Novel optimized utrophin micro-genes

Abstract

The present invention concerns a micro-utrophin comprising the structure NTDH1R1R2R3H2R22H4CRD and two further rod domains selected from the group consisting of: - the R4 to R10 rod domains of utrophin; - the R16 and R17 rod domains of dystrophin; a nucleic acid sequence encoding such a micro-utrophin, a recombinant adeno-associated virus (rAAV) vector comprising such a sequence, and their use for treating a muscular dystrophy such as Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD).

Description

[0001] NOVEL OPTIMIZED UTROPHIN MICRO-GENES

[0002] TECHNICAL FIELD

[0003] The present invention relates to novel optimized utrophin micro-genes which are useful in the treatment or prevention of dystrophic diseases, especially Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).

[0004] BACKGROUND OF THE INVENTION

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

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

[0007] There is no cure nor effective treatment available for DMD (Rodino-Klapac, L.R. et al., Curr Neurol Neurosci Rep, 2013. 13(3): 332) or BMD. Conventional therapies are limited to supportive care, which partially alleviates signs and symptoms, but does not directly target the disease mechanism nor reverse the phenotype.

[0008] The urgency to seek a cure for DMD has resulted in parallel efforts to develop therapies based on dystrophin gene restoration by exon skipping or termination codon read-through. Such dystrophin gene restoration-based therapeutic strategies are mutation dependent and consequently limited to a specific subset of patients (respectively 13 and 11% of the patients). In 1990, the description of a very mildly BMD patient, who had 46% of his dystrophin protein missing, paved the way to dystrophin-based gene therapy for DMD. Following extensive work and optimization, rAAV-mediated systemic gene therapy to deliver a truncated but partially functional micro-dystrophin (pDYS) gene is currently under investigation in clinical trials bringing hope for an effective therapy to the DMD community. Nevertheless, several challenges remain to be overcome, notably the preexisting circulating T cell immunity to dystrophin noted in some DMD boys and detected after pDYS treatment in some patients, in GRMD dogs and in mdx animals as well.

[0009] Utrophin was described as functional and structural autosomal paralogue of dystrophin able to compensate for the lack of dystrophin in DMD and to prevent the dystrophic pathology. Unlike dystrophin, utrophin is naturally expressed in dystrophic muscles and no immune response was observed with utrophin gene therapy. Besides, as demonstrated by the work of the Chamberlain team, the administration of a murine codon-optimized micro-utrophin (AR4-R21 / ACT) transgene to adult dystrophin' 7utrophin" (mdx:utrn ~) double-knockout mice may be a treatment option for DMD.

[0010] It appears that there is a great unmet clinical need to develop therapeutic solution applicable to all DMD patients irrespective to their genetic defect and immunologic background.

[0011] BRIEF SUMMARY OF THE INVENTION

[0012] The present invention, as defined in the claims, aims at alleviating or curing the devastating Duchenne muscular dystrophy (DMD) as well as Becker muscular dystrophy (BMD) by providing a robust alternative to pDYS approaches currently under investigation in clinic.

[0013] For the first time, the present invention offers a promising optimized utrophin-based gene therapy (pUTR) applicable to all Duchenne and Becker patients by increasing the dystrophin- related protein, utrophin. More specifically, the present invention is based on the use of recombinant adeno-associated virus (rAAV) to deliver and to express novel optimized micro- utrophin genes for gene replacement strategy.

[0014] In the frame of the present application, the expression “optimized micro-utrophin genes” or “optimized utrophin micro-genes” means that said genes encode micro-utrophins which have a better therapeutic activity than the previously described AR4-R21 / ACT micro-utrophin (see e.g. Odom et al., 2008, Mol Then, 16(9): 1539-45). The present invention therefore has various advantages, including the fact that the administration of pUTR as a transgene therapy product according to the invention does not induce any immune response against the exogenous transgene since this protein is naturally expressed in dystrophic muscle cells. Besides and based on the pUTR transgenes of the present invention, the efficiency of the pUTR can be ameliorated, e.g. by enhancing its binding to the actin cytoskeleton.

[0015] Definitions

[0016] Unless otherwise defined, all technical and scientific terms used therein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0017] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0018] “About” or “approximately” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0019] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0020] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a nonnative environment such as, for example, a host cell.

[0021] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0022] A “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or a RNA or a cDNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0023] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0024] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR and the like, and by synthetic means. As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0025] A protein may be “altered” and contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues as long as the biological activity is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid; positively amino acids may include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine, glycine and alanine, asparagine and glutamine, serine and threonine, and phenylalanine and tyrosine.

[0026] A “variant”, as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e. g. replacement of leucine with isoleucine. A variant may also have “non-conservative” changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art.

[0027] The expression “active fragment” refers to a portion or portions of a full length sequence that retain the biological function of the full length sequence encoding a protein, such as utrophin. “Identical” or “homologous” refers to the sequence identity or sequence similarity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or identical at that position. The percent of homology / identity between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched then the two sequences are 60% identical. Generally, a comparison is made when two sequences are aligned to give maximum homology / identity.

[0028] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include nonplasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. “Viral vector” refers to a non-replicating, non-pathogenic virus engineered for the delivery of genetic material into cells. In viral vectors, viral genes essential for replication and virulence are replaced with an expression cassette for the gene of interest. Thus, the viral vector genome comprises the expression cassette flanked by the viral sequences required for viral vector production.

[0029] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0030] The term “promoter” as used herein is defined as a nucleic acid sequence recognized by the transcriptional machinery of the cell, or introduced transcriptional machinery, required to initiate the specific transcription of a polynucleotide sequence, in particular of a gene of interest. As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence, which is required for expression of a gene product (i.e. the gene of interest) operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements, which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one, which expresses the gene product in a tissue specific manner.

[0031] The term “operably linked” as used above refers to the juxtaposition of the gene of interest with the sequences controlling its transcription. There may be additional residues between the promoter and the gene of interest so long as this functional relationship is preserved.

[0032] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0033] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell or when a repressor thereof is removed.

[0034] A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell preferentially if the cell is a cell of the tissue type corresponding to the promoter.

[0035] A “gene of interest” or “transgene” is a gene useful for a particular application, such as with no limitation, diagnosis, reporting, modifying, therapy and genome editing. For example, the gene of interest may be a therapeutic gene, a reporter gene or a genome-editing enzyme. In some embodiments, the gene of interest is a human gene.

