adeno-associated virus (AAV) vector is: i) AAV5'ITR (terminal inversion sequence), ii) Genetic regions controlled by promoters, iii) AAV3'ITR sequence Includes.

[0088] In addition, the second adeno-associated virus (AAV) vector is iv) AAV5'ITR (terminal inversion sequence) sequence, v) the gene portion, and favorably, the subsequent polyadenylation signal, vi) AAV3'ITR array Includes.

[0089] According to another specific embodiment, both the first and second adeno-associated virus (AAV) vectors are: - AAV5'ITR (terminal inversion sequence) sequence, - The gene portion controlled by the promoter, and favorably, the subsequent polyadenylation signal, - AAV3'ITR array Includes.

[0090] Such promoters may be natural or synthetic (artificial) promoters, and may be inductive or constitutive.

[0091] In one embodiment, the promoter is either a ubiquitous promoter or has low tissue specificity. For example, the expression vector may contain phosphoglycerate kinase 1 (PGK), EF1, ACTA1, β-actin, desmin, all MCK mutants, cardiac troponin, and CMV promoters.

[0092] In a preferred embodiment, the promoter sequence is selected to adequately control the expression of the nucleic acid sequence under its control, both in terms of expression level and tissue specificity.

[0093] In one embodiment, the expression vector includes a muscle-specific promoter. Such a promoter enables robust expression in skeletal muscle, in the diaphragm, and possibly in cardiac muscle, as well as in satellite cells. Examples of suitable promoters known to those skilled in the art include, for example, the desmin promoter, muscle creatine kinase (MCK) promoter, cleavage-type creatine kinase promoters, such as CK6, CK7, or CK8 promoters, the Syn promoter, MyoD, Myf5, Vcam, Pax3, and Pax7 satellite cell promoters. Another promoter is the synthetic promoter C5-12 (spC5-12). It is also possible to use hybrid promoters containing sequences from two or more transcriptional regulatory elements (see, for example, PCT / EP2019 / 053061).

[0094] Advantageously, the first nucleic acid sequence is placed under the control of a muscle-specific promoter. In other words, the first AAV vector further includes a muscle-specific promoter operably ligated to the nucleic acid sequence encoding the N-terminal portion of dystrophin.

[0095] As is well known in the art, there is no exhaustive list of other regulatory sequences that may be introduced into one AAV vector or another AAV vector. - Polyadenylation signal, favorably, polyadenylation signal at 3' of the sequence encoding a functional microdystrophin, - Sequences for transcript stabilization, e.g., introns, - Enhancer array, - miRNA target sequences that can inhibit the expression of functional dystrophin-coding sequences in non-target tissues where such expression is undesirable, for example, in non-target tissues where it may be toxic. That is the case.

[0096] In one embodiment, the intron is selected from the group consisting of human betaglobin b2 (or HBB2) introns, FIX introns, and chicken betaglobin introns, and the intron may be a modified intron, such as a modified HBB2 intron, a modified FIX intron, or a modified chicken betaglobin intron.

[0097] According to another embodiment, the polyadenylation signal is selected from the human betaglobin polyadenylation signal, the bovine growth hormone polyadenylation signal, the SV40 polyadenylation (pA) signal, or another naturally occurring or artificial polyadenylation signal.

[0098] According to a specific embodiment, the dual AAV vector system according to the present invention comprises the following elements: - CK8 promoter, favorably the CK8 promoter of sequence 23, and - SV40 polyA signal, favorably SV40 polyA signal of sequence number 24, - Chimeraintron, advantageously chimeraintron of sequence number 25 It contains at least one of them, or more favorably, all of them.

[0099] According to the first embodiment, such a dual AAV vector system is dedicated to the so-called duplication method. After in vivo recombination based on the duplication region shared by the two AAV vectors, the resulting mididystrophin is produced in high quantities. Therefore, the nucleic acid sequence encoding mididystrophin is divided into two parts: a 5' sequence encoding its N-terminal portion and a 3' sequence encoding its C-terminal portion. The 3' end of the 5' sequence and the 5' end of the 3' sequence are homologous or even identical so that homologous recombination can occur.

[0100] In a further embodiment, the present invention relates to a dual AAV vector system comprising two AAV vectors, The first AAV vector contains a first nucleic acid sequence encoding the N-terminal portion of mididystrophin between the 5' and 3' AAV ITRs. The second AAV vector contains a second nucleic acid sequence encoding the C-terminal portion of mididystrophin between the 5' and 3' AAV ITRs. The first and second nucleic acid sequences include overlapping regions that enable the production of the mididystrophin of the present invention by recombination. Regarding dual AAV vector systems.

[0101] Within the framework of the present invention, and as described below, the terms “overlapping region” (or “overlapping sequence”) and “homologous region” (or “sequence homology region”) have the same meaning and are used interchangeably.

[0102] These two AAV vectors have complementary sequences that form functional units upon recombination. As is well known to those skilled in the art, recombination occurs through the recognition of homologous sequences present in each AAV vector by the cellular DNA repair pathway.

[0103] Therefore, the gene portions of the two AAV vectors must meet the following requirements: - The gene portions of the first and second AAV vectors both include an open reading frame encoding the mididystrophin of the present invention, preferably an open reading frame encoding a mididystrophin of human origin. - Both the gene portions of the first and second AAV vectors contain homologous regions that enable the reconstruction of the open reading frame after homologous recombination. The following conditions must be met.

[0104] In this embodiment, the dual-vector system of the present invention implements a vector containing a sequence that enables recombination (homologous recombination), i.e., a duplicate vector. Therefore, the mididystrophin protein is reconstituted by performing homologous recombination by adding an appropriate duplicate region to each portion of the dystrophin gene introduced into each AAV vector.

[0105] In the dual-vector system according to this first embodiment, the first and second nucleic acid sequences represent a region of sequence homology that promotes intermolecular homologous recombination, and thus a large mididystrophin transgene is generated by recombining the two vectors of the dual AAV system, i.e., the nucleic acid sequences encoding the N-terminal and C-terminal portions of mididystrophin, respectively. In certain embodiments implementing this duplication system, the length of the region of sequence homology may vary considerably, as long as the size of the resulting insert (including 5' and 3'-ITR sequences and any expression regulatory sequences) is compatible with the size limitations of capsid formation within the AAV vector. Those skilled in the art are well aware of this size limitation and can adjust the sizes of both the first and second nucleic acid sequences, and therefore the size of the region of sequence homology, accordingly. Therefore, in certain embodiments, the sequence homology region is a polynucleotide sequence of the dystrophin gene having a length of less than 4599 nucleotides, for example, less than 4500, 4000, 3500, 3000, 2500, 2000, 1500, or 1000 nucleotides. In another particular embodiment, the sequence homology region is a polynucleotide sequence of the mididystrophin sequence having a length of at least 100 nucleotides, for example, at least 100, 200, 300, 400, 500, 600, 700, or 800 nucleotides. In yet another particular embodiment, the sequence homology region is a polynucleotide sequence of the dystrophin gene having a length of 500 to 1500 nucleotides, particularly 700 to 1500 nucleotides, favorably 800 to 1450 nucleotides.

