筋肉内及び心臓内でのSGCGの適切な発現を可能にする遺伝子療法発現系

Abstract

比較的重要な数の患者を対象として心臓表現型の観察結果を考慮しつつ、あるレベルのSGCG発現が心臓において必要とされることが十分に立証されたとしても(Calvoら、Neuromuscul. Disord. 2000; 10(8):560~6頁; Van der Kooiら、Heart 1998; 79(1):73~7頁)、内因的バランスを重視し、またあらゆる毒性を回避するために、心臓内では過度の過剰生成を引き起こさず、骨格筋内では適切なレベルでのSGCG発現を可能にする発現系を有することが極めて望ましい。【解決手段】本発明は、骨格筋内及び心臓内でのガンマサルコグリカン(SGCG)の適切な発現を可能にするプロモーターの制御下に置かれたSGCGをコードする配列を含む、全身投与用の発現系、並びに肢帯型筋ジストロフィーC型を処置するためのその使用に関する。

Description

[Technical Field]

[0001] The present invention is based on the recognition that it is beneficial to appropriately express SGCG (γ-sarcoglycan) in skeletal muscle and the heart, and moreover, that the quantity of SGCG protein in skeletal muscle should exceed or be equal to the quantity of SGCG protein in the heart. The present invention provides an expression system comprising a transgene and a promoter sequence, which avoids overproduction in the heart. The present invention then provides a useful and safe therapeutic tool for treating limb-girdle muscular dystrophy type 2C (LGMD2C) (newly named limb-girdle muscular dystrophy type R5 (LGMD R5)). Such an expression profile is also significant for other sarcoglycans, namely alpha (α) sarcoglycan (SGCA), beta (β) sarcoglycan (SGCB), and delta (δ) sarcoglycan (SGCD). [Background technology]

[0002] The term sarcoglycan disorders (SG) encompasses four distinct rare diseases belonging to a larger group of limb-girdle muscular dystrophy (LGMD): LGMD2C or γ-SG, LGMD2D or α-SG, LGMD2E or β-SG, and LGMD2F or δ-SG. Interestingly, the relative prevalence of each form varies considerably from region to region. For example, LGMD2F accounts for about 14% of SG in Brazil, while being extremely rare elsewhere (Moreira ES et al., J. Med. Genet. 2003; 40:E12), and LGMD2C is a form that occurs almost exclusively in North African and Roman populations (Bonnemann CG et al., Neuromuscul. Disord. 1998;8:pp. 193-197; Dalichaouche I. et al., Muscle Nerve. 2017;56:pp. 129-135; Piccolo F. et al., Hum. Mol. Genet. 1996;5:pp. 2019-2022; Ben Othmane K. et al., Am. J. Hum. Genet. 1995;57:pp. 732-734).

[0003] LGMD2C (LGMD R5) is caused by a mutation in the γ-sarcoglycan (SGCG) gene, which encodes γ-sarcoglycan. SGCG is a 35 kDa single-pass transmembrane glycoprotein consisting of a small intracellular domain at the N-terminus, a transmembrane domain, and a large extracellular domain containing an N-glycosylation site. Along with α, β, and δ-sarcoglycans, SGCG forms part of the sarcoglycan subcomplex present in striated muscle. This subcomplex is an important member of the dystrophin-associated glycoprotein complex (DGC) and plays a crucial role in maintaining connectivity between the submembrane cytoskeleton and the extracellular matrix. Mutations in any of the sarcoglycans disrupt DGC complex formation, leading to secondary deficiencies of other sarcoglycans on the muscle cell membrane at varying levels. Destabilization of this complex induces a loss of stability in the muscle cell membrane and a loss of protection of muscle fibers from contraction-induced damage (Petrof BJ et al., Proc. Natl. Acad. Sci. USA. 1993;90:3710~3714; Cohn RD, and Campbell KP, Muscle Nerve. 2000;23:1456~1471).

[0004] The loss of this protection leads to a genetic defect in LGMD2C, inducing a necrotic degenerative regeneration process and causing progressive muscle wasting. The disease is characterized by marked proximal weakness in the limbs (most often beginning in the lower extremities), generalized gastrocnemius hypertrophy, and early joint contractures. The frequency of respiratory dysfunction and dilated cardiomyopathy is variable. Clinical severity usually correlates with the amount of residual protein, and a genotype-phenotype correlation may also be observed. Null mutations are usually associated with the absence of protein and a severe Duchenne muscular dystrophy (DMD)-like phenotype, while missense mutations are associated with reduced protein levels and a milder LGMD-like phenotype (Semplicini C. et al., Neurology. 2015;84:1772-1781; Magri F. et al., Muscle Nerve. 2017;55:55-68).

[0005] To date, there are no available treatments for LMGD2C.

[0006] In recent years, gene therapy approaches to correct pathology have been demonstrated in a mouse model lacking γ-SG (Cordier L. et al., Mol. Ther. 2000;1:119-129). In 2012, the results of a Phase I-II clinical trial for LGMD2C involving intramuscular injection of AAV1 expressing the human γ-SG gene under the control of the desmin promoter were reported (Herson S. et al., Brain. 2012;135:483-492). Following this trial, Israeli et al. (Mol Ther Methods Clin Dev. 2019; 13:494-502) developed a gene therapy approach for Sgcg - / - We report the results of a dose-response study focusing on muscle repair after systemic administration of AAV2 / 8, which carries the same construct and expresses γ-SG under the control of the desmin promoter, in mice.

[0007] Meanwhile, International Publication No. 2019 / 152474 discloses a codon-optimized sequence encoding SGCG, which is supported by the AAVrh74 vector and expressed under the control of the MHCK7 promoter.

[0008] Therefore, gene replacement therapy based on SGCG is considered a promising treatment method for pathologies caused by SGCG deficiency. However, there is still a need for safe and effective treatment methods.

[0009] In relation to gene therapy, a safe expression system is defined as a system that ensures the production of therapeutically effective amounts of protein in target tissues, i.e., in tissues where the protein is needed, and that corrects abnormalities linked to deficiencies of natural proteins, particularly in essential and vital organs or tissues, without exhibiting any toxicity. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] International Publication No. 2019 / 152474 [Patent Document 2] International Publication No. 2007 / 000668 [Patent Document 3] U.S. Patent No. 7,282,199 [Patent Document 4] International Publication No. 2005 / 033321 [Patent Document 5] U.S. Patent No. 6,156,303 [Patent Document 6] International Publication No. 2003 / 042397 [Patent Document 7] International Publication No. 2003 / 123503 [Patent Document 8] International Publication No. 2019 / 193119 [Patent Document 9] European Patent No. 2020 / 061380 [Non-patent literature]

[0011] [Non-licensed Document 1] Moreira ESら, J. Med. Genet. 2003; 40:E12 [Non-licensed Document 2] Bonnemann CGら、Neuromuscul. Disord. 1998;8:193~197 pages [Non-licensed Document 3] Dalichaouche I.ら、Muscle Nerve. 2017;56:129~135 pages [Non-licensed Document 4] Piccolo F., Hum. Mol. Genet. 1996;5:2019~2022 pages [Non-licensed Document 5] Ben Othmane K., Am. J. Hum. Genet. 1995;57:732~734 pages [Non-licensed Document 6] Petrof BJら, Proc. Natl. Acad. Sci. USA. 1993;90:3710~3714 pages [Non-licensed Document 7] Cohn RD, and Campbell KP, Muscle Nerve. 2000;23:1456~1471 pages [Non-licensed Document 8] Semplicini C.ら, Neurology. 2015;84:1772~1781 pages [Non-licensed Document 9] Magri F.ら、Muscle Nerve. 2017; pages 55:55~68 [Non-licensed Document 10] Cordier L., Mol. Ther. 2000;1:119~129 pages [Non-licensed Document 11] Herson S.ら、Brain. 2012;135:483~492 pages [Non-licensed Document 12] Israeliら、Mol Ther Methods Clin Dev. 2019; 13:494~page 502 [Non-licensed Document 13] Calvo, Neuromuscul. Disord. 2000; 10(8):560~6 pages [Non-licensed Document 14] Van der Kooi, Heart 1998; 79(1): pages 73~7 [Non-licensed Document 15] Gaoら、The Journal of Clinical Investigation, 2015; 125(11): pages 4186~95 [Non-licensed Document 16] Wangら, 2008, Gene Therapy, Vol. 15, pages 1489~99 [Non-licensed Document 17] Piekarowiczら, 2017, European Society Of Gene & Cell Therapy conference, poster P096; HUMAN GENE THERAPY 28:A44 (2017), DOI: 10.1089 / hum.2017.29055.abstracts [Non-licensed Document 18] Corin, 1995, Proc. Natl. Acad. Sci., Vol. 92, pp. 6185-89. [Non-licensed Document 19] Blain, 2010, Human Gene Therapy, Vol. 21, pages 127~34 [Non-licensed Document 20] EW Martin, "Remington's Pharmaceutical Sciences" [Non-licensed Document 21] "Molecular Cloning: A Laboratory Manual", 4th edition (Sambrook, 2012) [Non-licensed Document 22] "Oligonucleotide Synthesis" (Gait, 1984) [Non-licensed Document 23] “Culture of Animal Cells” (Freshney, 2010) [Non-Patent Document 24] "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1997) [Non-Patent Document 25] "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987) [Non-Patent Document 26] "Short Protocols in Molecular Biology" (Ausubel, 2002) [Non-Patent Document 27] “Polymerase Chain Reaction: Principles, Applications and Troubleshooting”, (Babar, 2011) [Non-Patent Document 28] "Current Protocols in Immunology" (Coligan, 2002) [Non-Patent Document 29] Hack et al., J. Cell. Biol. 1998;142:1279~87 [Non-Patent Document 30] Goncalves et al., Mol Ther. 2011;19(7): pp. 1331-1341. [Non-Patent Document 31] Wang et al., Gene Therapy 2008;15:1489-99. [Non-Patent Document 32] Scheuermann et al., EMBO J. 2013; 32(13): pp. 1805-1816. [Overview of the project] [Problems that the invention aims to solve]

[0012] The present invention aims to alleviate or correct destructive pathologies linked to γ-sarcoglycan (SGCG) deficiency, such as limb-girdle muscular dystrophy type 2C (LGMD2C), by providing an expression system that ensures the production of an appropriate amount, i.e., a therapeutically effective amount of protein that is not toxic, within skeletal muscle and the heart. [Means for solving the problem]

[0013] Even if it is sufficiently established that a certain level of SGCG expression is necessary in the heart, taking into account observations of cardiac phenotypes in a relatively important number of patients (Calvo et al., Neuromuscul. Disord. 2000; 10(8): pp. 560-566; Van der Kooi et al., Heart 1998; 79(1): pp. 73-77), it is extremely desirable to have an expression system that does not cause excessive overproduction in the heart and enables appropriate levels of SGCG expression in skeletal muscle, in order to prioritize intrinsic balance and avoid any toxicity.