[0036] In some embodiments, the gene of interest is a functional version of a gene or a fragment thereof. The functional version of said gene includes the wild-type gene, a variant gene such as variants belonging to the same family and others, or a truncated version, which preserves the functionality of the encoded protein at least partially. A functional version of a gene is useful for replacement or additive gene therapy to replace a gene, which is deficient or non-functional in a patient. In other embodiments, the gene of interest is a gene which inactivates a dominant allele causing an autosomal dominant genetic disease. A fragment of a gene is useful as recombination template for use in combination with a genome editing enzyme. Alternatively, the gene of interest may encode a protein of interest for a particular application (for example an antibody or antibody fragment, a genome-editing enzyme) or a RNA. In some embodiments, the protein is a therapeutic protein including a therapeutic antibody or antibody fragment, or a genome-editing enzyme. In some embodiments, the RNA is a therapeutic RNA. The gene of interest is a functional gene able to produce the encoded protein, peptide or RNA in the target cells of the disease, in particular muscle cells. The RNA is advantageously complementary to a target DNA or RNA sequence or binds to a target protein. For example, the RNA is an interfering RNA such as a shRNA, a microRNA, a guide RNA (gRNA) for use in combination with a Cas enzyme or similar enzyme for genome editing, an antisense RNA capable of exon skipping such as a modified small nuclear RNA (snRNA) or a long non-coding RNA. The interfering RNA or microRNA may be used to regulate the expression of a target gene involved in muscle disease. The guide RNA in complex with a Cas enzyme or similar enzyme for genome editing may be used to modify the sequence of a target gene, in particular to correct the sequence of a mutated / deficient gene or to modify the expression of a target gene involved in a disease, in particular a neuromuscular disease. The antisense RNA capable of exon skipping is used in particular to correct a reading frame and restore expression of a deficient gene having a disrupted reading frame. In some embodiments, the RNA is a therapeutic RNA.

[0037] In some embodiments of the invention, said gene of interest encodes a therapeutic protein or therapeutic ribonucleic acid that can be any protein or ribonucleic acid providing a therapeutic effect to skeletal muscular cells. In another embodiment of the invention, the said therapeutic protein can be any protein providing a therapeutic effect outside of the skeletal muscular cells. The said therapeutic protein can indeed be secreted by the said cells.

[0038] The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the “normal” (expected) respective characteristic. Characteristics, which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

[0039] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. A subject can be a mammal, e.g. a human, a dog, but also a mouse, a rat or a nonhuman primate. In certain non-limiting embodiments, the patient, subject or individual is a human. A “disease” or a “pathology” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate. In contrast, a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject’s state of health.

[0040] A disease or disorder is “alleviated” or “ameliorated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced. This also includes halting progression of the disease or disorder. A disease or disorder is “cured” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is eliminated.

[0041] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of pathology or has not be diagnosed for the pathology yet, for the purpose of preventing or postponing the occurrence of those signs.

[0042] As used herein, “treating a disease or disorder” means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. Disease and disorder are used interchangeably herein in the context of treatment.

[0043] An “effective amount” of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. The phrase “therapeutically effective amount”, as used herein, refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or condition, including alleviating symptoms of such diseases. An “effective amount” of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound. DETAILED DESCRIPTION OF THE INVENTION

[0044] According to a first aspect, the present invention concerns a micro-utrophin (pUTR), advantageously a functional micro-utrophin, more advantageously of human origin.

[0045] In the frame of the invention, “micro-utrophin” means a peptide or protein which is shorter than the native or wild type utrophin. In the context of the invention, the terms “micro-utrophin” and “mini-utrophin” have the same meaning. In the rest of the application, the term “micro-utrophi- dystrophin” will be used.

[0046] According to a specific embodiment, a micro-utrophin according to the invention has a size of more than 30% or 35%, or even more than 40% of the size of the full-length utrophin (e.g. 3433 amino acids for the human version). In relation to the human version, this means that according to the invention, the micro-utrophin advantageously contains more than 1165 amino acids, still advantageously more than 1370 amino acids, e.g. 1380 aa (pUTRl), 1371 aa (pUTR2), or 1393 aa (pUTR3).

[0047] The structure of utrophin is well documented (see Figure 1C) and active fragments thereof have been disclosed (see Figure ID). As it would be understood in the art, an active fragment is a portion or portions of the full-length sequence that retain at least some of the biological functions of the full length sequence.

[0048] A “functional” truncated utrophin or micro-utrophin means that the corresponding peptide or protein is able to perform at least some of the functions of the wild-type utrophin protein and is able to alleviate, at least partially, one or more of the symptoms associated with the absence of a native dystrophin, especially fiber degeneration, inflammation, necrosis, replacement of muscle with scar and fat tissue, muscle weakness, respiratory and cardiac failure, as well as premature death.

[0049] It is preferred that the micro-utrophin according to the invention displays (to a greater or lesser extent) at least one of the properties disclosed in relation to the micro-utrophin and / or micro-dystrophins of the prior art, especially those disclosed by Yue, etal. (Mol Ther, 2006. 14(1): 79-87) or Kodippili et al. (Human Gene Therapy, 2018, 29 (3), 299-311).

[0050] Among others, preferred properties are:

[0051] - Binding with at least one DAP (“dystrophin associated proteins”), especially with syntrophin, dystrobrevin, nNOS and / or PAR-lb proteins; Recruitment of the DAP complex at the sarcolemma;

[0052] - Rescue of the microtubule network;

[0053] - Muscle protection from damage;

[0054] - Preservation of the global structure of the protein and organization of spectrin repeat (R) domains;

[0055] - Restoration of muscle structure and function. Of particular interest are the skeletal muscles, but also the cardiac muscle and the diaphragm;

[0056] - More generally, amelioration of muscular function, gait, cardiac function, respiratory function, survival, quality and / or expectancy of life.

[0057] As known in the art, said properties can be tested in vitro on various cells expressing dystrophin, e.g. on iPS derived human DMD myogenic cells, ex vivo on muscle fibres isolated from various animal models, or in vivo based on animal models or even on patients suffering from DMD or BMD. Animal models are e.g. the mdx mouse (Foster H. et al., Mol Ther, 2008. 16(11): p. 1825-32), the mdx4cNmouse (Decrouy et al., Gen Ther, 1997. 4(5): 401- 8), the D2.B10-mdx / J mouse (Coley et al., Human Molecular genetics, 2016. 25(1): 130-45), the CXMDj dog (Koo et al., J Gene Med, 2011. 13(9): 497-506) or the GRMD dog (Le Guiner et al., Mol Ther., 2014. 22(11): 1923-35). The mouse model is commonly used to test new constructs encoding microdystrophins. However, this model has drawbacks because the mouse displays a less severe form of the disease, without immune reactions. The other animal model is the dog which is considered more reliable to predict the therapeutic potential of a gene therapy product in humans. The rat model as disclosed by Larcher et al. (Pios One, 2014, 9(10), el 10371) is also very interesting since it displays cardiomyopathy.