[0106] According to a particular embodiment, the first nucleic acid sequence includes or comprises SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO: 19, and the second nucleic acid sequence includes or comprises SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 20.

[0107] In one embodiment, the first nucleic acid sequence may encode the N-terminal domain, H1, R1, R2, R3, H2, R8, R9, R10, R11, R12 and cleaved R13 of dystrophin, where the second nucleic acid sequence encodes the R9, R10, R11, R12, R13, R16, R17, R24, H4, CR domain and C-terminal domain of dystrophin (full-length or cleaved, preferably full-length as disclosed above).

[0108] According to a particular embodiment, the first nucleic acid sequence includes or comprises SEQ ID NO: 11, and the second nucleic acid sequence includes or comprises SEQ ID NO: 12.

[0109] In another embodiment, the first nucleic acid sequence may encode the N-terminal domain, H1, R1, H2, R8, R9, R10, R11, R12, R16, R17 and cleaved R20 of dystrophin, where the second nucleic acid sequence encodes the R6, R17, H3, R20, R21, R22, R23, R24, H4, CR domain and C-terminal domain of dystrophin (full-length or cleaved, preferably full-length as disclosed above).

[0110] According to further specific embodiments, the first nucleic acid sequence includes or comprises SEQ ID NO: 15, and the second nucleic acid sequence includes or comprises SEQ ID NO: 16.

[0111] In a further embodiment, the first nucleic acid sequence may encode the N-terminal domains of dystrophin, H1, R1, R2, R3, H2, R8, R9, R16, R17, H3, and R20, where the second nucleic acid sequence encodes the cleaved forms of dystrophin, R16, R17, H3, R20, R21, R22, R23, R24, H4, the CR domain, and the C-terminal domain (full-length or cleaved forms disclosed above, preferably full-length).

[0112] According to a particular embodiment, the first nucleic acid sequence includes or comprises SEQ ID NO: 19, and the second nucleic acid sequence includes or comprises SEQ ID NO: 20.

[0113] According to a second aspect, such a dual AAV vector system is dedicated to the so-called intein method. As is well 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 intein) is removed from a precursor protein by ligation of the C-terminal and N-terminal external proteins (called exteins) on both sides. This precursor protein contains three segments: an N-extein, followed by an intein, followed by a C-extein. After splicing occurs, the resulting protein contains an N-extein ligated to a C-extein.

[0114] In the case of a dual AAV vector, the first AAV enables the production of N-extin and subsequently the first portion of intein (the first fusion protein), and the second AAV enables the production of the second portion of intein and subsequently C-extin (the second fusion protein). The first and second fusion polypeptides are produced in cells, and the first and second portions of intein facilitate the conjugation of the first and second portions of extein, thereby delivering the extein to the cells.

[0115] Therefore, the nucleic acid sequence encoding mididystrophin can be divided into two parts: a 5' sequence encoding its N-terminal portion and a 3' sequence encoding its C-terminal portion. The 3' end of the 5' sequence fuses to the first part of the split intein, preferably its N-terminal portion, and the 5' end of the 3' sequence fuses to the second part of the split intein, preferably its C-terminal portion.

[0116] Examples of split-intanes are disclosed, for example, in WO2013 / 045632. Preferred split-intanes used within the framework of the present invention are GP41-1 and IMPDH1, favorably GP41-1.

[0117] The sequence of the N-terminal portion of GP41-1 is shown in sequence number 21.

[0118] The sequence of the C-terminal portion of GP41-1 is shown in sequence number 22.

[0119] In a further embodiment, the present invention relates to a dual AAV vector system comprising two AAV vectors, - The first AAV vector contains a first nucleic acid sequence between the 5' and 3' AAV ITRs that encodes the N-terminal portion of mididystrophin fused to the N-terminal portion of the split intein, - The second AAV vector contains a second nucleic acid sequence between the 5' and 3' AAV ITRs that encodes the C-terminal portion of split-intane fused to the C-terminal portion of mididystrophin. The present invention relates to a dual AAV vector system in which the first and second portions of the split-intein facilitate the joining of the first and second portions of the mididystrophin, thereby enabling the production of mididystrophin according to the present invention.

[0120] According to a particular embodiment, the first nucleic acid sequence includes or comprises SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO: 17, and the second nucleic acid sequence includes or comprises SEQ ID NO: 10, SEQ ID NO: 14, or SEQ ID NO: 18.

[0121] In one embodiment, the first nucleic acid sequence may encode the N-terminal domain, H1, R1, R2, R3, H2, R8, R9, R10, and cleaved R11 of dystrophin fused to the N-terminal portion of the GP41-1 gene, where the second nucleic acid sequence encodes the C-terminal portion of the GP41-1 gene fused to the remaining cleaved R11, R12, R13, R16, R17, R24, H4, CR domain, and C-terminal domain of dystrophin (full length or cleaved, preferably full length, as disclosed above).

[0122] According to a particular embodiment, the first nucleic acid sequence includes or comprises SEQ ID NO: 9, and the second nucleic acid sequence includes or comprises SEQ ID NO: 10.

[0123] In another embodiment, the first nucleic acid sequence may encode the N-terminal domain, H1, R1, H2, R8, R9, R10, R11, R12, R16, and cleaved R17 of dystrophin fused to the N-terminal portion of the GP41-1 gene, where the second nucleic acid sequence encodes the C-terminal portion of the GP41-1 gene fused to the remaining cleaved R17, H3, R20, R21, R22, R23, R24, H4, CR domain, and C-terminal domain (full length or cleaved, preferably full length, as disclosed above).

[0124] In a further specific embodiment, the first nucleic acid sequence includes or comprises SEQ ID NO: 13, and the second nucleic acid sequence includes or comprises SEQ ID NO: 14.

[0125] In a further embodiment, the first nucleic acid sequence may encode the N-terminal domains of dystrophin fused to the N-terminal portion of the GP41-1 gene, H1, R1, R2, R3, H2, R8, R9, R16, R17, and H3, where the second nucleic acid sequence encodes the C-terminal portion of the GP41-1 gene fused to the R20, R21, R22, R23, R24, H4, CR domain, and C-terminal domain of dystrophin (full-length or truncated, preferably full-length, as disclosed above).