[0014] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Technical terms used in the description are for the sole purpose of describing specific embodiments and are not intended to limit them.

[0015] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the articles. For example, "an element" means one or more elements.

[0016] When used herein, "about" or "approximately" is intended to encompass a variation of ±20%, ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from a given numerical value, and such variation is therefore appropriate for carrying out the disclosed method.

[0017] Scope: Throughout this disclosure, various aspects of the invention may be presented in range form. Range form should be understood as being solely for convenience and brevity, and should not be interpreted as an irrevocable limitation on the scope of the invention. Therefore, range descriptions should be considered to specifically disclose all conceivable subranges and individual numbers within that range. For example, a range description, e.g., 1-6, should be considered to specifically disclose subranges, e.g., 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0018] "Isolated" means that it has been altered or removed from its natural state. For example, nucleic acids or peptides that are naturally present in living animals are not in an "isolated" state, but the same nucleic acids or peptides that have been partially or completely separated from their naturally occurring coexisting substances are in an "isolated" state. Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, such as within a host cell.

[0019] In the context of this invention, the following abbreviations are used for commonly existing nucleic acid bases: "A" refers to adenosine, "C" to cytosine, "G" to guanosine, "T" to thymidine, and "U" to uridine.

[0020] The term "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 idiomatic expression "nucleotide sequence encoding a protein, or RNA or cDNA" may also include introns, to the extent that nucleotide sequences encoding proteins may contain introns in some versions.

[0021] "~coding" refers to the inherent characteristics of a particular sequence of nucleotides within a polynucleotide, such as a gene, cDNA, or mRNA, that serve as a template for the synthesis of defined sequences of nucleotides (i.e., rRNA, tRNA, and mRNA), defined sequences of amino acids, and other polymers and macromolecules having the biological properties derived therefrom, in biological processes. 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 strand, whose nucleotide sequence is identical to the mRNA sequence and is typically presented in sequence lists, and the non-coding strand used as a template for transcribing the gene or cDNA, can be said to code for the protein or other product of that gene or cDNA.

[0022] 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 that can be hydrolyzed to monomeric “nucleotides.” Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including conventional cloning techniques, PCR, and synthetic means, as well as recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes.

[0023] As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to compounds consisting of amino acid residues that are interchangeable and 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 may constitute a protein sequence or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the terms refer to both short chains, also commonly called, for example, peptides, oligopeptides, and oligomers in the art, and longer chains, also commonly called, for example, proteins (of which there are many types) in the art. Polypeptides include, among other things, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0024] Proteins can be "modified," but this can also involve silent changes, such as the deletion, insertion, or substitution of amino acid residues that result in functional equivalents. Planned amino acid substitutions can be made based on the similarity of residues in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity, as long as biological activity is preserved. For example, aspartic acid and glutamic acid can be cited as negatively charged amino acids; lysine and arginine can be cited as positively charged amino acids; and leucine, isoleucine, and valine, glycine and alanine, asparagine and glutamine, serine and threonine, and phenylalanine and tyrosine can be cited as amino acids with uncharged head groups having similar hydrophilic values.

[0025] As used herein, a “variant” refers to an amino acid sequence altered by one or more amino acids. A variant may have a “conservative” change in which the substituted amino acid has similar structural or chemical properties, such as a leucine-to-isoleucine substitution. A variant may also have a “non-conservative” change, such as a glycine-to-tryptophan substitution. Similar minor changes may include amino acid deletions or insertions, or both. Guidelines for determining whether an amino acid residue can be substituted, inserted, or deleted without invalidating biological or immunological activity can be found using computer programs well known in the art.

[0026] "Identical" or "homographic" refers to sequence identity or similarity between two polypeptides or two nucleic acid molecules. When a certain position in both sequences being compared is occupied by the same base or amino acid monomer subunit—for example, when a certain position in each of two DNA molecules is occupied by adenine—then the molecules are homologous or identical at that position. The percentage of homology / identity between two sequences is a function obtained by dividing the number of matching positions shared by the two sequences by the number of positions being compared, and multiplying the result by 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences are 60% identical. Generally, comparisons are performed when the two sequences are aligned to achieve maximum homology / identity.

[0027] A "vector" is a construct consisting of a substance that contains isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. A great many vectors are known in the art, including, but not limited to, ionic or amphiphilic compounds, plasmids, and linear polynucleotides associated with 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 transport 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 retrovirus vectors.

[0028] An "expression vector" refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence operatively linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by host cells or within an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (e.g., naked or liposome-containing types), and viruses incorporating recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0029] As used herein, the term “promoter” is defined as a DNA sequence recognized by a cellular synthetic mechanism or introduced synthetic mechanism that is required to initiate the specific transcription of a polynucleotide sequence.

[0030] As used herein, the term “promoter / control sequence” means a nucleic acid sequence required for the expression of a gene product that is operably linked to the promoter / control sequence. In some cases, this sequence may be a core promoter sequence, while in other cases, it may also include enhancer sequences and other regulatory elements required for the expression of the gene product. The promoter / control sequence may, for example, express a gene product in a tissue-specific manner.

[0031] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that codes for or defines a gene product, causes the production of that gene product within the cell under most or all physiological conditions.

[0032] A “reducible” promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, substantially causes the production of the gene product within the cell only when the corresponding inducer is present in the cell.

[0033] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defined by a gene, preferentially induces the production of a gene product within the cell if the cell is a tissue type corresponding to the promoter.

[0034] The term “abnormal,” when used in the context of an organism, tissue, cell, or component thereof, refers to an organism, tissue, cell, or component thereof that differs from an organism, tissue, cell, or component thereof that exhibits “normal” (expected) individual characteristics in at least one observable or detectable characteristic (e.g., age, treatment, date and time, etc.). A characteristic that is normal or expected for one cell or tissue type may be abnormal for a different cell or tissue type.

[0035] The terms “patient,” “subject,” and “individual” are used interchangeably herein and refer to any animal or its cells, regardless of whether they are suitable for the methods described herein in vitro or in situ. The subject may be a mammal, such as a human or dog, as well as a mouse, rat, or non-human primate. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0036] A "disease" or "pathology" is a health condition in which the subject is unable to maintain homeostasis, and if the disease does not improve, the subject's health will continue to deteriorate. In contrast, a "disorder" in a subject is a health condition in which the subject is able to maintain homeostasis, but the subject's health is less favorable than when the disorder is absent. Leaving a disorder untreated does not necessarily lead to further deterioration of the subject's health.

[0037] 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, decreases. This includes the cessation of 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, is eliminated.

[0038] "Therapeutic" treatments are administered to subjects exhibiting signs of pathology with the aim of reducing or eliminating such signs. "Prophylactic" treatments are administered to subjects who do not exhibit signs of pathology or who have not yet been diagnosed with such pathology with the aim of preventing or delaying the onset of such signs.

[0039] 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.

[0040] The “effective dose” of a compound is the amount of the compound sufficient to provide a beneficial effect to the target to which the compound is administered. The idiomatic term “therapeutic effective dose,” as used herein, refers to an amount that is sufficient or effective to prevent or treat a disease or condition (including delaying or preventing its onset, halting its progression, inhibiting, reducing or reversing its progression), including alleviating the symptoms of such a disease. The “effective dose” of a delivery medium is the amount sufficient to efficiently bind to or deliver the compound. [Modes for carrying out the invention]

[0041] This invention is based on the inventors' understanding that the endogenous quantity of SGCG in the heart is generally similar to or less than the endogenous quantity of SGCG in skeletal muscle. Therefore, the expression level of SGCG produced from expression systems (which is considerably higher in the heart than in skeletal muscle) may be harmful and should be avoided.

[0042] The present invention provides a technical solution to this newly identified problem, particularly the problem of overexpression in the heart, excluding the expression levels of SGCG transgenes, and more generally, sarcoglycans, in skeletal muscle.

[0043] Therefore, generally speaking, the present invention relates to an expression system for systemic administration, comprising a sequence encoding gamma sarcoglycan (SGCG) under the control of a promoter that enables appropriate expression of SGCG in skeletal muscle and in the heart.

[0044] In other words, the present invention relates to an expression system comprising a sequence encoding the SGCG protein, wherein the expression system is: - To express the protein at therapeutically acceptable levels in the target tissue, preferably within skeletal muscle and the heart; however - To avoid any potential cardiotoxicity, express proteins at appropriate levels in the heart compared to their expression levels in skeletal muscle. This makes it possible.

[0045] Within the framework of the present invention, an expression system is generally defined as a polynucleotide that enables the in vivo production of SGCG. According to one embodiment, the system comprises a nucleic acid encoding the SGCG protein, and regulatory elements (at least a promoter) required for its expression. Thus, the expression system can correspond to an expression cassette, or the expression cassette may be supported by a vector or plasmid. The term "expression system," as used herein, encompasses all embodiments.