[0058] As known in the art, the full length dystrophin (Fig. 1 A) is characterized by different domains: a N-terminal domain (NTD);

[0059] 4 hinge domains (Hl to H4);

[0060] 24 spectrin-like repeats or rod domains (R1 to R24); a cysteine-rich domain (CRD); a C-terminal domain (CTD).

[0061] As known in the art, the full length utrophin (Fig. 1C) is characterized by different domains: a N-terminal domain (NTD);

[0062] 4 hinge domains (Hl to H4);

[0063] 22 spectrin-like repeats or rod domains (R1 to R22); a cysteine-rich domain (CRD); a C-terminal domain (CTD). According to a particular embodiment, the sequence of the human utrophin is shown in SEQ ID NO: 1.

[0064] According to another embodiment, the human utrophin has the following structure:

[0065] - the NTD domain corresponding to amino acids 1 to 255 of SEQ ID NO: 1;

[0066] - the Hl hinge domain corresponding to amino acids 256 to 311 of SEQ ID NO: 1;

[0067] - the R1 rod domain corresponding to amino acids 312 to 416 of SEQ ID NO: 1;

[0068] - the R2 rod domain corresponding to amino acids 421 to 525 of SEQ ID NO: 1;

[0069] - the R3 rod domain corresponding to amino acids 532 to 636 of SEQ ID NO: 1;

[0070] - the H2 hinge domain corresponding to amino acids 637 to 689 of SEQ ID NO: 1;

[0071] - the R4 rod domain corresponding to amino acids 690 to 795 of SEQ ID NO: 1;

[0072] - the R5 rod domain corresponding to amino acids 801 to 901 of SEQ ID NO: 1;

[0073] - the R6 rod domain corresponding to amino acids 910 to 1012 of SEQ ID NO: 1;

[0074] - the R7 rod domain corresponding to amino acids 1019 to 1121 of SEQ ID NO: 1;

[0075] - the R8 rod domain corresponding to amino acids 1128 to 1229 of SEQ ID NO: 1;

[0076] - the R9 rod domain corresponding to amino acids 1236 to 1333 of SEQ ID NO: 1;

[0077] - the R10 rod domain corresponding to amino acids 1335 to 1436 of SEQ ID NO: 1;

[0078] - the R11 rod domain corresponding to amino acids 1438 to 1540 of SEQ ID NO: 1;

[0079] - the R12 rod domain corresponding to amino acids 1547 to 1648 of SEQ ID NO: 1;

[0080] - the R13 rod domain corresponding to amino acids 1653 to 1747 of SEQ ID NO: 1; the R14 rod domain corresponding to amino acids 1748 to 1848 of SEQ ID NO: 1; the R15 rod domain corresponding to amino acids 1849 to 1968 of SEQ ID NO: 1; the R16 rod domain corresponding to amino acids 1979 to 2080 of SEQ ID NO: 1;

[0081] - the R17 rod domain corresponding to amino acids 2087 to 2186 of SEQ ID NO: 1;

[0082] - the H3 hinge domain corresponding to amino acids 2188 to 2228 of SEQ ID NO: 1;

[0083] - the R18 rod domain corresponding to amino acids 2229 to 2332 of SEQ ID NO: 1;

[0084] - the R19 rod domain corresponding to amino acids 2349 to 2439 of SEQ ID NO: 1;

[0085] - the R20 rod domain corresponding to amino acids 2446 to 2555 of SEQ ID NO: 1;

[0086] - the R21 rod domain corresponding to amino acids 2562 to 2687 of SEQ ID NO: 1;

[0087] - the R22 rod domain corresponding to amino acids 2694 to 2796 of SEQ ID NO: 1; the H4 hinge domain corresponding to amino acids 2797 to 2810 of SEQ ID NO: 1;

[0088] - the CRD domain corresponding to amino acids 2812 to 3165 of SEQ ID NO: 1, including the WW domain (corresponding to amino acids 2812 to 2845 of SEQ ID NO: 1) and the ZZ domain (corresponding to amino acids 3065 to 3121 of SEQ ID NO: 1);

[0089] - the CTD domain corresponding to amino acids 3166 to 3433 of SEQ ID NO: 1. According to one embodiment, a micro-utrophin according to the invention has at least one domain lacking, advantageously at least one spectrin-like-repeat (R), compared to the full length utrophin.

[0090] A micro-utrophin (Fig. ID) according to the prior art has a structure defined as follows, from its N terminal end and in the following order: a N-terminal domain (NTD); the Hl hinge domain;

[0091] - the Rl, R2 and R3 rod domains; the H2 hinge domain; the R22 rod domain; the H4 hinge;

[0092] - the cysteine-rich domain (CRD).

[0093] In other words, it has the structure NTDH1R1R2R3H2R22H4CRD. According to a particular embodiment, the sequence of such a micro-utrophin is shown in SEQ ID NO: 3.

[0094] The micro-utrophins of the invention contain at least the spectrin-like-repeats of the micro- utrophin of the prior art, i.e. R1-R2-R3 and R22.

[0095] Advantageously, micro-utrophins according to the invention contain further rod (R) domains compared to the micro-utrophin of the prior art, advantageously at least one or even two chosen in the group consisting of R4, R5, R6, R7, R8, R9 and RIO of utrophin, advantageously of human utrophin, and / or the R16 and R17 of dystrophin, advantageously of human dystrophin. According to a preferred embodiment, micro-utrophins do not contain R11-R21 of utrophin, advantageously of human utrophin.

[0096] According to one aspect, micro-utrophins of the invention contain at least one key protein binding site, especially for F-actin and possibly for the nNOS protein.

[0097] According to one embodiment, micro-utrophins of interest contain the binding site of actin which was shown to lie in repeats 1 to 10 (R1-R10) of the rod domain of utrophin. According to a preferred embodiment, micro-utrophins of interest contain at least the Rl-3 rod domains of utrophin and at least two further rod domains among R4-10, advantageously two contiguous rod domains, e.g. the R4-5 or R9-10 rod domains of utrophin.