[0126] According to a particular embodiment, the first nucleic acid sequence includes or comprises SEQ ID NO: 17, and the second nucleic acid sequence includes or comprises SEQ ID NO: 18.

[0127] The nucleic acid sequence encoding mididystrophin is advantageously of human origin, but may be a canine, rat, mouse, or non-human primate sequence. In one embodiment, the nucleic acid sequence originates from the organism to which it is administered, and advantageously, for administration in humans, it originates from a human sequence.

[0128] In known forms, various methods exist for optimizing protein-coding sequences to increase mRNA levels (recombination and transcription) and / or protein levels (translation). Within the framework of the present invention, the sequences in the vectors of the dual AAV vector system of the present invention are advantageously optimized to increase recombination and / or increase the expression of mididystrophin polypeptide in vivo.

[0129] In certain embodiments, the sequence encoding the mididystrophin of the present invention is optimized. Sequence optimization is performed. - Introduction of a consensus Kozak sequence (GCCACC) prior to the AUG start codon in mRNA to improve translation initiation. - Codon optimization, preferably fitting to codon bias in Homo sapiens genes. - Increased GC content and decreased CpG island count [generally, areas with very high (>80%) or very low (<30%) GC content are avoided]. - Internal TATA box, chi region, ribosome entry site, AT-rich or GC-rich sequence extension region, RNA instability motif, repetitive sequence and RNA secondary structure reduction or suppression, - A decrease in the number of alternative open reading frames (ARFs), - Reduction in the number of splice donor and splice acceptor sites (potential sites) This may include several changes in the nucleic acid sequence, including [specific component].

[0130] Due to the degeneracy of the genetic code, different nucleic acid molecules may encode the same protein. Furthermore, it is well known that the genetic codes of different organisms are often biased to use one of several codons encoding the same amino acid more than others. Through codon optimization, a change is introduced into the nucleotide sequence that takes advantage of the codon bias present in a given cellular environment, such that the resulting codon-optimized nucleotide sequence is more likely to be expressed at relatively higher levels in a given cellular environment compared to a non-codon-optimized sequence. In a preferred embodiment of the present invention, such a sequence-optimized nucleotide sequence encoding functional mididystrophin is codon-optimized to improve its expression and stability in human cells compared to a non-codon-optimized nucleotide sequence encoding the same protein, for example, by taking advantage of a bias in human-specific codon utilization. In a particular embodiment, the entire sequence of mididystrophin is advantageously optimized in humans to improve its production by target or host cells.

[0131] For cloning purposes and the production of viral particles, the expression construct can be inserted into a plasmid suitable for selection, replication, and production of mididystrophin.

[0132] According to a particular embodiment of the present invention, the viral vector is an adeno-associated virus (AAV) vector.

[0133] Adeno-associated virus (AAV) vectors are becoming a powerful gene delivery tool for treating a variety of disorders. AAV vectors possess several characteristics that make them ideally suited for gene therapy, including lack of pathogenicity, moderate immunogenicity, and the ability to transduce postmittal cells and tissues in a stable and efficient manner. The expression of specific genes contained within AAV vectors can be specifically targeted to one or more cell types by selecting the appropriate combination of AAV serotype, promoter, and delivery method.

[0134] In one embodiment, the coding sequence is contained within the AAV vector. More than 100 naturally occurring AAV serotypes are known. Many naturally occurring mutants exist in AAV capsids, which allows for the identification and use of AAVs with properties particularly suited to dystrophin pathology. AAV viruses may be manipulated using conventional molecular biology techniques, which makes it possible to optimize these particles for cell-specific delivery of nucleic acid sequences, to minimize immunogenicity, to regulate stability and particle lifetime, for efficient degradation, and for precise delivery to the nucleus.

[0135] As mentioned above, the use of AAVs is a common form of exogenous DNA delivery because they are relatively non-toxic, provide efficient gene transfer, and can be easily optimized for specific purposes. Of the well-characterized AAV serotypes isolated from humans or non-human primates (NHPs), human serotype 2 is the first AAV developed as a gene transfer vector. Other currently used AAV serotypes include AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In addition, non-naturally engineered mutants and chimeric AAVs may also be useful.

[0136] Desired AAV fragments for assembly into vectors include cap proteins containing vp1, vp2, vp3 and a hypervariable region, rep proteins containing rep78, rep68, rep52, and rep40, and sequences encoding these proteins. These fragments can be readily utilized in various vector systems and host cells.

[0137] 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, an artificial AAV serotype includes, but is not limited to, an AAV having a capsid protein that does not exist naturally. Such an artificial capsid may be generated by any preferred technique using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with different selected AAV serotypes, discontinuous portions of the same AAV serotype, from a non-AAV viral source, or from heterogeneous sequences that may be obtained from a non-viral source (i.e., the capsid contains VP capsid proteins derived from at least two different AAV serotypes, or contains 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, but is not limited to, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. Furthermore, peptide (P) may be introduced into the capsid, for example, into the variable region of the cap gene, to potentially modify AAV orientation.

[0138] In one embodiment, a useful vector in the compositions and methods described herein contains at least a sequence encoding a selected AAV serotype capsid, e.g., an AAV9 capsid or a fragment thereof. In another embodiment, a useful vector contains at least a sequence encoding a selected AAV serotype rep protein, e.g., an AAV9rep protein or a fragment thereof. Such a vector may contain both AAVcap and rep proteins. In a vector providing both AAVrep and cap, both AAVrep and AAVcap sequences may be sequences of a single serotype origin, e.g., all of AAV9 origin. Alternatively, a vector may be used in which the rep sequence is from an AAV serotype different from the AAV serotype providing the cap sequence. In one embodiment, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or host cells and vectors). In another embodiment, these rep sequences are in-frame fused to cap sequences of different AAV serotypes to form a chimeric AAV vector.

[0139] In some embodiments, the AAV vector comprises genomes and capsids derived from different serotypes of AAV.

[0140] Therefore, exemplary AAVs or artificial AAVs include AAV2 / 8 (US 7,282,199), AAV2 / 5 (available from the National Institutes of Health), AAV2 / 9 (WO2005 / 033321), AAV2 / 6 (US 6,156,303), AAVrh10 (WO2003 / 042397), AAVrh74 (WO2003 / 123503), AAV9-rh74 hybrid or AAV9-rh74-P1 hybrid (WO2019 / 193119; WO2020 / 200499; EP20306005.8).