[0046] According to the present invention, target tissue is defined as a tissue or organ in which a protein plays a therapeutic role (particularly in cases where there is a defect in the native gene encoding this protein). According to a special embodiment of the present invention, target tissue includes striated skeletal muscle (hereinafter referred to as skeletal muscle), i.e., all muscles involved in motor function, as well as the diaphragm and smooth muscle. Non-limiting examples of target skeletal muscle include the tibialis anterior (TA), gastrocnemius, soleus, quadriceps, psoas, deltoid, diaphragm, gluteal muscles, extensor digitorum longus (EDL), biceps brachii, etc.

[0047] As described above, the heart is susceptible to damage in various diseases linked to SGCG deficiency and is therefore a potential target tissue. However, within the framework of this application, it has been shown that when SGCG is produced in excessively high quantities from existing expression systems, it can reach toxic levels in the heart. Therefore, in relation to gene transfer, expression systems should focus on appropriate SGCG expression within the heart and skeletal muscle, preferably endogenously, i.e., comparable to the profile observed for the native gene.

[0048] Therefore, even though SGCG plays a therapeutic role in the heart, if this protein becomes excessive in this tissue, it can be harmful or even fatal, and thus toxic, so its expression level should be strictly controlled.

[0049] Accordingly, in a particular embodiment, the present invention relates to a systemic expression system comprising a sequence encoding gamma sarcoglycan (SGCG) under the control of a promoter that enables appropriate expression of SGCG in skeletal muscle and in the heart.

[0050] According to the first characteristic, the expression system of the present invention includes a sequence encoding a gamma sarcoglycan (SGCG or γ-SG) corresponding to the transgene. In the context of the present invention, the term “transgene” means a sequence, preferably an open reading frame provided in trans using the expression system of the present invention.

[0051] In a particular embodiment, this sequence is a copy, identical, or equivalent of an endogenous sequence present in the genome of the body into which the expression system is introduced.

[0052] According to another embodiment, the endogenous sequence has one or more mutations that render the protein partially or completely nonfunctional, eliminate the protein (loss of expression or activity of the endogenous protein), or disrupt its arrangement in a desired intracellular compartment. In other words, the expression system of the present invention is intended to be administered to subjects having a defect in the copy of the protein-coding sequence and having a related pathology.

[0053] Therefore, the sequences possessed by the expression system of the present invention can be defined as encoding proteins that have therapeutic activity in the context of pathologies linked to SGCG deficiency. The concept of therapeutic activity is defined as follows, in relation to the term "therapeutably acceptable level".

[0054] The sequence encoding SGCG is a nucleic acid sequence or polynucleotide, also named ORF, which corresponds to an "open reading frame," and can be single-stranded or double-stranded DNA (deoxyribonucleic acid), RNA (ribonucleic acid), or cDNA (complementary deoxyribonucleic acid).

[0055] Advantageously, the sequence encodes a functional protein, i.e., a protein capable of ensuring its intrinsic or essential function, particularly in skeletal muscle. This suggests that proteins produced using the expression system of the present invention are properly expressed, positioned, and active.

[0056] In a preferred embodiment, the sequence codes for a native protein, which is preferably a human protein. The sequence may also be a derivative or fragment of this protein, provided that the derivative or fragment retains the desired activity. Preferably, the terms “derivative” or “fragment” refer to a protein sequence having at least 50%, preferably 60%, more preferably 70%, or still 80%, 85%, 90%, 95%, or 99% identity with the human SGCG sequence. This includes proteins of another origin (e.g., non-human mammals), or that have been truncated or mutated, but which are, for example, active proteins. Therefore, in the context of the present invention, the term “protein” is understood to mean a full-length protein, regardless of its origin, as well as its functional derivatives and fragments.

[0057] In the context of the present invention, the protein of interest is preferably human-derived SGCG, although mouse, rat, or canine versions (whose sequences are available in databases) may be used, for example.

[0058] According to a particular embodiment, the SGCG protein is a protein comprising or containing an amino acid sequence represented by SEQ ID NO: 1 (corresponding to a protein consisting of 291 aa) or SEQ ID NO: 2, which differs from SEQ ID NO: 1 at one position (one residue) and corresponds to its native variant.

[0059] According to certain embodiments, SGCG is a protein having the same function as natural human SGCG encoded by SEQ ID NO: 1 or SEQ ID NO: 2, in particular the ability to interact with α-, β-, and δ-sarcoglycans to form a portion of a sarcoglycan subcomplex (a member of the dystrophin-associated glycoprotein complex (DCG)), and / or the ability to at least partially alleviate one or more of the symptoms associated with defects in SGCG, in particular the LGMD2C phenotype disclosed above. SGCG may be its fragments and / or derivatives. According to one embodiment, the SGCG sequence has identity with the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 that exceeds or is equal to 50%, 60%, 70%, 80%, 90%, 95%, or still more than 99%. For example, Gao et al. (The Journal of Clinical Investigation, 2015; 125(11): pp. 4186-95) disclose so-called Mini-Gamma encoded by mRNA with exons 4-7 skipped.

[0060] Any sequences encoding these proteins, their functional therapeutic derivatives, or fragments may be introduced as part of the expression system of the present invention. For example, the corresponding nucleotide sequence (cDNA) is the sequence identified in International Publication No. 2019 / 152474.

[0061] According to certain embodiments, the sequence encoding SGCG includes or consists of the sequence of SEQ ID NO: 3, or corresponds to nucleotides 1186-2061 of the sequence of SEQ ID NO: 5, or nucleotides 1357-2232 of the sequence of SEQ ID NO: 6. Any sequence encoding the SGCG protein having 80%, 90%, 95%, or still greater than or equal to 99% identity with the sequence of SEQ ID NO: 3, and preferably the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, is also desirable.

[0062] This invention refers to SGCG proteins having disease-causing mutations in one or more target tissues, particularly in skeletal muscle and possibly in the heart.

[0063] Mutations in the SGCG gene can, in known ways, produce a full range of pathologies designated as limb-girdle muscular dystrophy type 2C (LGMD2C or LGMD R5). Clinical severity is usually correlated with the amount of residual protein, and correlations can be observed between genotype and phenotype: null mutations are usually associated with a severe Duchenne muscular dystrophy (DMD)-like phenotype, while missense mutations are associated with a milder LGMD-like phenotype. Therefore, strategies involving gene substitution or transposition, such as providing a trans sequence encoding natural therapeutic SGCG, may be helpful in treating the aforementioned pathology.

[0064] According to the present invention, also advantageously, the expression system or the promoter present in the expression system must enable the expression of SGCG protein at therapeutically acceptable levels in skeletal muscle and possibly in the heart. According to a preferred embodiment, and as reported in this application, the therapeutically acceptable level of SGCG corresponds to at least 30% (0.3 times) of the quantity of endogenous protein in the target tissue, particularly in skeletal muscle and possibly in the heart. In other words, and also advantageously, the ratio between the quantity of SGCG in skeletal muscle and the quantity of endogenous SGCG in the tissue is 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or greater than or equal to 1, or may still reach 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0065] In addition, according to another preferred embodiment, the expression system of the present invention, or the promoter present in the expression system, must enable the expression of SGCG in the heart at a toxicically acceptable level. According to a preferred embodiment, and as reported in this application, the toxically acceptable level of SGCG in the heart does not exceed 800% (8 times) the quantity of endogenous protein. In other words, and also advantageously, the ratio of the quantity of SGCG in the heart to the quantity of endogenous SGCG in the tissue is less than or equal to 20, 15, 10, or 9, and advantageously 8, 7, 6, 5, 4, 3, 2, or still less than or equal to 1.

[0066] In the context of this invention, the term "protein expression" can be understood as "protein production." Therefore, the expression system must enable the transcription and translation of the protein at the levels defined above. Correct folding and localization of the protein are also important.

[0067] The levels defined in the context of this invention, namely "therapeutably acceptable" and "toxically acceptable," relate to the quantity or number of proteins, as defined below, and their activity.

[0068] The amount of protein produced in a given tissue can be evaluated by immunoassay using an antibody targeting the protein, for example by Western blotting or ELISA, or by mass spectrometry. Alternatively, the corresponding messenger RNA can be quantified by PCR or RT-PCR, for example. This quantification can be performed in one or several tissue samples. Therefore, if the target tissue is skeletal muscle, it can be performed in one muscle type or several muscle types (e.g., quadriceps, diaphragm, tibialis anterior, triceps, etc.).

[0069] In the context of the present invention, the term “therapeutably acceptable level” refers to the fact that the proteins produced from the expression system of the present invention help to improve the pathological condition of a patient, particularly in terms of quality of life or lifespan. In relation to diseases affecting skeletal muscle, this relates to improving the muscle condition of a diseased subject or restoring a muscle phenotype similar to that of a healthy subject. As described above, muscle condition is mainly defined by muscle strength, size, tissue structure, and function, and can be evaluated by various methods known in the art, such as muscle biopsy, measurement of strength, muscle tone, volume, or mobility, clinical tests, medical imaging, biomarkers, etc.

[0070] Therefore, criteria that are helpful in evaluating the therapeutic benefit with respect to skeletal muscle and that can be evaluated at different time points after treatment include at least one of the following: - Extending life expectancy; - Improved muscle strength - Improvement of organizational structure; and / or - Improved diaphragm function.

[0071] In the context of this invention, the term “toxicly acceptable level” means that the proteins produced from the expression system of this invention do not cause significant changes in tissues, particularly histologically, physiologically, and / or functionally. In particular, protein expression must not be lethal. Toxicity in tissues is assessable histologically, physiologically, and functionally.

[0072] In specific cases of the heart, any protein toxicity can be assessed by morphological and cardiac function tests, by clinical laboratory tests, electrophysiology, imaging, biomarkers, monitoring of life expectancy, or by histological analysis including the detection of fibrosis and / or cellular infiltration and / or inflammation, for example, by staining with Sirius Red or hematoxylin (e.g., hematoxylin-eosin-saffran (HES) or hematoxylin-phloxine-saffron (HFS)).

[0073] Advantageously, the efficacy and / or toxicity levels of the expression system according to the present invention can be evaluated in vivo in animals, possibly in animals that have defects in the copy of a protein-coding gene and therefore suffer from a related pathology. Preferably, the expression system is administered systemically, for example, by intravenous (iv) injection.