[0098] According to one embodiment, the said at least 2 rod domains, advantageously the 2 rod domains are selected from the group consisting of R4, R5, R9 and RIO. According to one embodiment, the said at least 2 rod domains, advantageously the 2 rod domains are R4 and R5 or R9 and RIO.

[0099] According to another embodiment, micro-utrophins of interest contain the binding site of nNOS which was shown to lie in repeats 16 and 17 (R16, R17) of the rod domain of dystrophin (Lai et al., J. Clin. Invest., 2009. 119:624-635). According to a preferred embodiment, micro- utrophins of interest contain the R16-17 rod domains of dystrophin.

[0100] As known in the art, the sequence of the R16 and R17 rod domains is as shown in SEQ ID NO: 13.

[0101] According to a particular embodiment, the micro-utrophin has the structure CH1CH2H1R1R2R3H2R22H4CR of the utrophin and R16R17 of the dystrophin.

[0102] According to the invention, a micro-utrophin according to the prior art further comprises at least two other rod domains, advantageously two other rod domains, selected from the group consisting of:

[0103] - the R4 to RIO rod domains of utrophin;

[0104] - the R16 and R17 rod domains of dystrophin.

[0105] According to one embodiment, such a micro-utrophin further contains the R4 and R5 rod domains, advantageously from utrophin, more advantageously from human utrophin. In other words, such a micro-utrophin has the structure NTDH1R1R2R3H2R4R5R22H4CRD. According to a particular embodiment, the sequence of such a micro-utrophin is shown in SEQ ID NO: 5.

[0106] According to one embodiment, such a micro-utrophin further contains the R9 and RIO rod domains, advantageously from utrophin, more advantageously from human utrophin. In other words, such a micro-utrophin has the structure NTDH1R1R2R3H2R9R10R22H4CRD.

[0107] According to a particular embodiment, the sequence of such a micro-utrophin is shown in SEQ ID NO: 7. According to one embodiment, such a micro-utrophin further contains the R16 and R17 rod domains, advantageously from dystrophin, more advantageously from human dystrophin. In other words, such a micro-utrophin has the structure NTDH1R1R2R3H2R16R17R22H4CRD. In that case, the micro-utrophin can be considered as a hybrid / chimeric micro- dystrophin / utrophin.

[0108] According to a particular embodiment, the sequence of such a micro-utrophin is shown in SEQ ID NO: 9.

[0109] According to specific embodiments, micro-utrophins according to the invention lack the following rod domains:

[0110] R6 to R21. In other words, such a micro-utrophin contains R1 to R5 and R22;

[0111] R4 to R8 and R11 to R21. In other words, such a micro-utrophin contains R1 to R3, R9 to RIO and R22;

[0112] R4 to R21. In other words, such a micro-utrophin contains R1 to R3 and R22 of utrophin and R16 to R17 of dystrophin.

[0113] Advantageously, micro-utrophins according to the invention contain: a complete N-terminal domain (NTD), e.g. corresponding to amino acids 1 to 255 of SEQ ID NO: 1 or of SEQ ID NO: 3 a complete cysteine-rich (CRD) domain, e.g. corresponding to amino acids 2812 to 3165 of SEQ ID NO: 1 a partial or full-length C-terminal domain, e.g. the full-length C-terminal domain corresponding to amino acids 3166 to 3433 of SEQ ID NO: 1 at least one hinge (H) domain, chosen in the group consisting of Hl, H2, H3 and H4, advantageously at least Hl and H2, more advantageously Hl, H2 and H4.

[0114] According to one embodiment, the micro-utrophin protein is deprived of the Rl l, R12, R13, R14, R15, R18, R19, R20, and R21 rod domains of the utrophin gene and of the C-terminal domain (CTD) of the utrophin gene.

[0115] According to specific embodiments, the micro-utrophin according to the invention is: a AR6-21 / ACTD micro-utrophin (further named pUTRl), advantageously comprising or consisting of SEQ ID NO: 5 (Fig. IE); or a AR4-8 / AR11-21 / ACTD micro-utrophin (further named pUTR2), advantageously comprising or consisting of SEQ ID NO: 7 (Fig. IF); or a AR4-15 / AR18-21 / ACTD micro-utrophin with the R16 and R17 rod domains of dystrophin (further named pUTR3), advantageously comprising or consisting of SEQ ID NO: 9 (Fig. 1G).

[0116] According to one embodiment, a micro-utrophin is “substantially identical”, that is, is about 60% identical, preferably about 70% identical, more preferably about 80% identical, even more preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even more preferably about 99% identical to the micro-utrophins disclosed therein, especially those of sequence SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9, advantageously of SEQ ID NO: 7.

[0117] According to a further aspect, the present invention relates to an isolated synthetic nucleic acid molecule or a nucleic acid sequence encoding a micro-utrophin protein as disclosed above.

[0118] In one embodiment, said nucleic acid sequence is an ORF (“Open Reading Frame”. Advantageously, said sequence corresponds to a cDNA. However, e.g. single- or doublestranded DNA or RNA can be used.

[0119] According to one embodiment, the nucleic acid sequence encoding a micro-utrophin according to the invention comprises or consists of sequence SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, advantageously SEQ ID NO: 8.

[0120] According to another embodiment, the nucleic acid sequence encoding a micro-utrophin according to the invention is “substantially identical”, that is, is about 60% identical, preferably about 70% identical, more preferably about 80% identical, even more preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even more preferably about 99% identical to the sequence SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, advantageously of SEQ ID NO: 8.

[0121] The nucleic acid sequence encoding the functional micro-utrophin is advantageously of human origin but can also be a canine, a rat or a murine sequence. In one embodiment, the nucleic acid sequence originates from the organism it will be administered to (e.g., a human sequence in humans).

[0122] According to a specific embodiment, the nucleic acid sequence encoding the micro-utrophin according to the invention is optimized for use in a given subject, advantageously in humans. As examples, this optimized sequence can be modified as follows:

[0123] The sequence is modified to include a consensus Kozak sequence before AUG start codon within mRNA, to improve initiation of translation. The sequence is optimized based on transfer RNA frequencies in human and GC content is increased to promote RNA stability. As a result, and in a specific case, codon optimization for humans advantageously leads to 63% of codons being modified and the GC content increased to over 60%. This of course depends on the original (before optimization) micro- utrophin sequence and the target host.