[0141] According to one embodiment, the AAV is an AAV of serotype 2, 5, 8, or 9, or AAVrh74. Advantageously, the claimed vector comprises a capsid selected from the group consisting of AAV8 capsid, AAV9 capsid, AAV9-rh74 capsid, and AAV9-rh74-P1 capsid.

[0142] According to a particular embodiment, the AAV is serotype 9 AAV (AAV9).

[0143] In the AAV vector 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.

[0144] Advantageously, and as mentioned above, the target nucleic acid sequence is inserted between the ITR ("terminal inversion sequence") sequences of the AAV vector. Typically, the ITR sequences originate from AAV2 or AAV9, advantageously AAV2.

[0145] As is well known in the art, recombinant viral particles can be obtained, for example, by triple transfection of 293HEK cells, by herpes simplex virus lines, by baculovirus lines, or by using specific cell lines. Vector titers are usually expressed as viral genome per ml (vg / ml).

[0146] Furthermore, the present invention relates to cells, particularly muscle cells, that are genetically modified using the dual AAV vector system disclosed above.

[0147] In another aspect, the present invention relates to a composition, advantageously a therapeutic composition or pharmaceutical, comprising the dual AAV vector system disclosed above and possibly other active molecules (other gene therapy products, chemical molecules, peptides, proteins, etc.) specifically for the treatment of the same or a different disease.

[0148] Furthermore, the present invention provides a pharmaceutical composition comprising a dual AAV vector system, or a first or second AAV vector of the said system. Such a composition comprises a therapeutically effective amount of a therapeutic agent (the nucleic acid or vector of the present invention) and a pharmaceutically acceptable carrier. In certain embodiments, the term “pharmaceutically acceptable” means approved by a federal or state regulatory authority or listed in the United States or European Pharmacopoeia or other generally recognized pharmacopoeias for use in animals and humans. The term “carrier” refers to a diluent, adjuvant, excipient, or medium through which the therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oil, and oils may include oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Also, saline and aqueous dextrose and glycerol solutions can be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, and ethanol.

[0149] Furthermore, the composition may optionally contain a small amount of wetting agent or emulsifier, or a pH buffer. These compositions may take the form of solutions, suspensions, emulsions, sustained-release formulations, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions contain a therapeutically effective amount of the therapeutic agent, preferably in a purified form, together with a suitable amount of carrier to provide a form for appropriate administration to the subject.

[0150] In preferred embodiments, the composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizer and a local anesthetic, such as lidocaine, to relieve pain at the injection site.

[0151] In one embodiment, the composition according to the present invention is suitable for administration in humans. The composition is preferably in liquid form, and is advantageously a physiological saline composition, more advantageously a phosphate-buffered saline (PBS) composition or Ringer's lactate solution.

[0152] The amount of the therapeutic agent of the present invention (i.e., expression system, vector, or cells) effective in treating the target disease can be determined by standard clinical techniques. In addition, in vivo and / or in vitro assays may be used to aid in predicting the optimal dose range. Furthermore, the exact dose used in the formulation should be determined by the judgment of the practitioner and the circumstances of each patient, depending on the route of administration, the physical characteristics of the individual being considered, such as sex, age, and weight, concurrent drug treatments, other factors, and the severity of the disease.

[0153] A suitable administration should allow for the delivery of a therapeutically effective dose of the gene therapy product to target tissues, particularly skeletal muscle, and potentially the heart and diaphragm. In the context of this invention, if the gene therapy product is a viral vector, the therapeutic dose is defined as the amount of viral particles containing the transgene (vg for the viral genome) administered per kilogram (kg) of the subject.

[0154] For treatments involving the administration of viral vectors, such as AAV vectors, a typical dose of the vector is at least 1 x 10¹⁶ per kilogram of body weight. 8 Vector genome (vg / kg), e.g., at least 1 x 10⁻¹⁶ 9 vg / kg, at least 1x10 10 vg / kg, at least 1x10 11vg / kg, at least 1x10 12 vg / kg, at least 1x10 13 vg / kg, at least 1x10 14 vg / kg, at least 10 15 The dosage is vg / kg. In particular, the dosage is 5x10 11 vg / kg to 10 14 vg / kg, for example 1, 2, 3, 4, 5, 6, 7, 8 or 9x10 13 vg / kg and can be. Also, for example 1, 2, 3, 4, 5, 6, 7, 8 or 9x10 12 Lower dosages of vg / kg may be contemplated to avoid potential toxicity and / or immune responses. As is known to those skilled in the art, the lowest possible dosage that gives satisfactory results in respect of efficiency is preferred.

[0155] The available routes of administration are topical (local), enteral [although having a systemic effect, delivered through the gastrointestinal (GI) tract] or parenteral [although having a systemic effect, delivered by a route other than the GI tract]. The preferred routes of administration of the compositions disclosed herein are parenteral, including intramuscular administration (i.e., into the muscle) and systemic administration (i.e., into the circulatory system). In this context, the term "injection" (or "perfusion" or "infusion") encompasses intravascular, particularly intravenous (IV), intramuscular (IM), intraocular, intrathecal or intracerebral administration. Injections are usually performed using a syringe or a catheter.

[0156] In one embodiment, the systemic delivery of the composition comprises administering the composition near the local treatment site, i.e., into a vein or artery near the weakened muscle. In certain embodiments, the present invention includes local delivery of a composition that produces a systemic effect. This route of administration is usually referred to as "regional (local-regional) infusion", "administration by isolated limb perfusion" or "high-pressure intravenous limb perfusion" and has been used and been successful as a gene delivery method in muscular dystrophy.

[0157] In one embodiment, the composition is administered separately to the limbs (locally) by infusion or perfusion. In other words, the present invention includes intravascular administration routes under pressure, i.e., local delivery of the composition to the legs and / or arms by vein (transvenous) or artery. This is usually achieved by allowing local diffusion of the product being infused while using a tourniquet to temporarily restrict blood circulation, as disclosed, for example, by Toromanoff et al. (2008).

[0158] In one embodiment, the composition is injected into the limbs of the subject. If the subject is human, the limbs may be arms or legs. According to one embodiment, the composition is administered to the lower part of the subject's body, for example, below the knee, or to the upper part of the subject's body, for example, below the elbow.

[0159] A preferred method of administration according to the present invention is systemic administration. Systemic injection opens a breakthrough for injection into the entire body to reach all the muscles of the subject's body, including the heart and diaphragm, and thus for the practical treatment of these systemic and still incurable diseases. In certain embodiments, systemic delivery includes delivery of the composition to the subject such that the composition is accessible throughout the subject's entire body.