[0074] According to the present invention, the expression system also preferably includes at least one sequence that enables appropriate expression of SGCG in skeletal muscle and in the heart.

[0075] According to another embodiment, the expression system according to the present invention comprises a sequence encoding gamma sarcoglycan (SGCG) under the control of a promoter, enabling appropriate expression of SGCG in skeletal muscle and cardiac region.

[0076] Preferably, the expression system of the present invention includes a promoter sequence, preferably located at 5' of the transgene and functionally linked thereto, which controls the transcription of the protein-coding sequence. Preferably, the expression system of the present invention ensures therapeutically acceptable levels of expression for the protein in skeletal muscle and possibly in the heart, as defined above, and toxically acceptable levels in the heart.

[0077] In the characteristic scheme according to the present invention, such promoters should further ensure the proper expression of SGCG in the heart and skeletal muscle, for example, in TA muscle.

[0078] Within the framework of the present invention, the term “adequate” is equivalent to “appropriate,” “adapted,” or “balanced,” and advantageously means that the expression profile is comparable to the intrinsically observed profile for native genes. As reported in the examples, the quantity of SGCG protein in the heart should advantageously not exceed the quantity of SGCG protein in skeletal muscle. As already described, the quantity can be assessed by any technique known in the art, for example, by evaluating the intensity of the corresponding band in Western blotting.

[0079] As observed in relation to endogenous genes, the quantity of SGCG produced in skeletal muscle from the expression system according to the present invention is advantageously greater than or equal to the amount produced in the heart.

[0080] This can be evaluated by calculating the ratio between the amount of SGCG in the heart and the amount of SGCG in skeletal muscle, such as TA muscle.

[0081] According to one embodiment, this ratio shall not exceed 5. Favorably, this ratio should be 4, 3, 2, or still less than or equal to 1. More favorably, this ratio is less than 1.

[0082] Conversely, the aforementioned ratio can be expressed as the ratio between the amount of SGCG in skeletal muscle, for example, in TA muscle, and the amount of SGCG in the heart.

[0083] According to one embodiment, this ratio shall not be less than 0.2. More favorably, this ratio shall be greater than or equal to 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or still greater than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. More favorably, this ratio shall be at least 0.9 or still equal to 1.

[0084] This may include inducible or constitutive natural or synthetic (artificial) promoters. Similarly, promoters can be of any origin (including human), either of the same or different origin as the transgene.

[0085] This includes any promoter exhibiting the above-defined expression profiles within skeletal muscle and heart, for example: - Muscle creatine kinase promoters, particularly truncated MCK promoters having double (dMCK) or triple (tMCK) tandem MCK enhancers, or derivatives of CK6 promoters (Wang et al., 2008, Gene Therapy, Vol. 15, pp. 1489-1499); - Muscle hybrid (MH) promoter (Piekarowicz et al., 2017, European Society of Gene & Cell Therapy conference, poster P096; HUMAN GENE THERAPY 28:A44 (2017), DOI: 10.1089 / hum.2017.29055.abstracts); - For example, promoters containing at least one UpStream Enhancer (USE) sequence, such as those identified in the troponin I promoter sequence (Corin et al., 1995, Proc. Natl. Acad. Sci., Vol. 92, pp. 6185-89), or possibly 3x3 or 4x4 copy types thereof with a 100bp deletion (ΔUSE; Blain et al., 2010, Human Gene Therapy, Vol. 21, pp. 127-34). The DeltaUSEx3 (DUSEx3) promoter and DeltaUSEx4 (DUSEx4) promoter are of particular interest; - Promoter of the gamma sarcoglycan gene; - Skeletal muscle alpha-actin (ACTA1) promoter or its derivative version It includes.

[0086] According to certain embodiments, such a promoter is neither, for example, the desmin promoter of the sequence of SEQ ID NO: 13, nor, for example, the CK8 promoter of the sequence of SEQ ID NO: 14. According to another embodiment, such a promoter is not, for example, the MHCK7 promoter disclosed in International Publication No. 2019 / 152474.

[0087] Advantageously, the promoter used within the framework of the present invention is a tMCK promoter. According to a preferred embodiment, the tMCK promoter has the sequence represented by SEQ ID NO: 4.

[0088] Promoter sequences corresponding to or fragments thereof derived from the aforementioned sequence, but having similar promoter activity, particularly with respect to tissue specificity and possibly efficacy, are also subject to the present invention. Preferably, the terms “derivative” or “fragment” refer to sequences having at least 60%, preferably 70%, more preferably 80%, or still 90%, 95%, or 99% identity with the aforementioned sequence and advantageously SEQ ID NO: 4. Promoter sequences that enable appropriate SGCG expression in the heart and skeletal muscle are particularly targeted, as defined above.

[0089] According to a particular embodiment, the present invention relates to an expression system comprising a sequence encoding SGCG, preferably the sequence of sequence 3, or a derivative or fragment thereof, under the control of a promoter having the sequence of sequence 4, as defined above.

[0090] Advantageously, the expression system of the present invention has the following corresponding sequence: - Nucleotides 1-2061 of Sequence ID No. 5; or - Nucleotides 172-2232 of Sequence ID No. 6 Includes.

[0091] According to certain embodiments, the target promoter is further selected for its ability to enable low or no expression in non-target tissues (i.e., tissues in which SGCG does not have a therapeutic effect or in which SGCG is not naturally expressed). As described above, and also advantageously, muscle (smooth muscle and skeletal muscle) and the heart are excluded from the non-target tissues. Conversely, the liver can be considered a non-target tissue.

[0092] According to certain embodiments, promoters that enable appropriate expression of SGCG in skeletal muscle and heart are inactive or have low activity in non-target tissues, such as the liver. Alternatively, the expression system according to the present invention further includes sequences that enable prevention or reduction of SGCG expression in non-target tissues, particularly the liver.

[0093] In the context of the present invention, the technical term "prevent expression" preferably refers to a case where expression is not observed (even if the sequence is not present), while the technical term "reduce the level of expression" refers to a case where the expression is reduced (or decreased) by providing the sequence.

[0094] Advantageously, the sequence has the ability to prevent or reduce the expression level of SGCG in non-target tissues where protein expression may be toxic or undesirable. This effect is particularly: - Regarding the transcription level of protein-coding sequences; - relating to transcripts resulting from the transcription of protein-coding sequences (e.g., via their degradation); - Regarding the translation of transcripts into proteins It can be implemented based on various mechanisms.

[0095] Such sequences are preferably, for example, from the following group: - MicroRNA; - Endogenous small interfering RNA or siRNA; - Small molecular weight fragments of transfer RNA (tRNA); - RNA in the intergenetic region; - Ribosomal RNA (rRNA); - Small nuclear RNA (snRNA); - Nucleolar small RNA (snoRNA); - piwi RNA that interacts with proteins (piRNA) It is a target for small RNA molecules selected from among them.

[0096] According to one embodiment, this sequence does not affect SGCG expression in target tissues, particularly in skeletal muscle and the heart.

[0097] Preferably, such sequences are selected for their efficacy in tissues where protein expression is inactive or even toxic. Since the efficacy of these sequences may vary depending on the tissue, it may be necessary to combine some of these sequences (selected for their efficacy within the said tissue).

[0098] According to a preferred embodiment, this sequence is a target sequence for microRNA (miRNA). As is well known, such appropriately selected sequences are useful for specifically repressing gene expression in selected tissues.

[0099] Accordingly, according to a particular embodiment, the expression system of the present invention includes a target sequence of a microRNA (miRNA) expressed or present in tissues where the expression of the protein is not therapeutically active and / or toxic, such as the liver. Preferably, the quantity of this miRNA present in the target tissue, particularly skeletal muscle and the heart, is less than the quantity present in tissues where SGCG is useless or even toxic, or this miRNA may not even be expressed in the target tissue. According to a particular embodiment, the target miRNA is not expressed in skeletal muscle and possibly in the heart. According to another particular embodiment, the target miRNA is specifically or even exclusively expressed in the liver.

[0100] As is well known to those skilled in the art, the presence or expression level of miRNA in a given tissue can be evaluated by PCR, preferably RT-PCR, or by Northern blotting.

[0101] Different miRNAs, as well as their target sequences and tissue specificities, are known to those skilled in the art and are described, for example, in International Publication No. 2007 / 000668. An example of a miRNA expressed in the liver is miR-122.

[0102] According to a particular embodiment, the expression system according to the present invention does not contain any target sequences of miRNAs expressed in the heart, such as miR208a.

[0103] According to the present invention, the expression system or expression cassette includes elements necessary for the expression of the transgene. In addition to the sequences defined above, such a system may include other sequences, for example: - Sequences for transcript stabilization, for example, intron 2 / exon 3 (modified) of the gene encoding human β-globin (HBB2), e.g., nucleotides 734-1179 of SEQ ID NO: 5, or nucleotides 905-1350 of SEQ ID NO: 6. As shown in the sequence, the HBB2 intron is advantageously followed by a consensus Kozak sequence (GCCACC) placed before the AUG start codon in the mRNA to improve translation initiation; - Preferably, a polyadenylation signal at the 3' end of the sequence encoding SGCG, such as polyA of the gene in question, SV40, or polyA of beta-hemoglobin (HBB2). As a preferred example, polyA of HBB2 corresponds to nucleotides 2072-2833 of SEQ ID NO: 5 or nucleotides 2243-3004 of SEQ ID NO: 6; - Enhancer array It may include.

[0104] The expression system according to the present invention can be introduced into cells, tissues, or the body, particularly into humans. The introduction can be carried out ex vivo or in vivo by methods known to those skilled in the art, for example, by transfection or transduction. In another embodiment, the present invention thus encompasses cells or tissues of preferred human origin, including the expression system of the present invention.