[0124] According to another aspect, the present invention concerns an expression construct (or “expression cassette” or “insert” or “expression system”) comprising the nucleic acid sequence encoding the functional micro-utrophin of the invention.

[0125] According to one embodiment, the expression construct comprises the nucleic acid sequence encoding the transgene of interest, in the present case a micro-utrophin (pUTR), the 2 ITR sequences of an AAV vector, but also all the sequences required for a proper expression of said MD, i.e. regulatory elements.

[0126] According to common knowledge in the art, the size of the insert should not largely exceed the wild-type AAV genome length. For example, AAV2 contains 2 ITR sequences of 145 bp each and has a genome of 4682 pb (including the ITR sequences).

[0127] In a particular embodiment, the expression construct to be introduced in the AAV vector has a length of less than 5 kb, such as less than 4.9, 4.8, 4.7, 4.6 or 4.5 kb.

[0128] To advantage, and to limit the constraint of the size of the AAV packaging, the nucleic acid sequences encoding the micro-utrophin correspond to exons. In other words, they are preferably cDNA fragments.

[0129] According to a specific embodiment, the nucleic acid sequence is placed under the control of regulatory sequence(s).

[0130] In particular, the expression of said nucleic acid is governed by a promoter, usually located upstream said sequence.

[0131] Such promoters can be natural or synthetic (artificial) promoters, inducible or constitutive.

[0132] In one embodiment, the promoter is a ubiquitous promoter or has a low tissue-specificity. As an example, the expression vector can harbor the phosphoglycerate kinase 1 (PGK), EFl, ACTA1, P-actin, Desmin, all MCK variants, cardiac Troponin and CMV promoter. In a preferred embodiment, the promoter sequence is chosen in order to adequately govern the expression of the nucleic acid sequence placed under its control, in terms of expression level, but also of tissue specificity.

[0133] In one embodiment, the promoter is a muscle specific promoter. Such a promoter allows a robust expression in the skeletal muscles, in the diaphragm and possibly in the cardiac muscle.

[0134] Examples of suitable promoters known by the skilled person are e.g. the desmin promoter, the muscle creatine kinase (MCK) promoter, truncated creatine kinase promoters such as e.g. the CK6, CK7 or CK8 promoter, or derivatives thereof such as e.g. the MHCK7 promoter, the Syn promoter, the CMV promoter, MyoD, Myf5, Vcam, Pax3 and Pax7 satellite cell promoter. Another promoter is the synthetic promoter C5-12 (spC5-12).

[0135] Another example is the H3 promoter, advantageously of sequence SEQ ID NO: 11.

[0136] As known in the art, a non-exhaustive list of other possible regulatory sequences to be introduced in an AAV vector is:

[0137] - a polyadenylation signal, advantageously in 3’ of the sequence encoding the functional micro-utrophin;

[0138] - sequences for transcript stabilization, e.g. intron;

[0139] - enhancer sequences ;

[0140] - miRNA target sequences, which can inhibit the expression of the sequence encoding the micro-utrophin in non target tissues, in which said expression is not desired, for example where it can be toxic.

[0141] According to one aspect, the intron is selected from the group consisting of a human beta globin b2 (or HBB2) intron, a FIX intron and a chicken beta-globin intron, wherein said intron is optionally a modified intron such as a modified HBB2 intron, a modified FIX intron, or a modified chicken beta-globin intron.

[0142] According to another embodiment, the polyadenylation signal is selected among the human beta globin polyadenylation signal, the bovine growth hormone polyadenylation signal, the SV40 polyadenylation (pA) signal, or another naturally occurring or artificial polyadenylation signal.

[0143] An example of such a polyadenylation signal is the SV40 poly A, advantageously of sequence SEQ ID NO: 12. As previously mentioned, and for recombinant AAV production, the nucleic acid sequence encoding the microdystrophin is inserted between the ITR (« Inverted Terminal Repeat ») sequences of an AAV vector, advantageously the ITR of an AAV of serotype 2 (ITR2).

[0144] According to another aspect, the present invention relates to a plasmid or a vector comprising the nucleic acid sequence or the expression construct as disclosed above.

[0145] According to a preferred embodiment, the expression system of the invention includes a vector having a suitable tropism, in this case higher for the target tissue(s), advantageously the skeletal muscles and the heart, than for the tissues where the expression of the protein is not desired.

[0146] Advantageously, such a vector is a viral vector. Viral vectors commonly used in gene therapy in mammals, including humans, are known to those skilled in the art. Such viral vectors are preferably chosen from the following list: vector derived from the herpes virus, baculovirus vector, lentiviral vector, retroviral vector, adenoviral vector and adeno-associated viral vector (AAV).

[0147] According to a specific embodiment of the invention, the viral vector is an adeno-associated viral (AAV) vector.

[0148] Adeno-associated viral (AAV) vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, moderate immunogenicity, and the ability to transduce post-mitotic cells and tissues in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.

[0149] In one embodiment, the encoding sequence is contained within an AAV vector. More than 100 naturally occurring serotypes of AAV are known. Many natural variants in the AAV capsid exist, allowing identification and use of an AAV with properties specifically suited for dystrophic pathologies. AAV viruses may be engineered using conventional molecular biology techniques, making it possible to optimize these particles for cell specific delivery of nucleic acid sequences, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivery to the nucleus. As mentioned above, the use of AAVs is a common mode of exogenous delivery of DNA as it is relatively non-toxic, provides efficient gene transfer, and can be easily optimized for specific purposes. Among the serotypes of AAVs isolated from human or non-human primates (NHP) and well characterized, human serotype 2 is the first AAV that was developed as a gene transfer vector. Other currently used AAV serotypes include AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. In addition, non-natural engineered variants and chimeric AAV can also be useful.

[0150] Desirable AAV fragments for assembly into vectors include the cap proteins, including the vpl, vp2, vp3 and hypervariable regions, the rep proteins, including rep 78, rep 68, rep 52, and rep 40, and the sequences encoding these proteins. These fragments may be readily utilized in a variety of vector systems and host cells.

[0151] Such fragments may be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, without limitation, AAV with a non-naturally occurring capsid protein. Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vpl capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV serotype, noncontiguous portions of the same AAV serotype, from a non-AAV viral source, or from a non- viral source (i.e. its capsid comprises VP capsid proteins derived from at least two different AAV serotypes, or comprises at least one chimeric VP protein combining VP protein regions or domains derived from at least two AAV serotypes). An artificial AAV serotype may be, without limitation, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid. Moreover, a peptide (P) can be introduced in said capsids, for example into a variable region of the cap gene, possibly to modify the AAV tropism.