[0160] In a preferred embodiment, systemic administration is carried out by intravascular injection of the composition, i.e., intravascular (intravenous or intra-arterial) administration. In one embodiment, the composition is administered intravenously via a peripheral vein.

[0161] In certain embodiments, the treatment comprises a single dose of the composition.

[0162] In one embodiment, the presence of a gene therapy product and / or the expression of functional micro-eutrophin, as well as the associated therapeutic benefits, are observed over the entire lifetime of the subject, for up to one month, or three months, or six months, or even one year, two years, five years, ten years, or more.

[0163] In accordance with the present invention, the subject is preferably a human, but may also be a mouse, rat, non-human primate, or dog.

[0164] Subjects who can benefit from the compositions of the present invention include all patients who have been diagnosed with muscular dystrophy or who are at risk of developing such muscular dystrophy. Accordingly, subjects to be treated may be selected based on the identification of mutations or deletions in the dystrophin gene by any method known to those skilled in the art, including sequencing of the dystrophin gene, and / or through the evaluation of dystrophin expression levels or activity levels by any method known to those skilled in the art. Thus, the subjects include both subjects who already exhibit symptoms of dystrophic disease and subjects who are at risk of developing the disease. In one embodiment, the subjects include subjects who already exhibit symptoms of dystrophic disease. In another embodiment, the subjects include patients who are able to walk and patients who are in the early stages of immobility.

[0165] Such compositions are particularly intended for gene therapy in subjects, especially for the treatment of diseases caused by deficiencies of the proteins specified above, in particular dystrophin.

[0166] Such compositions are particularly intended for the treatment of neuromuscular diseases, especially muscular dystrophy involving defective dystrophin, i.e., dystrophic diseases.

[0167] In the context of this invention, “dystrophy disease” means a disease linked to a defect in the dystrophin gene. This defect may be a deletion or mutation resulting in low levels of expression or absence of expression, introduction of a stop codon in the open reading frame, or production of an inactive protein. Preferred dystrophy diseases are Duchenne and Becker muscular dystrophy (DMD / BMD) caused by mutations in the dystrophin gene. Such mutations may result in the absence or low levels of dystrophin expression, or the production of a partially or completely inactive, possibly cleaved, protein.

[0168] According to one embodiment, the present invention relates to a dual AAV vector system disclosed above, or a composition comprising the AAV vector, for use in the treatment of dystrophy diseases. According to another embodiment, the present invention relates to the use of the AAV vector disclosed above, or a composition comprising the AAV vector, for the preparation of a pharmacopoeia for the treatment of dystrophy diseases.

[0169] In other words, the present invention provides a method for treating dystrophy diseases, particularly DMD and BMD, in a subject, comprising administering to the subject a dual AAV vector system or a composition comprising the system disclosed above.

[0170] A first objective of the present invention is to provide a safe (non-toxic) and non-immunogenic treatment. A further objective is to provide an efficient treatment that can delay, slow, or prevent the onset of disease and, potentially, improve the patient's phenotype, which can be easily monitored at the clinical level. In this context, the AAV vectors and compositions according to the present invention are - To improve muscle function (skeletal muscle is of particular interest, but cardiac muscle and diaphragm are also of interest), - To improve walking motion, - To improve cardiac function, - To improve respiratory function, - To extend survival, and more generally to improve quality of life and life expectancy. It can be used.

[0171] Improvements to the aforementioned functions may be described in the following examples or evaluated by methods known to those skilled in the art.

[0172] More generally and according to further embodiments, the present invention is - To increase muscle strength, muscle endurance and / or muscle mass in the subject, - To reduce fibrosis in the target population, - To reduce contraction-induced injury in the target population. - To treat muscular dystrophy in the subject, - To reduce degenerated or necrotic fibers in subjects suffering from muscular dystrophy. - To reduce inflammation in subjects suffering from muscular dystrophy, - To reduce the levels of creatine kinase (or any other dystrophy marker) in subjects suffering from muscular dystrophy. - To treat muscle fiber atrophy and hypertrophy in subjects suffering from muscular dystrophy. - To reduce dystrophic calcification in subjects suffering from muscular dystrophy. - To reduce fat infiltration in the subject, - To reduce central nucleation in the target. It is useful for [something].

[0173] The present invention will be carried out using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the scope of the art of those skilled in the art, unless otherwise indicated. Such techniques are fully described in 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), and "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of polynucleotides and polypeptides of the present invention and can therefore be considered in the manufacture and implementation of the present invention. Techniques particularly useful for specific embodiments are described in the following sections.

[0174] Each and all patent, patent application and publication disclosures cited herein are incorporated herein by reference in their entirety.

[0175] Without further explanation, those skilled in the art will likely be able to produce and utilize the compounds of the present invention and carry out the claimed methods using the above description and the following exemplary examples.

[0176] Experimental Examples The present invention will be described in further detail by reference to the following experimental examples and accompanying drawings. These examples are provided for illustrative purposes only and are not intended to be limiting. [Brief explanation of the drawing]