[0105] The expression system according to the present invention, in this case the isolated nucleic acid, can be administered into a target in the form of naked DNA. To facilitate the introduction of this nucleic acid into cells, it can be combined with different chemical means, such as colloidal dispersion systems (polymer complexes, nanocapsules, microspheres, beads) or lipid systems (oil-in-water emulsions, micelles, liposomes).

[0106] Alternatively, according to another preferred embodiment, the expression system of the present invention comprises a plasmid or a vector. 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 selected from the following list: herpesvirus-derived vectors, baculovirus vectors, lentivirus vectors, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors (AAVs).

[0107] According to a particular embodiment of the present invention, the viral vector containing the expression system is an adeno-associated virus (AAV) vector.

[0108] Adeno-associated virus (AAV) vectors have become 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 reliably and effectively transduce postmittal cells and tissues. By selecting the appropriate combination of AAV serotype, promoter, and delivery method, the expression of specific genes contained within an AAV vector can be specifically targeted to one or more types of cells.

[0109] In one embodiment, the coding sequence is contained within the AAV vector. More than 100 naturally occurring serotypes of AAV are known. Many native variants exist for AAV capsids, making it possible to identify and use AAVs with properties particularly suitable for dystrophy pathology. Conventional molecular biology techniques can be used to engineer AAV viruses, and these particles can be optimized to deliver nucleic acid sequences cell-specifically, minimize immunogenicity, regulate stability and particle lifetime, degrade efficiently, and deliver precisely to the nucleus.

[0110] As described above, AAV vectors are relatively non-toxic, enable efficient gene transfer, and can be easily optimized for specific purposes, making their use a common mode of exogenous DNA delivery. Among the serotypes of AAVs isolated from humans or non-human primates (NHPs) and well-characterized, human serotype 2 was the first AAV developed as a gene transfer vector. Other AAV serotypes currently in use include AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh74, AAV11, and AAV12. In addition, non-naturally engineered variants and chimeric AAVs may also be useful.

[0111] Desired AAV fragments for incorporation into vectors include cap proteins containing vp1, vp2, vp3, and a highly variable region, rep proteins containing rep 78, rep 68, rep 52, and rep 40, and sequences encoding these proteins. These fragments can be readily utilized in various vector systems and host cells.

[0112] Such fragments may be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements derived from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, non-limited to, AAVs having non-spontaneous capsid proteins. Such artificial capsids may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with heterogeneous sequences that may be obtained from different selected AAV serotypes, discontinuous portions of the same AAV serotype, or non-AAV viral sources or non-viral sources. Artificial AAV serotypes may, non-limited to, be chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Accordingly, exemplary or artificial AAVs include, among others, AAV2 / 8 (U.S. Patent No. 7,282,199), AAV2 / 5 (available from the National Institutes of Health), AAV2 / 9 (International Publication No. 2005 / 033321), AAV2 / 6 (U.S. Patent No. 6,156,303), AAVrh10 (International Publication No. 2003 / 042397), AAVrh74 (International Publication No. 2003 / 123503), AAV9-rh74 hybrid, or AAV9-rh74-P1 hybrid (International Publication No. 2019 / 193119), and AAV variants disclosed in PCT / European Patent No. 2020 / 061380. In one embodiment, a vector useful in the compositions and methods described herein contains at least a sequence encoding a selected AAV serotype capsid, such as AAV8 capsid or a fragment thereof. In another embodiment, a useful vector contains at least a sequence encoding a selected AAV serotype rep protein, such as an AAV8 rep protein or a fragment thereof. Optionally, such a vector may contain both AAV cap proteins and rep proteins. In a vector providing both AAV rep and cap, both the AAV rep sequence and the AAV cap sequence may originate from a single serotype, for example, all AAV8. Alternatively, a vector may be used in which the rep sequence originates from a single AAV serotype (different from the serotype providing the cap sequence).In one embodiment, the rep sequence and cap sequence are expressed from a different source (e.g., another vector or host cell and vector). In another embodiment, these rep sequences are in-frame fused to cap sequences of different AAV serotypes to form a chimeric AAV vector, such as AAV2 / 8 (U.S. Patent No. 7,282,199).

[0113] According to one embodiment, the composition comprises AAV of serotype 2, 5, 8, or 9, or AAVrh74. Advantageously, the claimed vector is an AAV8 or AAV9 vector, particularly an AAV2 / 8 or AAV2 / 9 vector.

[0114] 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.

[0115] Advantageously, the polynucleotide encoding SGCG is inserted between the ITR (inverted terminal repeat) sequences of the AAV vector. Typical ITR sequences correspond to nucleotides 1-145 (5' ITR sequence) and nucleotides 3005-3149 (3' ITR sequence) of SEQ ID NO: 6.

[0116] Recombinant viral particles can be obtained by any method known to those skilled in the art, for example, by simultaneous transfection of 293 HEK cells, by herpes simplex virus lines, and by baculovirus lines. Vector titers are usually expressed as viral genome count / 1 mL (vg / mL).

[0117] In one embodiment, the vector advantageously includes a control sequence, in particular a promoter sequence, as described above.

[0118] With respect to the polynucleotide encoding the sequence of SEQ ID NO: 1, the vector of the present invention may include the sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6.

[0119] According to a preferred embodiment, the expression system of the present invention comprises a vector having suitable tropism, in this case, higher tropism towards target tissues, advantageously skeletal muscle and heart, and possibly smooth muscle, than towards tissues where protein expression may be toxic.

[0120] Further embodiments of the present invention relate to the following: - Cells containing the expression system of the present invention, or vectors containing the expression system, as disclosed above. The cells can be any type of cell, namely prokaryotic or eukaryotic cells. The cells can be used for vector proliferation or further introduced into a host or subject (e.g., transplantation). The expression system or vector can be introduced into cells by any means known in the art, for example, by conversion, electroporation, or transfection. Cell-derived vesicles can also be used. - As disclosed above, advantageously, a non-human gene-transformed animal containing the expression system of the present invention, a vector containing the expression system, or a cell containing the expression system or the vector.

[0121] Another aspect of the present invention relates to a composition comprising an expression system, vector, or cells as disclosed above, for use as a pharmaceutical.

[0122] According to one embodiment, the composition comprises at least the gene therapy product (expression system, vector, or cell) and optionally other active molecules (other gene therapy products, chemical molecules, peptides, proteins, etc.) that are specialized for the treatment of the same disease or another disease.

[0123] According to certain embodiments, the expression system according to the present invention is used in combination with the use of anti-inflammatory drugs, such as corticoids.

[0124] The present invention then provides pharmaceutical compositions comprising the expression system, vector, or cells of the present invention. Such compositions comprise a therapeutically effective amount of the therapeutic agent (the expression system, vector, or cells 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 animal and human use. The term “carrier” refers to a diluent, adjuvant, excipient, or medium administered together with the therapeutic agent. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including liquids of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, or sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are also available as liquid carriers, particularly for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol.

[0125] The compositions may also contain, if desired, trace amounts of wetting agents or emulsifiers, or pH buffers. These compositions can take the form of solutions, suspensions, emulsions, sustained-release formulations, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions contain a therapeutically effective amount of a therapeutic agent, preferably in a purified form, along with a suitable amount of carrier (to provide a form for appropriate administration to the target).

[0126] In preferred embodiments, the composition is formulated according to a routine procedure as a pharmaceutical composition configured 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 include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site.

[0127] In one embodiment, the composition according to the present invention is suitable for administration to humans. The composition is preferably in liquid form, advantageously in the form of a saline composition, more advantageously in the form of a phosphate-buffered saline (PBS) composition, or in the form of a Ringer's lactate solution.

[0128] The amount of the therapeutic agent of the present invention (i.e., expression system, vector, or cells) that would be effective in treating the target disease can be determined by standard clinical techniques. In addition, in vivo and / or in vitro assays may be optionally employed to help predict the optimal dosage range. The exact dose adopted in formulation should also depend on the route of administration, body weight, and disease severity, and should be determined according to expert judgment and the individual patient's condition.

[0129] 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 possibly the heart. In the context of the present invention, when the gene therapy product is a viral vector containing a polynucleotide encoding human SGCG, the therapeutic dose is defined as the number of viral particles containing the SGCG sequence (vg in the case of a viral genome) administered per kilogram (kg) of the subject.

[0130] Available routes of administration are topical, enteral (systemic effect, delivered via the gastrointestinal (GI) tract), or parenteral (systemic effect, delivered via a route other than the GI tract). Preferred routes of administration for the compositions disclosed herein are parenteral, including intramuscular (i.e., intramuscular) and systemic (i.e., circulatory) administration. In this context, the term “injection” (or “perfusion” or “infusion”) includes intravascular, particularly intravenous (IV), intramuscular (IM), intraocular, intrathecal, or intracerebral administration. Injections are typically performed using a syringe or catheter.

[0131] In one embodiment, systemic delivery of the composition involves administering the composition intravenously or into an artery near a local treatment site, i.e., near a weakened muscle. In certain embodiments, the present invention includes local delivery of a composition that produces a systemic effect. This route of administration is commonly referred to as “regional (locally regional) infusion,” “separated limb perfusion,” or “hypertensive intravenous limb perfusion,” and has been used successfully as a gene delivery method in muscular dystrophy.

[0132] In one embodiment, the composition is administered to a separated limb (locally) by infusion or perfusion. In other words, the present invention involves local delivery of the composition into the leg and / or arm by an intravascular administration route, i.e., vein (transvenous) or artery, under pressure. This is typically achieved by temporarily suspending blood circulation using a tourniquet, while allowing local diffusion of the product being infused, as disclosed, for example, by Toromanoff et al. (2008).

[0133] In one embodiment, the composition is injected into the limbs of the subject. When the subject is human, the limbs may be arms or legs. According to one embodiment, the composition is administered into the lower body of the subject, for example, below the knee, or into the upper body of the subject, for example, below the elbow.

[0134] A preferred method of administration according to the present invention is systemic administration. Systemic injection is a method of injecting into the entire body so that it reaches all the muscles of the subject's body, including the heart and diaphragm, thus enabling the practical treatment of such systemic and still incurable diseases. In certain embodiments, systemic delivery includes delivery of the composition to the subject so that the composition is accessible throughout the subject's entire body.