[0152] In one embodiment, the vectors useful in the compositions and methods described herein contain, at a minimum, sequences encoding a selected AAV serotype capsid, e.g., an AAV9 capsid, or a fragment thereof. In another embodiment, useful vectors contain, at a minimum, sequences encoding a selected AAV serotype rep protein, e.g., AAV9 rep protein, or a fragment thereof. Optionally, such vectors may contain both AAV cap and rep proteins. In vectors in which both AAV rep and cap are provided, the AAV rep and AAV cap sequences can both be of one serotype origin, e.g., all AAV9 origin. Alternatively, vectors may be used in which the rep sequences are from an AAV serotype, which differs from that which is providing the cap sequences. In one embodiment, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or a host cell and a vector). In another embodiment, these rep sequences are fused in frame to cap sequences of a different AAV serotype to form a chimeric AAV vector. In some embodiments the AAV vector comprises a genome and a capsid derived from AAVs of different serotypes.

[0153] Thus exemplary AAVs, or artificial AAVs, include AAV2 / 8 (US 7,282,199), AAV2 / 5 (available from the National Institutes of Health), AAV2 / 9 (W02005 / 033321), AAV2 / 6 (US 6,156,303), AAVrhlO (W02003 / 042397), AAVrh74 (W02003 / 123503), AAV9-rh74 hybrid or AAV9-rh74-Pl hybrid (WO2019 / 193119; W02020 / 200499; EP20306005.8).

[0154] According to one embodiment, the AAV is of serotype 2, 5, 8 or 9, or an AAVrh74. Advantageously, the claimed vector comprises a capsid selected from the group consisting of: AAV8 capsid, AAV9 capsid, AAV9-rh74 capsid and AAV9-rh74-Pl capsid.

[0155] According to a particular embodiment, the AAV is an AAV of serotype 9 (AAV9).

[0156] In the AAV vectors used in the present invention, the AAV genome may be either a single stranded (ss) nucleic acid or a double stranded (ds) / self complementary (sc) nucleic acid molecule.

[0157] Advantageously and as mentioned above, the nucleic acid sequence of interest is inserted between the ITR (« Inverted Terminal Repeat ») sequences of the AAV vector. Typically, ITR sequences originate from AAV2 or AAV9, advantageously AAV9.

[0158] Thus, according to one aspect, the present invention relates to a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid molecule as disclosed above.

[0159] As known in the art, recombinant viral particles can be obtained, e.g., by tri -transfection of 293 HEK cells, by the herpes simplex virus system and by the baculovirus system, or using specific cell lines. The vector titers are usually expressed as viral genomes per ml (vg / ml).

[0160] Further aspects of the invention concern:

[0161] A cell comprising the expression system of the invention, or a vector comprising said expression system, as disclosed above.

[0162] The cell can be any type of cells, i.e. prokaryotic or eukaryotic. The cell can be used for propagation of the vector or can be further introduced (e.g. grafted) in a host or a subject. The expression system or vector can be introduced in the cell by any means known in the art, e.g. by transformation, electroporation or transfection. Vesicles derived from cells can also be used. A transgenic animal, advantageously non-human, comprising the expression system of the invention, a vector comprising said expression system, or a cells comprising said expression system or said vector, as disclosed above.

[0163] Another aspect of the invention relates to a composition comprising an expression system, a vector or a cell, as disclosed above, for use as a medicament.

[0164] According to another aspect, the present invention relates to a composition, advantageously a pharmaceutical composition or a drug, comprising an AAVr vector as disclosed above.

[0165] According to an embodiment, the composition of the invention may comprise other active molecules (other gene therapy products, chemical entities, peptides, proteins, ...), dedicated to the treatment of the same disease or another disease.

[0166] The present invention then provides pharmaceutical compositions comprising an expression system, a vector or a cell of the invention. Such compositions comprise a therapeutically effective amount of the therapeutic, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. or European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.

[0167] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, sustained-release formulations and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to release pain at the site of the injection.

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

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

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

[0171] In case of a treatment comprising administering a viral vector, such as an AAV vector, to the subject, typical doses of the vector are of at least IxlO8vector genomes per kilogram body weight (vg / kg), such as at least IxlO9vg / kg, at least IxlO10vg / kg, at least IxlO11vg / kg, at least IxlO12vg / kg at least IxlO13vg / kg, at least IxlO14vg / kg, at least 1015vg / kg. Specifically, the dose can be between 5.1011vg / kg and 1014vg / kg, e.g. 1, 2, 3, 4, 5, 6, 7, 8 or 9.1013vg / kg. A lower dose of e.g. 1, 2, 3, 4, 5, 6, 7, 8 or 9.1012vg / kg can also be contemplated in order to avoid potential toxicity and / or immune reactions. As known by the skilled person, a dose as low as possible giving a satisfying result in term of efficiency is preferred. Available routes of administration are topical (local), enteral (system-wide effect, but delivered through the gastrointestinal (GI) tract), or parenteral (systemic action, but delivered by routes other than the GI tract). The preferred route of administration of the compositions disclosed herein is parenteral which includes intramuscular administration (i.e. into the muscle) and systemic administration (i.e. into the circulating system). In this context, the term “injection” (or “perfusion” or “infusion”) encompasses intravascular, in particular intravenous (IV), intramuscular (IM), intraocular, intrathecal or intracerebral administration. Injections are usually performed using syringes or catheters.

[0172] In one embodiment, systemic delivery of the composition comprises administering the composition near a local treatment site, i.e. in a vein or artery nearby a weakened muscle. In certain embodiments, the invention comprises the local delivery of the composition, which produces systemic effects. This route of administration, usually called “regional (loco-regional) infusion”, “administration by isolated limb perfusion” or “high-pressure transvenous limb perfusion” has been successfully used as a gene delivery method in muscular dystrophy.

[0173] According to one aspect, the composition is administered to an isolated limb (loco-regional) by infusion or perfusion. In other words, the invention comprises the regional delivery of the composition in a leg and / or arm by an intravascular route of administration, i.e. a vein (transvenous) or an artery, under pressure. This is usually achieved by using a tourniquet to temporarily arrest blood circulation while allowing a regional diffusion of the infused product, as e.g. disclosed by Toromanoff et al. (2008).