[0177] [Figure 1] Schemes for various types of dystrophines. A / Scheme for full-length dystrophines. B / Scheme for LP1 mididystrophines (ΔR4~R7ΔR14~R15ΔR18~R23) according to the present invention. C / Scheme for LP2 mididystrophines (ΔR2~R7ΔR13~R15ΔR18~R19) according to the present invention. D / Scheme for LP3 mididystrophines (ΔR4~R7ΔR10~R15ΔR18~R19) according to the present invention. [Figure 2] Production of LP1 mididystrophin using the overlap method. A / General scheme. B / Scheme for the first AAV vector in a dual AAV vector system. C / Scheme for the second AAV vector in a dual AAV vector system. D / Scheme for the mididystrophin thus obtained. [Figure 3] Production of LP1 mididystrophin using the intein method. A / General scheme. B / Scheme for the first AAV vector in a dual AAV vector system. C / Scheme for the second AAV vector in a dual AAV vector system. D / Scheme for the mididystrophin thus obtained. [Figure 4] Production of LP2 mididystrophin using a redundant method. A / General scheme. B / Scheme for the first AAV vector in a dual AAV vector system. C / Scheme for the second AAV vector in a dual AAV vector system. D / Scheme for the mididystrophin thus obtained. [Figure 5] Production of LP2 mididystrophin using the intein method. A / General scheme. B / Scheme for the first AAV vector in a dual AAV vector system. C / Scheme for the second AAV vector in a dual AAV vector system. D / Scheme for the mididystrophin thus obtained. [Figure 6] Production of LP3 mididystrophin using a redundant method. A / General scheme. B / Scheme for the first AAV vector in a dual AAV vector system. C / Scheme for the second AAV vector in a dual AAV vector system. D / Scheme for the mididystrophin thus obtained. [Figure 7] Production of LP3 mididystrophin using the intein method. A / General scheme. B / Scheme for the first AAV vector in a dual AAV vector system. C / Scheme for the second AAV vector in a dual AAV vector system. D / Scheme for the mididystrophin thus obtained. [Figure 8] Quantitative analysis of dystrophin production. Western blot analysis using the JESS system of gastrocnecmius muscle from wild-type mice (WT) injected with saline buffer, mdx mice (saline) injected with saline buffer, mdx mice (Midi-Dys 1) injected with the dual AAV vector system of the present invention to ensure LP1 production by the intaining method, mdx mice (Midi-Dys 2) injected with the dual AAV vector system of the present invention to ensure LP2 production by the intaining method, and mdx mice (Micro-dys) injected with an AAV vector to ensure MD1 production as disclosed in the prior art. [Figure 9A] Quantification of dystrophin-positive fibers. The percentage (%) of dystrophin-positive muscle fibers in the gastrocnemius (A) and tibialis anterior (TA) muscles (B) of wild-type mice (WT) injected with physiological saline buffer (n=5), mdx mice (physiological saline) injected with physiological saline buffer (n=5), mdx mice (Midi-Dys 1) injected with the dual AAV vector system of the present invention to ensure LP1 production (n=5), mdx mice (Midi-Dys 2) injected with the dual AAV vector system of the present invention to ensure LP2 production (n=5), and mdx mice (Micro-dys) injected with an AAV vector to ensure MD1 production as disclosed in the prior art (n=5) is shown. [Figure 9B] Same as above. [Figure 10A] Quantitative analysis of fibrosis and calcium deposition in the gastrocnemius muscle. Percentages (%) of collagen deposition regions (A) and calcium deposition regions (B) are shown for wild-type mice (WT) (n=5) injected with physiological saline buffer (n=5), mdx mice (physiological saline) (n=5) injected with physiological saline buffer (Midi-Dys 1) (n=5) injected with the dual AAV vector system of the present invention to ensure LP1 production (Midi-Dys 2) (n=5) injected with the dual AAV vector system of the present invention to ensure LP2 production (Midi-Dys 2) (n=5), and mdx mice (Micro-dys) (n=5) injected with an AAV vector to ensure MD1 production as disclosed in the prior art. [Figure 10B] Same as above. [Examples]

[0178] Experimental example material and method 1 / In vitro trial of AAV9 encoding LP1, LP2, LP3, and μDys using muscle organoids derived from induced pluripotent stem cells of DMD patients. AAV production and organoid infection Recombinant AAV is produced at Genethon using the AAV9 serotype. Purification is performed using affinity chromatography, and titration is performed by ddPCR using ITR primers. The AAV9-CK8-GFP construct is used for infection optimization. The microdystrophin transgene used in the study, under the control of the CK8 promoter, is an optimized version of the construct used in Genethon's preclinical and clinical trials, with deletions from spectrin-like repeat sequences 4-23 and total C-terminal excision, referred to here as μDys (see WO2015 / 197869). Infection of organoids is performed on day 7 by delivering AAV9 particles to differentiation medium at two different doses, 1E+10vg / organoid (low dose) and 5E+10vg / organoid (high dose). The medium is replaced 24 hours after infection and changed daily until day 14.

[0179] MYOrganoid generation MYOrganoid is generated from iPSCs by adapting the protocol (Tiburcy et al. 2020) described for the cardiac tissue to be manipulated. In particular, committed hIPSCs (hIPSC-committed) (24 hours after doxycycline induction) 1.25 x 10⁻¹⁵ 6 The cells are resuspended in 77 μl of growth medium supplemented with hES cell Recovery (Stemgent), and then molded into a hydrogel composed of 40 μl of 6 mg / ml bovine collagen solution (Sigma-Aldrich), 17.8 μl of Matrigel low growth factor (Corning) 10% v / v (3), 40 μl of 2X DMEM (Gibco) (4), and 5.2 μl of NaOH 1.5N (5). For the generation of MYOrganoid containing fibroblasts, 1.25 x 10⁶ immortalized fibroblasts are used. 5 The cells are placed in an iPSC-committed cell mixture (1:10) and then encapsulated in a hydrogel. The hydrogel is placed in a 48-well plate TM5 MyrPlate (Myriamed) (static spreading device) containing a pair of flexible poles in each well to support the ring-shaped proliferation of the tissue to be manipulated. After polymerization at 37°C for 1 hour, culture medium is added and left for 24 hours. On day 2 of the 3D cycle, the proliferation medium (SKM02) is replaced with differentiation medium (SKM03plus), and this replacement is continued daily until day 14.

[0180] Muscle strength analysis Functional analysis will be performed 14 days after 3D formation. Contraction experiments will be conducted using the MyoDynamics Muscle Strip System 840 MD (DMT Technologies) and CS4 stimulator (DMT Technologies). All functional analyses will be performed in Tyrode's solution supplemented with 25 mM NaHCO3 at 37°C, 5% CO2 / 95% O2. The optimal muscle length will be determined by gradually stretching the muscle until no further increase in single-contraction tension is possible. Functional tests will be performed under isometric and eccentric conditions. The organoids will be electrically stimulated at 30V for 250 pulses with a 4ms width and a frequency of 125Hz for both isometric and eccentric contractions. For the eccentric analysis, the organoid will be stretched by 1 mm at 6.5 cm / sec during muscle contraction. For each organoid, one isometric contraction, ten eccentric contractions, and one isometric contraction will be performed. Data collection and analysis are performed using a Powerlab device and LabChart software (AD Instruments), respectively. Fatigue is expressed as the percentage drop force between the first and last isometric contractions. Force is normalized by CSA (cross-sectional area) and expressed in mN / mm². 2 It is expressed as follows.

[0181] Protein expression analysis using WB and IHC Western blot analysis Reconstituted mididystrophin was monitored by size selection (range 260-280 kDa) using Western blotting. Homogenization extracted organoid proteins into RIPA buffer supplemented with a protease inhibitor cocktail (EDTA-free, Roche) and benzoase. Subsequently, total protein was quantified using the BCA method with the Pierce BCA protein assay kit (Invitrogen) according to the manufacturer's instructions. Protein detection was performed by capillary Western blotting with JESS protein simple (Bio-techne) according to the manufacturer's instructions. Microdystrophin was detected using antibodies DysB and Dys2, which recognize the N-terminus and C-terminus, respectively, and its expression was quantified by total protein normalization.