[0135] According to a preferred embodiment, systemic administration occurs via intravascular injection of the composition, i.e., intravascular (intravenous or intra-arterial) administration. According to one embodiment, the composition is administered by intravenous injection through a peripheral vein.

[0136] Systemic administration is typically carried out under the following conditions: - A flow rate of 1 to 10 mL / min, preferably 1 to 5 mL / min, for example, 3 mL / min; - The total volume to be injected can vary between 1 and 20 mL of vector preparation per kg of subject, but is preferably 5 mL. The volume injected should not exceed 10% of the total blood volume and is preferably about 6%.

[0137] When delivered systemically, the composition is preferably 10 15 vg / kg or still 10 14 vg / kg or less or equal thereto, preferably 10 10 , 10 11 or still 10 12 vg / kg or more or equal thereto and is administered at a dose. Specifically, the dose can be 5.10 12 vg / kg to 10 14 vg / kg, for example 1, 2, 3, 4, 5, 6, 7, 8, or 9.10 13 vg / kg. For example, low doses of 1, 2, 3, 4, 5, 6, 7, 8, or 9.10 12 vg / kg can also be considered to avoid potential toxicity and / or immune reactions. As is known to those skilled in the art, the lowest possible dose that gives satisfactory results with respect to efficacy is preferred.

[0138] In certain embodiments, the treatment comprises a single administration of the composition.

[0139] Such compositions are particularly intended for use in gene therapy, especially for treating limb-girdle muscular dystrophy type 2C (LGMD2C or LGMD R5) or γ-sarcoglycanopathy in a subject.

[0140] Subjects who would benefit from the compositions of the present invention include all patients who have been diagnosed with such a disease or who are at risk of developing such a disease. Subjects to be treated may then be selected by any method known to those skilled in the art, including sequencing of the SGCG gene, and / or by any method known to those skilled in the art, based on the identification of mutations or deletions in the SGCG gene. Thus, the subjects include both subjects who are already exhibiting symptoms of such a disease and subjects who are at risk of developing such a disease. In one embodiment, the subjects include subjects who are already exhibiting symptoms of such a disease. In another embodiment, the subjects include walking patients and early non-walking patients.

[0141] More generally and according to further embodiments, the expression system according to the present invention is: - To increase muscle strength, muscle endurance, and / or muscle mass in the subject; - To reduce fibrosis in the subject; - To reduce contraction-induced injury in the subject; - To treat muscular dystrophy in the subject; - To reduce fibrous degeneration or necrosis 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 them.

[0142] According to one embodiment, the present invention relates to a method for treating such a condition, comprising the step of administering a gene therapy product (expression system, vector, or cells) as disclosed above to a target.

[0143] Advantageously, the expression system is administered systemically in the body, particularly in animals, advantageously in mammals, and more preferably in humans.

[0144] In putting the present invention into practice, unless otherwise indicated, prior art in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology that is well understood by those skilled in the art will be employed. Such techniques are described without exception in the literature, for example, "Molecular Cloning: A Laboratory Manual," 4th edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology" and "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 can be applied to the generation of polynucleotides and polypeptides of the present invention and may therefore be considered when realizing and practicing the present invention. Techniques particularly useful for specific embodiments are discussed in the following sections.

[0145] Patents, patent applications, and disclosures of publicly available materials cited herein are all incorporated herein by reference in their entirety.

[0146] Without further explanation, those skilled in the art will likely be able to prepare and utilize the compounds of the present invention and practice the claimed methods using the foregoing description and the following illustrative examples. [Examples]

[0147] Experimental Examples The present invention is further described by reference to the following experimental examples and accompanying drawings. These examples are presented for illustrative purposes only and are not intended to be limiting.

[0148] In particular, the present invention is illustrated in relation to an AAV8 vector containing a sequence encoding SGCG under the control of the tMCK promoter. [Brief explanation of the drawing]

[0149] [Figure 1] A / This figure shows the Western blot detection of γ-sarcoglycan (SGCG) expression in the tibialis anterior muscle (TA) and heart of mice or monkeys using a γ-sarcoglycan antibody (Ab203113 - Abcam). B / This figure shows a graphical representation of SGCG expression in each tissue based on the signals detected in (A). Statistical ANOVA test: (*) indicates a P value less than 0.05 (statistically significant). ns: No significant difference [Figure 2] This figure shows the luciferase activity of the GFP-Luc transgene, after normalization by total protein content in TA muscle and heart, obtained from C57Bl6 albino mice injected with AAV9-prom-GFP-Luc(Des, CK8, and tMCK). [Figure 3]This figure shows the vector genome copy number (VGCN) per diploid genome, measured by QPCR, in tissues (TA, heart, and liver) obtained from three groups of SGcg- / - mice that were intravenously injected with an AAV8 vector carrying SGCG under the control of a desmin promoter (AAV8-Des-SGCG), a CK8 promoter (AAV8-CK8-SGCG), or a tMCK promoter (AAV8-tMCK-SGCG). [Figure 4A] This figure shows the SGCG mRNA levels after normalization by RT-QPCR in tissues (TA, heart, and liver) obtained from three groups of Sgcg- / - mice that were intravenously injected with an AAV8 vector carrying SGCG under the control of a desmin promoter (AAV8-Des-SGCG), a CK8 promoter (AAV8-CK8-SGCG), or a tMCK promoter (AAV8-tMCK-SGCG), after normalization to the endogenous P0 level as measured by RT-QPCR. [Figure 4B] This figure shows the ratio of the relative abundances of SGCG / P0 mRNA and VGCN within each tissue. [Figure 4C] This figure shows the relative abundance of cardiac SGCG mRNA compared to TA muscle. The dotted line corresponds to a ratio of 1 (same expression level in the heart and TA muscle). Statistical ANOVA test: (*) indicates a P value less than 0.05 (statistically significant). [Figure 5] A / This figure shows the results of Western blot detection of human γ-sarcoglycan expression in the TA muscle and heart of 5 mice from each group (Sgcg- / - mice intravenously injected with an AAV8 vector carrying SGCG under the control of a desmin promoter (AAV8-Des-SGCG), a CK8 promoter (AAV8-CK8-SGCG), or a tMCK promoter (AAV8-tMCK-SGCG)) using human-specific γ-sarcoglycan antibody (Ab203112-Abcam). B / This figure shows a graphical representation of SGCG expression in each tissue (Ht: heart; TA: tibialis anterior muscle) based on the signals detected in (A). Statistical ANOVA test: (*) indicates a P value less than 0.05 (statistically significant). ns: no significant difference [Figure 6]This figure shows anti-SGCG immunohistochemistry performed in the TA and heart of SGCG- / - mice that were intravenously injected with an AAV8 vector carrying SGCG under the control of a desmin promoter (AAV8-Des-SGCG), a CK8 promoter (AAV8-CK8-SGCG), or a tMCK promoter (AAV8-tMCK-SGCG). Scale bar = 100 μm [Figure 7] A / This figure shows the correlation between the percentage of SGCG expression and the percentage of centronucleated fibers. Black dots correspond to muscle obtained from WT mice, and white dots correspond to muscle obtained from KO-Sgcg mice. Gray dots correspond to muscle obtained from KO-Sgcg injected with different AAV transduction efficiency levels (5e12vg / kg, 1e13vg / kg, and 5e13vg / kg of AAV8-Des-SGCG). B / This figure shows Western blot detection of γ-sarcoglycan expression in TA muscle and cardiac WT mice intravenously injected with PBS or AAV8-Des-SGCG (3e14vg / kg) (using γ-sarcoglycan antibody (Ab203113-Abcam)). C / This figure shows a graphical representation of SGCG expression in each tissue (Ht: heart; TA: tibialis anterior muscle) based on the signals detected in (B). [Figure 8] A / This figure shows the results of Western blot detection of human γ-sarcoglycan expression in the TA muscle and heart of rats (Sprague Dolly system, intravenously injected with AAV8 vectors carrying SGCG under the control of tMCK promoter (AAV8 tMCK), desmin promoter (AAV8 desmin), and MHCK7 promoter (AAV8 MHCK7)) from each group (using human-specific γ-sarcoglycan antibody (Ab203112-Abcam)). B / This figure shows a graphical representation of SGCG expression in each tissue (heart; TA: tibialis anterior muscle) based on the signals detected in (A). Statistical Student's Test: (*) indicates a P-value less than 0.05, and (***) indicates a P-value less than 0.001 (statistically significant). ns: No significant difference. [Figure 9]This figure shows the rMyh6 / rMyh7 molecular ratio measured by RT-QPCR transcripts obtained from three groups of Sprague-Dolly rats that were intravenously injected with PBS or an AAV8 vector carrying SGCG under the control of tMCK promoter (AAV8-tMCK-SGCG), desmin promoter (AAV8-desmin-SGCG), and MHCK7 promoter (AAV8-MHCK7-SGCG). Statistical ANOVA test: (**) represents a P-value less than 0.001.

[0150] Materials and methods: Animal models The animal studies were conducted in accordance with the latest European legislation on animal husbandry and experimental methods (2010 / 63 / EU) and were approved by the Ethics Committee of the Centre d'Exploration et de Recherche Fonctionnelle Experimentale in Evry, France (Protocol APAFIS DAP 2018-024-B#19736).

[0151] Sgcg - / - The mouse strain (Hack et al., J. Cell. Biol. 1998;142:pp. 1279-1287) was used in this study. These mice were reared in a pure C57BL / 6J background by mating them 10 times on a C57BL / 6J background. C57Bl / 6J and C57Bl6 albino mice were ordered from the Charles River Facility. Monkey-derived samples were provided by Inserm UMR 1089, Atlantic Gene Therapies, Institut de Recherche Therapeutique (IRT 1) Universite de Nantes (France), and Silabe (67207 Niederhausbergen, France).

[0152] One-month-old male Sprague Dolly rats were also used in this study.

