Secreted splice variants of klotho for treating muscle disorders

Abstract

The present invention provides a polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence at least 85% identical to SEQ ID NO: 1, for use in the prevention and / or treatment of a muscle disease or disorder. The present invention also provides a nucleic acid sequence encoding the polypeptide, a gene construct comprising the nucleic acid sequence, or an expression vector comprising the gene construct, for said use. The polypeptide, nucleic acid sequence, gene construct, or expression vector of the present invention may be administered in the form of a pharmaceutical composition, together with at least one pharmaceutically acceptable excipient, diluent, or carrier. The present invention also provides non-therapeutic methods for improving muscle function and / or increasing muscle mass in a subject.

Description

[Technical Field]

[0001] This application claims the benefit of European Patent Application No. 22383173.6, filed December 2, 2022.

[0002] [Technical field] The present invention relates to the field of medicine, and in particular to medical methods for preventing and / or treating muscle disorders. The compounds of the present invention are particularly useful in treating age-related muscle disorders.

[0003] [Background technology] Aging represents a stage of life associated with an increased incidence of morbidity and increased risk of mortality due to the accumulation of molecular alterations and tissue dysfunction, promoting the decline of tissue protective systems.

[0004] Muscle tissue is one of the systems highly affected during aging and is characterized by phenotypes such as decreased muscle strength and resistance, sarcopenia, muscle fibrosis, and decreased muscle regenerative capacity. Skeletal muscle condition is an important indicator of animal health and can directly affect survival by increasing the likelihood of falls and physical injury in aging animals.

[0005] The tissue-level features of sarcopenia have been extensively described, including reduced myofibril size, increased intramuscular fat accumulation, preferential atrophy of type II (fast-twitch) muscle fibers, and dysfunction. However, our understanding of the cellular mechanisms underlying sarcopenia remains lacking, a weakness that has hindered the development of targeted and specific interventions.

[0006] Currently, approaches to the treatment and prevention of sarcopenia largely focus on prescribing exercise and dietary modifications, strategies that have shown moderate success. Furthermore, no pharmacological interventions for sarcopenia are currently on the market.

[0007] Previous reports have investigated the possible relationship between full-length Klotho and muscle function, but very little is known about the secreted isoform of Klotho, s-KL, and its potential role in muscle function and diseases.

[0008] Thus, despite the efforts made to date, there remains a need for effective and safe treatments for muscle disorders, particularly age-related muscle disorders.

[0009] [Summary of the Invention] The present inventors have developed a novel therapy for the prevention and / or treatment of muscle disorders based on the administration of the secreted splicing isoform of Klotho, s-KL.

[0010] The Klotho gene expresses two major transcripts: full-length Klotho mRNA and alternatively spliced ​​Klotho mRNA. Full-length Klotho mRNA transcribes a 135-kDa, single-pass transmembrane protein called m-KL. The extracellular domain of m-KL can be released from the membrane by protease-mediated shedding to generate soluble, circulating processed Klotho (p-KL, 130 kDa), sometimes simply called soluble Klotho, which has two active domains. Alternatively spliced ​​Klotho mRNA displays a premature stop codon and generates a secreted protein, s-KL (70 kDa), which contains only one of the active domains and an extra 15 amino acids at the C-terminus of s-KL. Although the same abbreviation (s-KL) is sometimes used in the prior art to refer to soluble Klotho, a processed version of the full-length transmembrane Klotho, and secreted Klotho, a splicing isoform, these two isoforms exhibit completely different structures, sizes, and biological activities. Indeed, full-length Klotho has been described to be involved in FGF23 receptor binding, PTH synthesis, regulation of parathyroid growth, and alteration of vitamin D metabolism and calcium ion blood levels, whereas the secreted splicing isoform (s-KL) of Klotho has not.

[0011] The present invention is based exclusively on the use of secreted Klotho, a splicing isoform whose biological function remains unknown, and the abbreviation s-KL is used herein to refer exclusively to secreted Klotho.

[0012] As shown in the following Examples, the present inventors surprisingly found that administration of s-KL improved not only the physical performance of aged animals (Fig. 2a-d) but also the condition of aged muscle tissue (Fig. 3a-b). In particular, administration of s-KL increased the number of muscle fibers and reduced the percentage of fibrous tissue in muscle.

[0013] Furthermore, we found that muscles of animals treated with s-KL displayed a higher number of muscle stem cells and increased proliferative capacity, which resulted in a higher regenerative capacity in muscles, especially when treatment was performed during adult development (Figure 4a-h).

[0014] All of these effects of s-KL were highly unexpected in light of the prior art, since it has previously been shown that intracerebroventricular administration of s-KL in mice did not affect locomotor function in mice (Masso A. et al., "Secreted αKlotho isoform protects against age-dependent memory deficits," Mol Psychiatry, 2018, Vol. 23(9), pp. 1937-1947).