[0182] immunocytochemistry On day 14, MYOrganoid is fixed overnight in 4% methanol-free PFA. For whole-mount staining, the fixed MYOrganoid is permeabilized, stained, and dehydrated using the MACS Clearing Kit (Miltenyi) according to the manufacturer's instructions. Subsequently, the whole-mount stained organoid is imaged at 10X magnification using a confocal microscope (LEICA STED SP8).

[0183] For staining of cross-sectional or longitudinal sections, fixed MYOrganoids are dehydrated over-day using a sucrose gradient (7.5%–30%) and embedded in OCT matrix in plastic molds. After 24 hours, the embedded organoids are processed into 15 μm thick sections using a cryostat (LEICA). The slices are then dried and fixed again with 4% methanol-free PFA (Invitrogen). The fixed sections are then blocked with a serum cocktail (5% goat serum and 5% fetal bovine serum) and stained overnight at +4°C with dystrophin primary antibody. The slices are then washed three times in PBS and hybridized with AlexaFluor secondary antibody depending on the host species of the primary antibody. The stained slides are then covered with Fluoromont+Dapi (Ref) and 1.5H glass slides. For imaging, the sections are scanned with an AxioScan microscope and a confocal Leica SP8.

[0184] 2 / Evaluation of the therapeutic efficiency of pre-selected constructs in vitro in the invasive DMD mouse model DBA2-mdx. Mididystrophin design and viral particle preparation The mididystrophin (LP1 and LP2, see below) protein sequences are derived from the dystrophin muscle isoform (Dp427m; reference sequence NM_004006.3) and optimized by GeneArt Services (Life Technologies) to remove rare human codons, CpGs, and possible alternative ORFs.

[0185] Gene fragments were produced using GeneArt gene synthesis (Life Technologies) and cloned into plasmids containing the CK8 promoter and poly(A) SV40. The plasmids were amplified using an endotoxin-free method.

[0186] For recombinant AAV production, HEK293-T cells (obtained from Stanford University School of Medicine) cultured in suspension were transfected with three plasmids encoding adenovirus helper protein, AAV Rep and Cap proteins, and an ITR flanking transgene expression cassette.

[0187] Three days after transfection, cells were harvested, chemically dissolved, and treated with benzonase (Merck-Millipore, Darmstadt, Germany). After filtration, the viral capsid was purified by affinity chromatography, formulated in sterile PBS, and the vector stock was stored at -80°C. The titer of the AAV vector was determined using digital droplet polymerase chain reaction (ddPCR). Viral particles were treated with DNase I at 37°C for 30 minutes (Invitrogen, MA, USA), and then viral DNA was amplified using ddPCR Supermix (Biorad, CA, USA) as a probe and poly(A) SV40-specific primers.

[0188] Capillary Western blot Capillary Western blotting of Midi-Dys (LP1 and LP2) and Micro-Dys (MD1) was performed using the Simple Western® Jess system (ProteinSimple, Bio-Techne) according to the manufacturer's instructions, using the 60-440 kDa isolation module (ProteinSimple SMW004) and the anti-mouse detection module (Protein Simple DM-002). Briefly, protein samples consisting of mididystrophin or microdystrophin proteins were obtained from muscle lysates. The proteins were extracted in RIPA buffer, and 2.5 ng was used for dystrophin detection in mdx mice injected with the medium, AAV-mididystrophin, or AAV-microdystrophin. DysB primary antibody was used to recognize the N-terminal portion of dystrophin (Leica, IL, USA), and Mancho-11 primary antibody (DSHB, IA, USA) was used to recognize the C-terminal portion of dystrophin.

[0189] Animal management and use DBA2(B6;129S4-DBA2 tm1Cpr / J, system number 000671) and DBA2-mdx(D2.B10-Dmd mdx / J) Mice were supplied by Jackson Laboratory. All animal procedures were approved by the National Ethics Committee, C2EA 51 (Evry, France) and the French Ministry of Research (MESRI), and the National Consensus Number (APAFIS 38006) was received. Dual mididystrophin (LP1 and LP2) AAV9 vectors were administered to 4-week-old Dba2_mdx and Dba2_WT male mice by post-orbital injection at a dose of 2E+13vg / Kg for each vector (total dose 4E+13vg / Kg), and microdystrophin AAV9 vectors were administered in 2x10⁻¹⁶ doses. 13AAV9 was injected at a dose of vg / kg. Mice injected with AAV9 were compared to Dba2_mdx mice (negative control) and Dba2 WT mice (positive control) injected with PBS. Seven weeks after injection, muscle and serum were collected for molecular and histological analysis. Five mice were tested per experimental condition.

[0190] tissue staining Skeletal muscle was collected and frozen in isopentane cooled in liquid nitrogen. Frozen cross-sectional sections (8-10 μm) were prepared from the frozen muscle, air-dried, and stored at -80°C. The muscle sections were prepared for Sirius Red and Red Alizarin tissue staining. The sections were visualized using an Axioscan Z1 automated slide scanner (Zeiss, Germany) with a Plan APO 10X / 0.45 NA objective lens.

[0191] Sections were immunostained overnight at 4°C with primary antibodies specific to dystrophin (N-terminus, DysB, LEICA) and laminin (ThermoFisher, MA, USA). After three washes with PBS, muscle sections were incubated at room temperature for 1 hour with goat secondary antibody conjugated with Alexa Fluor 488 or 594 dye (Molecular Probe, 1:1000 dilution). Sections were then mounted using DAPI-Fluoromount-G (Southern Biotech) and visualized using a LEICA TCS-SP8 confocal microscope (Leica, IL, USA) with a 63X APO CS2 1.4 NA objective lens, or using an Axioscan Z1 automated slide scanner (Zeiss, Germany) with a Plan APO 10X / 0.45 NA objective lens.

[0192] Fibrosis and calcium deposition quantification Cross-sectional sections stained with Sirius Red and Alizarin 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 then used to quantify fibrous and calcified areas.

[0193] Quantitative analysis of dystrophin-positive fibers Muscle sections were stained with laminin to label the cell membranes. The Cellpose2 cyto2 model 10 was fine-tuned using images of manually labeled muscle fibers based on laminin staining. This fine-tuning was performed using hyperparameters set to 200 epochs, a learning rate of 0.05, and a weight decay of 0.0001. The labeled dataset was carefully adjusted to allow the model to segment muscle fibers while simultaneously ignoring regions with poor staining accuracy.

[0194] After fine-tuning, these models were used to extract muscle fiber masks. Muscle fiber masks were reconstructed from the full scan images using the Cellpose package 10. These reconstructed masks were then converted into Regions of Interest (ROIs) using the Labels_To_Rois.py FIJI plugin 11, with each ROI corresponding to an individual muscle fiber.