[0153] Expression cassettes and AAV-mediated gene transfer Three different AAV cassettes were designed using the same ITR sequence, the transgene GFP-Luc, and poly(A) HBB2. The promoter was the only element that differed between the constructs. This study compared the human desmin (Des) promoter (SEQ ID NO: 13), the CK8 promoter (Goncalves et al., Mol Ther. 2011;19(7): pp. 1331-41; SEQ ID NO: 14), and the tMCK promoter (Wang et al., Gene Therapy 2008;15: pp. 1489-99; SEQ ID NO: 4). Serotype 9 was used to generate recombinant adeno-associated viruses (AAV9-prom-GFP-Luc).

[0154] Using the same promoter, we also designed three other AAV cassettes, including one with an SGCG transgene (see SEQ ID NO: 6 in relation to the tMCK promoter). In addition, we further tested the MHCK7 promoter (SEQ ID NO: 15), as disclosed in International Publication No. 2019 / 152474, in this context. Serotype 8 was used to generate recombinant SGCG adeno-associated viruses (AAV8-prom-SGCG).

[0155] TaqMan® probes specific to primer pairs and poly(A) HBB2 sequences: FWD: 5'-CCAGGCGAGGAGAAACCA-3' (Sequence ID 7), REV: 5'-CTTGACTCCACTCAGTTCTCTTGCT-3' (SEQ ID NO: 8), and Probe: 5'-CTCGCCGTAAAACATGGAAGGAACACTTC-3' (Sequence ID 9) The viral genome was quantified using the TaqMan® real-time PCR assay.

[0156] Different vectors were injected via a single systemic intravein dose into male 1-month-old C57Bl6 albino mice to express the GFP-Luc transgene, or into female 5-week-old Sgcg- / - mice to repair γ-sarcoglycan expression in the muscle. The dose of the injected vector was standardized according to the mouse's body weight using 5e13vg / kg of AAV9-prom-GFP-Luc, or 5e12vg / kg, 1e13vg / kg, 5e13vg / kg, or 3e14vg / kg of AAV8-prom-SGCG. The mice were killed 3 or 2 weeks after treatment, and tissue was collected. The tibialis anterior (TA) muscle was selected as a representative skeletal muscle.

[0157] Furthermore, three AAV8 vectors (MHCK7-hSGCG, desmin-hSGCG, and tMCK-hSCGC) were intravenously injected into the tail vein of one-month-old male Sprague-Dolly rats at a dose of 3e14vg / kg. Another group of rats injected with PBS was included as a control. One month after injection, the rats were killed. Cardiac and tibial anterior (TA) muscle tissue was collected.

[0158] Quantitative determination of luciferase by luciferase assay The samples were first homogenized with 500 μL of assay buffer containing 0.2% Triton X-100 and the protease inhibitor cocktail PIC (Roche) (Tris / phosphate, 25 mM; glycerol, 15%; DTT, 1 mM; EDTA, 1 mM; MgCl2, 8 mM). 10 μL of lysate was loaded into flat-bottom wells of a white, opaque 96-well plate. An Enspire spectrophotometer was used to quantify luminescence. Assay buffer containing D-luciferin (167 μM; Interchim) and ATP (40 nM) (Sigma-Aldrich) was delivered to each well of the plate using a pumping system. Relative luminescence (RLU) signals were measured after delivery of D-luciferin and ATP, respectively, with a 2-second delay between each sample. BCA protein quantification (Thermo Scientific) was performed to standardize the protein quantities in each sample. The results were expressed as RLU levels standardized by protein content.

[0159] Histological and immunohistochemical analysis 8-micrometer transverse frozen sections were cut from TA muscle or heart frozen with liquid nitrogen-cooled isopentane. The transverse frozen sections were then blocked for 1 hour in PBS containing 20% ​​fetal bovine serum (FCS) and incubated overnight at 4°C with a rabbit monoclonal primary antibody targeting human γ-sarcoglycan protein (Abcam - ab203112). After washing with PBS, the sections were incubated for 1 hour at room temperature with a goat anti-rabbit secondary antibody conjugated with AlexaFluor 594 dye (Thermo Fisher Scientific).

[0160] After washing with PBS, the sections were mounted using Fluoromount-G and DAPI (Southern Biotech) and visualized using a fluorescence microscope (Zeiss Axiophot2). Complete imaging of all sections was finally performed using an AXIOSCAN microscope (Zeiss).

[0161] To determine the number of centrally nucleated fibers, sections were labeled with rabbit anti-laminin antibody (DAKO-Z0097) using goat anti-rabbit antibody conjugated with AlexaFluor 488 dye (Thermo Fisher Scientific) as a secondary antibody, and then mounted using Fluoromount-G and DAPI (Southern Biotech). Image acquisition of all sections was finally performed using an AXIOSCAN microscope (Zeiss). The number of centrally nucleated fibers (CNF / mm²) was then determined. 2 Morphometric analysis of skeletal muscle, which defines the skeletal muscle, was performed as follows: To segment nuclei and fibers, FIJI software is used to process post-scan RGB images including 8-bit channel laminin immunofluorescence and DAPI staining captured at 10x magnification. Nuclear segmentation is performed based on DAPI intensity using global binarization (IsoData) and particle analysis. Fibers are segmented based on laminin staining using the MorphoLib plugin-type "Morphological Segmentation" tool (boundary image option) and the ImageJ particle analysis tool (object roundness > 0.2, object size filter depending on muscle type and species).

[0162] The target regions (ROIs) of nuclei and fibers are converted into spatial objects using R software (RimageJROI, spatstat, and the sp library), and intrafibrous nuclei are identified from the intersections of the nuclei and fiber objects. For intrafibrous nuclei, the distance to the fiber centroid and the nearest neighbor of the membrane is calculated.

[0163] Artifacts are removed by filtering based on size, shape, and fluorescence intensity (nerves are identified as fibers, spited fibers, or adhesive fibers).

[0164] Based on the distance between the nucleus and the nearest neighbor (compared to the ferret fiber diameter, or absolute distance, user choice), centronucleating fibers are identified.

[0165] Measurement of viral genome copy number (VGCN) in tissues Genomic DNA was extracted from frozen tissue using the NucleoMag pathogen kit (Macherey Nagel) in conjunction with a KingFisher robot (Thermo Fisher Scientific) as directed by the manufacturer. The vector genome copy number was determined from 20 ng of genomic DNA using qPCR. DNA samples of plasmids carrying one copy per amplicon were sequentially diluted, and these dilutions were used as standard curves. Real-time PCR was performed using a LightCycler 480 (Roche) with 0.2 μM primers and 0.1 μM probes, following the protocol of Absolute QPCR Rox Mix (Thermo Fisher Scientific). The sequence located at poly(A) HBB2 on the cassette was used to quantify the viral genome. The primer pairs and Taqman® probes specific to the poly(A) HBB2 sequence were identical to those disclosed above (SEQ ID NOs. 7-9).

[0166] Ubiquitous acidic ribosomal phosphoprotein (P0) was used for the quantification of genomic DNA. The primer pairs and Taqman® probes used for P0 amplification were: FWD: 5'-CTCCAAGCAGATGCAGCAGA-3' (Sequence ID 10), REV: 5'-ATAGCCTTGCGCATCATGGT-3' (SEQ ID NO: 11), and Probe: 5'-CCGTGGTGCTGATGGGCAAGAA-3' (Sequence ID 12) That was the case.

[0167] The number of diploid genomes is half the number of copies of the P0 gene. The level of tissue transduction is determined by the number of VGCN per diploid genome.

[0168] Quantification of mRNA Total RNA extraction from frozen tissue was performed according to the NucleoSpin® RNA Set protocol (Macherey Nagel) for NucleoZOL. The extracted RNA was eluted in 60 μl of RNase-free water, and residual DNA was removed by treatment with the TURBO® DNase kit (Ambion). Total RNA was quantified using a Nanodrop spectrophotometer (ND8000 Labtech).

[0169] To quantify the expression of the transgene, 1 μg of RNA was reverse transcribed using the RevertAid H-minus reverse transcriptase kit (Thermo Fisher Scientific) and a mixture of random oligonucleotides and oligo-dT. Real-time PCR was performed using a LightCycler 480 (Roche) with a commercially available primer set and a probe for quantifying human γ-sarcoglycans (Hs00165089_m1; Thermo Fisher Scientific). In mouse samples, ubiquitous acidic ribosomal phosphoprotein (P0) was used to standardize data between samples and to quantify VGCN as previously described.

[0170] Each experiment was performed redundantly. Quantitative cycle (Cq) values ​​were calculated using LightCycler® 480 SW 1.5.1 with the 2nd Derivative Max method. RT-qPCR results, expressed as raw Cq, were standardized relative to P0. Relative expression levels were calculated using 2 -ΔCt The calculation was performed using the Livak method.

[0171] Measurement of the transfer ratio Myh6 / Myh7 Myh6 and Myh7 transcripts were quantified by RT-QPCR using a commercially available primer set and probes for quantifying rMyh6 (Rn00691721_g1; Thermo Fisher Scientific) and rMyh7 (Rn01488777_g1; Thermo Fisher Scientific). The results are expressed as the molecular weight ratio of Myh6 transcript to Myh7 transcript.

[0172] Western blot analysis Frozen sections of tissue (liver, heart, or TA muscle) approximately 1 mm in size were solubilized in radioactive immunoprecipitation (RIPA) buffer containing a protease inhibitor cocktail. Protein extracts were quantified by BCA (bicinchoninic acid) protein assay (Pierce). 30 μg of total protein was processed for Western blotting analysis using anti-γ-sarcoglycan antibodies (human-specific: Ab203112, and for common recognition of mouse, human, and monkey morphologies: Abcam; Ab203113).

[0173] The fluorescence signal of the secondary antibody was read on the Odyssey imaging system, and the band intensity was measured using Odyssey application software (LI-COR Biosciences, version 2.1).