[0015] This unusual combination of effects that s-KL exerts on muscle tissue, discovered by the present inventors, clearly positions s-KL as a useful therapeutic agent for improving muscle function and treating muscle disorders, particularly age-related muscle degeneration.

[0016] Thus, in a first aspect, the present invention provides a polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, for use in the prevention and / or treatment of a muscle disease or disorder, in particular by improving muscle function.

[0017] In a second aspect, the present invention provides a nucleic acid sequence encoding a polypeptide or a variant thereof as defined in the first aspect for use in the prevention and / or treatment of a muscle disease or disorder, in particular by improving muscle function.

[0018] In a third aspect, the present invention provides a genetic construct comprising a nucleic acid sequence as defined in the second aspect operably linked to an expression promoter for use in the prevention and / or treatment of a muscle disease or disorder, particularly by improving muscle function.

[0019] In a fourth aspect, the present invention provides an expression vector comprising a genetic construct as defined in the third aspect for use in the prevention and / or treatment of a muscle disease or disorder, in particular by improving muscle function.

[0020] In a fifth aspect, the present invention provides a host cell transformed or transfected with a nucleic acid sequence defined in the second aspect, a genetic construct defined in the third aspect, or an expression vector defined in the fourth aspect, for use in the prevention and / or treatment of a muscle disease or disorder, in particular by improving muscle function.

[0021] In a sixth aspect, the present invention provides a polypeptide consisting of the sequence of SEQ ID NO: 1 or a variant thereof consisting of a sequence which is at least 85% identical to SEQ ID NO: 1, or a nucleic acid sequence encoding the polypeptide or variant thereof, for use in reducing muscle fibrosis.

[0022] In a seventh aspect, the present invention provides a non-therapeutic method for improving muscle function and / or increasing muscle mass in a subject comprising administering to the subject a polypeptide consisting of the sequence of SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, or a nucleic acid sequence encoding the polypeptide or variant thereof.

[0023] In an eighth aspect, the present invention provides a non-therapeutic method for increasing muscle regeneration capacity in a subject, comprising administering to the subject a polypeptide consisting of the sequence of SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, or a nucleic acid sequence encoding the polypeptide or variant thereof.

[0024] In a ninth aspect, the present invention provides a non-therapeutic method for improving the physical condition or performance of a subject comprising administering to the subject a polypeptide consisting of the sequence of SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, or a nucleic acid sequence encoding the polypeptide or variant thereof. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1 shows that s-KL treatment efficiently increased s-KL protein concentrations. a) Schematic of experimental design. b) Analysis of s-KL gene expression in the liver of males (left panel) and females (right panel). c) Quantification of total s-KL protein concentrations in the serum of males (left panel) and females (right panel). Analysis was performed using samples from a subset of animals euthanized at 24 months of age. In (b), data are expressed as fold changes compared to null-treated animals. Mean ± standard error (SEM), n = 4, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001. [Figure 2] Treatment with s-KL improved the physical performance of aged animals in physical tests. a) Results of the accelerating rotarod test, expressed as the maximum speed at which the animals could run. b-c) Results of the horizontal bar test, expressed as the time it took for the animals to fall off the bar in males (b) and females (c). d) Results of the grip strength test. Results represent the average force the animals were able to exert in each of the three trials performed. Data are presented as mean ± standard error of the mean (SEM), n = 8-11 animals, *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 3] Figure 1 shows that s-KL treatment reduced age-related muscle fibrosis in mice. a) Quantification of fiber size in soleus muscle. b) Quantification of the percentage of fibrous area in soleus muscle. Data are expressed as mean ± standard error of mean (SEM), n = 4, *p < 0.05, **p < 0.01. [Figure 4-1]Figure 1: Aged muscles of treated animals displayed greater regenerative capacity. a-b) Quantification of graft muscle fibers as mean fiber size (a) and frequency of various fiber sizes (b). c-e) Quantification of Pax7, Ki67, and double Pax7 + Ki67 positive cells found in the grafts. f-h) Quantification of MyoD, Ki67, and double MyoD + Ki67 positive cells found in the grafts. Data are presented as mean ± standard error of the mean (SEM), n = 8-11, *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 4-2] Figure 1: Aged muscles of treated animals displayed greater regenerative capacity. a-b) Quantification of graft muscle fibers as mean fiber size (a) and frequency of various fiber sizes (b). c-e) Quantification of Pax7, Ki67, and double Pax7 + Ki67 positive cells found in the grafts. f-h) Quantification of MyoD, Ki67, and double MyoD + Ki67 positive cells found in the grafts. Data are presented as mean ± standard error of the mean (SEM), n = 8-11, *p < 0.05, **p < 0.01, ***p < 0.001.

[0026] [Detailed Description of the Invention] In this application, all terms used herein should be understood in their ordinary meaning as known in the art unless otherwise stated. Other, more specific definitions for certain terms used in this application are set forth below and are intended to be applied uniformly throughout the specification and claims, unless a definition expressly set forth otherwise provides a broader definition.