[0195] Muscle sections were co-labeled with laminin for membrane labeling and with dystrophin for further analysis. Similar to the initial labeling, a region of interest (ROI) was generated based on the membrane labeling and subsequently used to quantify the dystrophin signal using FIJI macros.

[0196] Quantitative analysis of serum biomarkers A custom-made sandwich ELISA assay was used for serum MYOM3 quantification. First, a 1:10 dilution of polyclonal MYOM3 antibody (Proteintech, 17692-1-AP) was spread onto a 96-well plate and incubated overnight at 4°C. After incubation, the plate was washed three times with PBST and then blocked with a saturated solution of 3% BSA in PBS. Subsequently, diluted serum samples were added to the plate and incubated at room temperature for 2 hours. For detection, a monoclonal antibody coupled with SULFO-TAG (MSD) (Proteogenix, REF:51-H1-B4) was used, and the plate was incubated for a further 2 hours at room temperature. After incubation, the plate was washed three times with washing buffer (0.05% Tween 20 in PBS). The absorbance of SULFO-TAG was measured using a MESO Quickplex SQ 120 (MSD). To quantify the MYOM3 concentration in serum samples, a set of concentration-restricted MYOM3 peptides (His-tagged, Proteogenix) was included in the same experiment and used as a baseline for calculations.

[0197] Creatine kinase (CK) measurement CK quantification was initiated with 10 μl of mouse serum using a colorimetric assay with the FUJI DRI-CHEM nx500 system (DMV Imaging), which is used to measure creatine phosphokinase concentration.

[0198] Muscle strength assessment For the avoidance test, mice were placed in a 30 cm long tube, with their tails attached to a horizontal tension transducer. In response to a gentle pinching stimulus on the tail, the mice attempted to escape from the tube. Short force peaks were recorded using the force transducer, and typically 15 pinching stimuli were performed. The top 5 tensile tensions were averaged and divided by the mouse's body weight. Due to memory effects, the test could only be performed once during the mouse's lifetime. Data were reported as the maximum peak normalized to body weight and the average of the 5 peaks. In the bilimb grip strength test, grip strength was measured using a grip strength meter (Bioseb, https: / / www.bioseblab.com / , accessed December 1, 2021; France Grip Test 25N). Three independent measurements were performed, and the average grip strength normalized to body weight was calculated.

[0199] statistical analysis All data were analyzed using GraphPad Prism 9.5.1 software. Parametric tests, such as t-tests and ANOVA, were used for statistical comparisons. To compare two groups, the variances were first compared using F-tests. If there was no difference in variances, statistical comparisons were performed using unpaired t-tests. To compare multiple groups, we used one-way ANOVA and Tukey 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. Figures show the mean ± standard deviation.

[0200] result Construction of AAV vectors Different recombinant AAV2 / 9 vectors were constructed using either a dual AAV vector "intene" or "duplication" method.

[0201] Both methods are well known in the art. The "intei" method is disclosed, for example, in WO01 / 29243, and the "duplication" method is used for the production of pseudodystrophin in WO2020 / 193636.

[0202] Table 1: List of dystrophin tested [Table 1-1] [Table 1-2]

[0203] Production and activity of mini / mididystrophin Figure 8 shows that, at the protein level, the prior art microdystrophins are expressed at 2.5 to 4 times better levels than the present invention's mididystrophins (LP1 and LP2) produced using the intein method.

[0204] In terms of distribution and activity, Figures 9 and 10 show that both mididystrophins of the present invention (LP1 and LP2) and prior art microdystrophin (MD1) exhibit similar profiles, and in particular, equal therapeutic efficacy.

[0205] Taking into account the different amounts of protein present in muscle, it can be concluded that the mididystrophin according to the present invention performs better when expressed at the same protein level as the prior art microdystrophin.

Claims

1. Mididystrophin comprising at least the rod domains R1, R8, R9, R10, R11, R12, R16, R17, and R24 of dystrophin.

2. The mididystrophin according to claim 1, lacking R4, R5, R6, and R7 rod domains (ΔR4 to R7), R14, and R15 rod domains (ΔR14 to R15), and R18, R19, R20, R21, R22, and R23 rod domains (ΔR18 to R23).

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

4. The mididystrophin according to claim 2 or 3, having the sequence of sequence number 1 or a sequence having at least 90% identity thereto.

5. The mididystrophin according to claim 1, lacking R2, R3, R4, R5, R6, and R7 rod domains (ΔR2 to R7), lacking R13, R14, and R15 rod domains (ΔR13 to R15), and lacking R18 and R19 rod domains (ΔR18 to R19).

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

7. The mididystrophin according to claim 5 or 6, having the sequence of sequence number 2 or a sequence having at least 90% identity thereto.

8. A nucleic acid sequence encoding mididystrophin according to any one of claims 1 to 7, wherein the sequence is advantageously selected from the group consisting of sequence number 4, sequence number 5, sequence number 6 and sequences having at least 70% identity with them.

9. A dual AAV vector system containing two AAV vectors, - The first AAV vector contains a first nucleic acid sequence encoding the N-terminal portion of mididystrophin between the 5' and 3' AAV ITRs. - The second AAV vector contains a second nucleic acid sequence encoding the C-terminal portion of mididystrophin between the 5' and 3' AAV ITRs. The first and second nucleic acid sequences include overlapping regions that enable the recombinant production of mididystrophin according to any one of claims 1 to 7. Dual AAV vector system.

10. The dual AAV vector system according to claim 9, wherein the first nucleic acid sequence has the sequence of SEQ ID NO: 11 or SEQ ID NO: 15, and the second nucleic acid sequence has the sequence of SEQ ID NO: 12 or SEQ ID NO:

16.

11. A dual AAV vector system containing two AAV vectors, - The first AAV vector contains, between the 5' and 3' AAV ITRs, a first nucleic acid sequence encoding the N-terminal portion of mididystrophin, which is favorably fused to the N-terminal portion of a split-intane derived from the GP41.1 gene. - The second AAV vector comprises a second nucleic acid sequence fused between the 5' and 3' AAV ITRs, which preferably originates from the GP41.1 gene and encodes the C-terminal portion of the split-intane, The first and second portions of the split-intein facilitate the bonding of the first and second portions of the mididystrophin, thereby enabling the production of the mididystrophin according to any one of claims 1 to 7. Dual AAV vector system.

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

14.

13. Cells transduced with the dual AAV vector system according to any one of claims 9 to 12, preferably muscle cells.

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

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

Images