[0174] In one embodiment, the composition is injected in a limb of the subject. When the subject is a human, the limb can be the arm or the leg. According to one embodiment, the composition is administered in the lower part of the body of the subject, e.g. below the knee, or in the upper part of the body of the subject, e.g., below the elbow.

[0175] A preferred method of administration according to the invention is systemic administration. Systemic injection opens the way to an injection of the whole body, in order to reach the entire muscles of the body of the subject including the heart and the diaphragm and then a real treatment of these systemic and still incurable diseases. In certain embodiments, systemic delivery comprises delivery of the composition to the subject such that composition is accessible throughout the body of the subject.

[0176] According to a preferred embodiment, systemic administration occurs via injection of the composition in a blood vessel, i.e. intravascular (intravenous or intra-arterial) administration. According to one embodiment, the composition is administered by intravenous injection, through a peripheral vein.

[0177] In a specific embodiment, the treatment comprises a single administration of the composition.

[0178] In one embodiment, the presence of the gene therapy product and / or the expression of the functional micro-utrophin, as well as the associated therapeutic benefits, are observed for up to 1 month, or 3 months or 6 months or even 1 year, 2 years, 5 years, 10 years, or even more the whole life of the subject.

[0179] According to the invention, the subject is preferably a human, but can also be a mouse, a rat, a non-human primate, or a dog.

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

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

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

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

[0184] According to another embodiment, the invention concerns the use of a pUTR, a rAAV or a composition as disclosed above for the preparation of a medicament for the treatment of a dystrophic disease.

[0185] In other words, the present invention provides a method for treating a dystrophic disease, especially DMD and BMD, in a subject, comprising administrating, advantageously by systemic administration, to the subject a pUTR, a rAAV or a composition as disclosed above.

[0186] A first target of the invention is to provide a safe (not toxic) and non-immunogenic treatment. A further aim is to provide an efficient treatment which allows to postpone, slow down or prevent the development of the disease, and possibly to ameliorate the phenotype of the patient which can be easily monitored at the clinical level. In a subject, AAV vectors and compositions according to the invention can be used: for ameliorating muscular function. Of particular interest are the skeletal muscles, but also the cardiac muscle and the diaphragm; for ameliorating gait; for ameliorating cardiac function; for ameliorating respiratory function; for prolonging survival, more generally to ameliorate the quality and the expectancy of life.

[0187] The amelioration of said functions can be assessed by the methods described in the examples below or known from the skilled person.

[0188] More generally and according to further embodiments, the invention is useful for: increasing muscular force, muscular endurance and / or muscle mass in a subject; reducing fibrosis in a subject; reducing contraction-induced injury in a subject;

[0189] - treating muscular dystrophy in a subject; reducing degenerating fibers or necrotic fibers in a subject suffering from muscular dystrophy; reducing inflammation in a subject suffering from muscular dystrophy; reducing levels of creatine kinase (or any other dystrophic marker) in a subject suffering from muscular dystrophy; - treating myofiber atrophy and hypertrophy in a subject suffering from muscular dystrophy; decreasing dystrophic calcification in a subject suffering from muscular dystrophy; decreasing fatty infiltration in a subject; decreasing central nucleation in a subject.

[0190] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of Animal Cells” (Freshney, 2010); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1997); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Short Protocols in Molecular Biology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles, Applications and Troubleshooting”, (Babar, 2011); “Current Protocols in Immunology” (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

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

[0192] EXPERIMENTAL EXAMPLES

[0193] The invention is further described in detail by reference to the following experimental examples and the attached figures. These examples are provided for purposes of illustration only and are not intended to be limiting.

[0194] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. FIGURES

[0195] Figure 1 : Domain organization of different forms of dystrophin and utrophin:

[0196] A / Scheme of the full-length dystrophin

[0197] B / Scheme of the AR4-23 / ACTD micro-dystrophin (see e.g. WO2015 / 197869)

[0198] C / Scheme of the full-length utrophin

[0199] D / Scheme of the AR4-21 / ACTD micro-utrophin (SEQ ID NO: 3) according to the prior, e.g. in W02005 / 049850 (pUTRb)

[0200] E / Scheme of the AR6-21 / ACTD micro-utrophin (SEQ ID NO: 5) according to the invention (pUTRl)

[0201] F / Scheme of the AR4-8 / AR11-21 / ACTD micro-utrophin (SEQ ID NO: 7) according to the invention (pUTR2).

[0202] G / Scheme of the AR4-15 / AR18-21 / ACTD micro-utrophin (SEQ ID NO: 9) according to the invention (pUTR3).

[0203] ACBD: Actin Binding Domain nNOS: neuronal nitric oxide synthase

[0204] DG : Dystroplycoprotein complex

[0205] SYN : Syntrophin

[0206] NTD : N-terminal region

[0207] CRD : Cysteine-rich domain

[0208] CTD : C -terminal domain

[0209] Figure 2: Activity tests in wild-type mice (wt), mutant mice mdx) and mdx mice treated with micro-utrophins (pUTRb and pUTRl-3)

[0210] (A) Total activity

[0211] (B) Total distance

[0212] (C) Total upright position

[0213] (D) Maximal speed

[0214] Figure 3: Muscle function assays in wild-type mice (wt), mutant mice mdx) and mdx mice treated with micro-utrophins (pUTRb and pUTRl-3)

[0215] (A) Grip test assay

[0216] (B) Specific tetanic force assay

[0217] (C) Specific twitch force assay Figure 4: Individual and general recovery score of the mdx mice treated with micro-utrophins (pUTRb and pUTRl-3) in comparison to the sick mdx animals (0%) and the wild type C57 mice (100%)

[0218] MATERIALS AND METHODS

[0219] Animals

[0220] Different mouse models were used in this study: C57BL / 10 (wild-type) and bl 10 mdx (lacking the dystrophin gene). All mouse procedures were done according to protocol approved by the Ethic Committee at the CERFE of Evry animal facility and under appropriate biological containment.

[0221] Tested micro-utrophins

[0222] Preparation of rAAV9-pUTR

[0223] The expression cassettes including the AAV9 ITR, the H3 promoter, the human pUTR cDNA and the SV40 PolyA were generated.

[0224] Adeno-associated virus vectors were produced following a triple transfection protocol using 293 cells in suspension and purified by affinity chromatography.

[0225] The rAAV production was performed using the Genethon facilities. Administration

[0226] All rAAVs were systemically injected with a total 1.1012vg dose for a duration of 10 weeks in non-immunocompetent 4-weeks old bll Q.mdx. At 14 weeks of age, functional assays were run to evaluate the full in vivo potential of all pUTR transgenes.