[0174] statistical analysis Statistical analysis was performed using GraphPad Prism version 6.04 (GraphPad Software, San Diego, CA). Statistical analysis was performed using statistical ANOVA or Student's test as shown. Data are expressed as mean ± SD. P-values ​​less than 0.05 were considered statistically significant (*).

[0175] result: I / Endogenous SGCG expression profiles in mice and monkeys: To define the relative proportion of endogenous SGCG between cardiac and skeletal muscle in different species, the relative abundance of SGCG protein was investigated in different tissues of wild-type mice or monkeys (TA muscle, representing skeletal muscle, and the heart).

[0176] Figure 1 clearly shows that in mice, SGCG is produced at similar levels in the TA muscle and heart. In monkeys, a mammalian model corresponding to humans, the amount of SGCG in the heart is observed to be dramatically lower than the amount of SGCG in the TA muscle.

[0177] II / Evaluation of different promoters in C57BL6 mice: We conducted tests to identify expression constructs that exhibit expression profiles as similar as possible to those observed for endogenous genes in the cardiac and TA muscle, i.e., expression levels in the TA muscle that are similar or even higher than those in the cardiac muscle.

[0178] For this purpose, we tested different promoters known to possess muscle activity using the reporter gene GFP-Luc.

[0179] We conducted experiments comparing the desmin promoter, the CK8 promoter, and the tMCK promoter. The desmin promoter was selected because it corresponds to the promoter tested by Israeli et al. (Mol Ther Methods Clin Dev. 2019; 13:494-502) (whose effectiveness in muscle activity repair was reported).

[0180] From Figure 2: - The AAV9-CK8-GFP-Luc vector is more effective for transduction in both cardiac and TA muscle; - AAV9-tMCK-GFP-Luc appears to have weaker promoter strength, but is more equilibrium between cardiac and skeletal muscle expression. It becomes clear.

[0181] The tMCK promoter is a promising candidate, and it is clear that it will ensure appropriate expression in the heart and TA muscle, as observed for endogenous genes in mice and monkeys. Conversely, the desmin and CK8 promoters induce much higher expression in the heart than observed in TA muscle, which may be associated with cardiotoxicity.

[0182] III / Validation of the tMCK promoter in Sgcg- / - mice: To verify these observations, further studies were conducted comparing three different SGCG AAV8 vectors administered intravenously to SGCG-deficient mice. Promising tMCK promoters were compared to the two other promoters tested above, namely the desmin promoter and the CK8 promoter.

[0183] First, the effectiveness of transduction was compared among the three constructs. As shown in Figure 3, the level of transduction was the same for identical tissues, so no bias was observed in the infectivity of the three vectors. The liver was clearly the organ with the highest transduction (approximately 1 VGCN / diploid genome). Transduction in the heart and TA muscle was similar, reaching approximately 0.01 VGCN / diploid genome. With such low infection levels, there is no risk of reaching a saturation effect that could interfere with subsequent analyses.

[0184] Next, the transcriptional activity of the three promoters was compared. SGCG mRNA levels in the TA muscle did not differ significantly among the three mouse groups. Conversely, tMCK promoter activity was considerably lower in the heart compared to the other two mouse groups, showing a statistically significant difference, at least compared to the CK8 promoter. The number of transduced cells in the liver was very high, and SGCG mRNA levels were also high (Figure 4A).

[0185] By standardizing mRNA SGCG levels using VGCN, it was confirmed that tMCK promoter activity was significantly different from both Des promoter and CK8 promoter activity (Figure 4B).

[0186] Ultimately, the cardiac SGCG mRNA ratio to TA muscle obtained using the tMCK promoter (approximately 0.6) showed higher equivalence to the endogenous state (Figure 4C), meaning that expression was higher in TA muscle than in the heart.

[0187] These observations were confirmed by investigating SGCG protein expression in these different tissues: As is clear from Figure 5, the amount of transgene protein was significantly higher in the heart than in the TA muscle in mice injected with the AAV8-CK8-SGCG vector and the AAV8-Des-SGCG vector, whereas this was not the case for mice injected with the AAV8-tMCK-SGCG vector: the amount of SGCCG did not differ significantly between the heart and the TA muscle. In addition, it should be noted that the level of SGCG protein in the TA muscle was similar regardless of the promoter used. Based on these results, it is confirmed that tMCK has an appropriate expression profile, i.e.: - In TA muscle, it exhibits high activity similar to that of desmin and the CK8 promoter; - In the heart, it exhibits lower activity than desmin and the CK8 promoter. Direct observation of TA and cardiac tissue (Figure 6) confirmed that SGCG expression within the TA muscle did not differ significantly among the three mouse groups. Conversely, hearts obtained from mice injected with the AAV8-tMCK-SGCG vector exhibited fewer positive fibers compared to the other two mouse groups.

[0188] IV / Determination of the critical volume of SCGC in muscle and heart: To determine the minimum therapeutically effective dose of SGCG in muscle and the maximum non-toxic dose of SGCG in the heart, further experiments were conducted using the AAV8-Des-SGCG vector, which was shown above to produce appropriate levels of expression in TA muscle but excessive levels of expression in the heart, and is therefore likely toxic.

[0189] From Figure 7A, we can conclude that in order to reach an acceptable level of centronucleation (comparable to or slightly exceeding the levels observed in the muscle of WT mice, i.e., up to 20%), the expression system should be able to express at least 30% of the normal SGCG level in skeletal muscle.

[0190] On the other hand, Figures 7B and 7C clearly show that the aforementioned system, which is likely toxic in the heart, results in intracardiac SGCG levels eight times higher than those observed in the hearts of wild-type mice.

[0191] Evaluation of different promoters in rats: V-1 / Protein SGCG expression profile: The experiments described above in mice were further carried out in rats by adding the MHCK7 promoter (AAV8-MHCK7-SGCG vector) as a new test promoter.

[0192] Figure 8 clearly shows that in rats, the amount of transgene protein was significantly higher in the heart than in the TA muscle in the groups of rats injected with the AAV8 desmin-SGCG vector and the AAV8 MHCK7-SGCG vector.

[0193] Conversely, when the AAV8 tMCK-SGCG vector was used, the transgene protein was expressed to a similar degree in both the TA muscle and the heart.

[0194] Note that the expression profile ratios obtained using the AAV8 desmin-SGCG vector and the AAV8 tMCK-SGCG vector are similar in mice and rats.

[0195] V-2 / Effects on the heart: The transcription ratio Myh6 / Myh7 is a good indicator for detecting changes in cardiac tissue associated with stress-induced pathological conditions within the heart (Scheuermann et al., EMBO J. 2013; 32(13): pp. 1805-1816).

[0196] Figure 9 shows that this ratio did not change significantly in the group of rats injected with the AAV8 tMCK-SGCG vector (8.3) compared to the PBS control group (10.2). From the same figure, it is even clearer that the ratio was significantly reduced in the hearts of rats injected with the AAV8 desmin-SGCG vector (1.8), even if not statistically different. Finally, the ratio was significantly reduced in the hearts of rats injected with the AAV8 MHCK7-SGCG vector (0.8) compared to rats in the PBS control group and the AAV8-tMCK group.

[0197] Overall, the tMCK promoter promotes equal expression between cardiac and skeletal muscle, while the other two promoters (desmin and MHCK7) result in higher SGCG expression in the cardiac than in skeletal muscle, as observed in both rats and mice. In addition, only the tMCK promoter preserves the proper Myh6 / Myh7 ratio, while this ratio is altered in the other two promoters, suggesting cellular stress in the cardiac.

[0198] conclusion As is well known in the art, the two most important organs that need to be targeted for treatment of LGMD2C patients are skeletal muscle and the heart.

[0199] Based on measurements of endogenous SGCG protein in wild-type mice and monkeys, it was concluded that cardiac expression should be at the same level as, or even lower than, skeletal muscle expression.

[0200] In relation to these different aspects, the AAV8-tMCK-SGCG vector was confirmed to be a very promising candidate. Expression levels were significantly lower in the heart compared to the other three promoters. Furthermore, in TA muscle, the expression level of the transgene was close to that obtained using the AAV8-Des-SGCG vector (a vector widely described as effective for skeletal muscle transduction and muscle activity repair (see, for example, Israeli et al., Mol Ther Methods Clin Dev. 2019; 13: pp. 494-502)).

Claims

1. An expression system for systemic administration comprising a sequence encoding gamma sarcoglycan (SGCG) under the control of a promoter that enables the expression of SGCG in skeletal muscle and in the heart, wherein the ratio of SGCG expression in skeletal muscle to SGCG expression in the heart is greater than or equal to 0.

9.

2. The expression system according to claim 1, which enables the expression of SGCG in skeletal muscle in a quantity greater than or equal to 0.3 times the endogenous quantity.

3. The expression system according to claim 1 or 2, which enables the expression of SGCG in the heart in a quantity less than or equal to eight times the endogenous quantity.

4. The expression system according to any one of claims 1 to 3, wherein the promoter is preferably a tMCK promoter of the sequence of SEQ ID NO:

4.

5. The expression system according to any one of claims 1 to 4, wherein the SGCG protein has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO:

1.

6. The expression system according to claim 5, wherein the sequence encoding the SGCG protein has the sequence of SEQ ID NO:

3.

7. An expression system according to any one of claims 1 to 6, comprising SEQ ID NO: 5 or SEQ ID NO:

6.

8. An expression system according to any one of claims 1 to 7, comprising a viral vector, preferably an adeno-associated viral vector (AAV) of serotype 8 or 9.

9. The expression system according to claim 8, comprising an AAV2 / 8 or AAV2 / 9 vector.

10. A pharmaceutical composition comprising the expression system described in any one of claims 1 to 9.

11. An expression system according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10, for use in gene therapy.

12. An expression system according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10 for use in the treatment of pathologies caused by SGCG deficiency.

13. The expression system or pharmaceutical composition for use according to claim 12, wherein the pathology caused by SGCG deficiency is limb-girdle muscular dystrophy type C (LGMD2C or LGMD R5).

14. An expression system or pharmaceutical composition for use according to claim 12 or 13, which is administered systemically, preferably by intravenous injection.

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