[0227] Functional assays

[0228] Movements measurement

[0229] Experiment on a vigilant rodent allowing to measure different parameters of movements and activity of the rodent.

[0230] Equipment:

[0231] An LE8811 infrared actimeter from Bioseb comprising:

[0232] • a computer

[0233] • 2 infrared detection frames LE8815

[0234] • 1 frame support LE8817

[0235] • 1 container of dimensions (acquisition zones): 450 x 450 x 300 mm

[0236] • 1 electronic control box

[0237] • Actitrack software for transferring values to PC with LE8811 connection cable

[0238] Mice are put one by one in the acquisition zones. The analysis is carried out over one hour and a half of acquisition. Thanks to the detection frames, the following parameters are measured:

[0239] - Total activity

[0240] The percentage of inactivity is measured.

[0241] The percentage of activity is calculated with the following formula: 100 - percentage or inactivity.

[0242] - Total distance

[0243] Distance traveled by the mouse during the measurement.

[0244] - Total upright position

[0245] Number of upright positions during the measurement.

[0246] - Maximal speed

[0247] Maximal speed reached during the measurement. Grip strength test assay

[0248] Mice were tested to determine peak paw grip strength, positioned horizontally with their forepaws on a grip bar using a grip strength meter (MK-380 M; Muromachi Kikai Co., Ltd., Japan), and pulled back slowly and steadily until mice released their grip. This was repeated six times, and peak force for the four-limb paws was measured. Grip strength was normalized for mouse body weight measured on the same date as grip strength.

[0249] In situ ex vivo functional test in tibialis anterior (TA) muscle (tetanic force and twitch force) Tetanic force: repetitions of electrical stimulation leading to a summation and fusion of tremors. The myogram presents a plateau appearance.

[0250] Twitch force: this corresponds to a contraction followed by a relaxation of a muscle in response to a single, short-lasting isolated stimulation of the nerve.

[0251] For the measurement of contractile properties, mice were anaesthetized (e.g. with xylazine) and intact tibialis anterior (TA) muscles were dissected and placed in an experimental organ bath. Muscles were stimulated by an electric field generated between two platinum electrodes placed longitudinally on either side of the muscle (e.g. square wave pulses 25 V, 0.2 ms in duration, 150 Hz). Muscles were adjusted to the optimum length (Lo) for the development of isometric twitch force and a 5 min recovery period was allowed between stimulations. Optimal muscle length (Lo) and stimulation voltage were determined from micromanipulation of muscle length and a series of twitch contractions that produced maximum isometric twitch force.

[0252] Recovery score

[0253] The percentage recovery score was calculated as described on the TREAT-NMD website (TREAT -NMD SOP M.l. l_001 : http: / / www.treat-nmd.eu / downloads / file / sops / dmd / MDX / DMD M.1.1 001.pdf).

[0254] All counting was performed blinded and no fibres were specifically selected or removed to generate the results.

[0255] RESULTS

[0256] As shown on Figure 2, the mdx mouse presents significant defects in total activity, total distance, total upright position, and maximal speed compared to wild-type animal. In all functional assays, significant benefits were noted with the rAAVs-pUTR treatments.

[0257] As shown on Figure 3, the mdx mice present significant defect compared to wild-type (wt) animal in all assays. As previously observed, the pUTR are efficient transgenes to restore force in mdx animals. Based on all the results generated, a recovery score was established to situate all mice / groups in comparison to mdx animals (0%) and the wild-type mice (100%). Figure 4 presents the recovery scores per assays and the general recovery scores obtained so far for all groups of pUTRs.

[0258] Based on these data, it appears that the pUTRs according to the invention are more efficient that the pUTR of the prior art to rescue defects in mdx mice.

[0259] CONCLUSION

[0260] This study brings the proof of concept that the H3-pUTRl-3 therapeutic cassette coding for optimized micro-utrophins according to the invention and encapsulated in the AAV9 capsid provides clinical benefit in the mouse model of the Duchenne myopathy after systemic intravenous administration.

[0261] Overall, these data present the benefits of newly optimized pUTR gene therapy (pUTRl-3) in comparison to the initial pUTR transgene (pUTRb). All optimized pUTR outcompete the initial pUTR construct. As dystrophin-based gene therapies is currently facing important immune responses issues, notably against the dystrophin transgene, not naturally expressed in DMD, an optimized, similarly efficient micro-utrophin gene therapy with a safer immunologic profile (utrophin is naturally expressed in DMD) could offer an innovative gene therapy solution to treat DMD.

Claims

CLAIMS1. A micro-utrophin comprising the structure NTDH1R1R2R3H2R22H4CRD and two further rod domains selected from the group consisting of:- the R4 to RIO rod domains of utrophin;- the R16 and R17 rod domains of dystrophin.

2. The micro-utrophin according to claim 1 having the structure NTDH1R1R2R3 H2R9R10R22H4CRD.

3. The micro-utrophin according to claim 2 having the sequence SEQ ID NO: 7.

4. The micro-utrophin according to claim 1 having the structure NTDH1R1R2R3 H2R4R5R22H4CRD.

5. The micro-utrophin according to claim 4 having the sequence SEQ ID NO: 5.

6. The micro-utrophin according to claim 1 having the structure NTDH1R1R2R3H2R16R17R22H4CRD.

7. The micro-utrophin according to claim 6 having the sequence SEQ ID NO: 9.

8. A nucleic acid sequence encoding the micro-utrophin according to claims 1 to 7.

9. The nucleic acid sequence according to claim 8 comprising or consisting of a sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 6, SEQ ID NO: 10, and a sequence having at least 90% identity thereto.

10. A recombinant adeno-associated virus (rAAV) vector comprising the nucleic acid sequence according to claim 8 or 9.

11. The rAAV according to claim 10, wherein the nucleic acid sequence is operably linked to the H3 promoter, advantageously of sequence SEQ ID NO: 11.

12. The rAAV according to claim 10 or 11, wherein the AAV is of serotype 9.

13. A composition comprising, in a pharmaceutically acceptable carrier, the rAAV according to any of claims 10 to 12.

14. The rAAV according to any of claims 10 to 12 or the composition according to claim 13, for use as a medicament.

15. The rAAV according to any of claims 10 to 12 or the composition according to claim 13 for use in treating a dystrophic disease, in particular a Duchenne Muscular Dystrophy (DMD) or a Becker Muscular Dystrophy (BMD).

Images