[0027] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more." Unless otherwise indicated, definite articles used herein, such as "the," also include the plural of the noun in question.

[0028] As mentioned above, in a first aspect, the present invention provides a polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, for use in the prevention and / or treatment of a muscle disease or disorder, in particular by improving muscle function.

[0029] This aspect can also be formulated as the use of a polypeptide as defined above for the manufacture of a medicament for the prevention and / or treatment of a muscle disease or disorder. The present invention also relates to a method for the treatment and / or prevention of a muscle disease or disorder, comprising administering a therapeutically effective amount of a polypeptide as defined above, together with a pharmaceutically acceptable excipient or carrier, in a subject, including a human, in need thereof.

[0030] In more particular embodiments of the first aspect of the invention, the polypeptide consists of SEQ ID NO: 1 or a variant thereof that consists of a sequence that is at least 85%, 86%, 87%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to SEQ ID NO: 1. In even more particular embodiments, the polypeptide consists of SEQ ID NO: 1 or a variant thereof that consists of a sequence that is at least 88% or 98% identical to SEQ ID NO: 1.

[0031] In more particular embodiments of the first aspect of the invention, the polypeptide consists of the sequence of SEQ ID NO:1 or a variant thereof consisting of a sequence that is at least 85%, 86%, 87%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to SEQ ID NO:1, wherein the variant substantially maintains or improves the muscle therapeutic effect of SEQ ID NO:1.

[0032] In another embodiment of the first aspect of the invention, the polypeptide consists of the sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0033] Protein and polypeptide variants are well understood to those of skill in the art and can involve amino acid sequence modifications, which typically fall into one or more of three classes: substitutional, insertional, or deletional variants.

[0034] In the present invention, the term "identity" refers to the percentage of residues that are identical in two sequences when the sequences are optimally aligned. If a position in a first sequence is occupied by the same amino acid residue as the corresponding position in a second sequence during optimal alignment, the sequences are said to be identical at that position. The percentage of identity determines the number of identical residues over a defined length in a given alignment. Thus, the level of identity between two sequences ("percent sequence identity") is measured by the ratio of the number of identical positions shared by the sequences to the number of compared positions (i.e., percent sequence identity = (number of identical positions / total number of compared positions) x 100). Gaps, i.e., positions in the alignment where a residue is present in one sequence but not in the other, are considered as positions with non-identical residues and are counted as compared positions.

[0035] By way of example, a polypeptide having an amino acid sequence that is at least, for example, 95% identical to the reference amino acid sequence of SEQ ID NO: 1 intends that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may contain up to 5 amino acid changes for each 100 amino acids of the reference amino acid sequence of SEQ ID NO: 1. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These changes in the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, either individually among residues in the reference sequence, or scattered in one or more contiguous groups within the reference sequence.

[0036] Several mathematical algorithms for rapidly obtaining optimal alignments and calculating identity between two or more sequences are known and are incorporated into several available software programs. For purposes of the present invention, sequence identity between two amino acid sequences is preferably determined using default settings using an algorithm based on global alignment, such as the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol., 48:443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite; Rice et al., 2000, Trends Genet., 16:276-277) or the BLAST global alignment tool (Altschul et al., "Basic local alignment search tool," 1990, J. Mol. Biol., 215, 403-410). Local alignments can also be used if the sequences being compared are of substantially the same length.

[0037] Polypeptides having at least 88% percent identity with either SEQ ID NO: 1 or SEQ ID NO: 2 include s-KL of mammals other than mouse and human.

[0038] SEQ ID NO: 1 is the amino acid sequence of a transcript from alternative splicing of the α-Klotho human gene, which contains the KL1 domain sequence, has an estimated weight of 70 kDa, and has a specific secretion signal consisting of a 15-amino acid tail not found in the m-KL transcript. The α-Klotho human gene is located in chromosome 13NC_000013.11 (33016063..33066145) of the human genome assembly GRCh38 (24.12.2013) maintained by the Genome Reference Consortium. SEQ ID NO: 1 is derived from the cDNA corresponding to SEQ ID NO: 3, which is derived from an alternatively spliced ​​transcript of a 5012-base pair mRNA sequence with GenBank database accession number NM_004795, version 3, as of May 3, 2014.

[0039] SEQ ID NO:2 is the amino acid sequence of a transcript from alternative splicing of the α-Klotho mouse gene, which contains the KL1 domain sequence, an estimated weight of 70 kDa, and a specific secretory signal consisting of a 15-amino acid tail not found in the m-KL transcript. The α-Klotho mouse gene is located in chromosome 5 (150,952,607-150,993,809) of the UCSC Genome Browser of the Mouse July 2007 (NCBI37 / mm9) assembly of the mouse genome. SEQ ID NO:2 is derived from the corresponding cDNA of SEQ ID NO:4, which in turn is derived from the alternatively spliced ​​transcript of the 5124 base pair mRNA sequence with GenBank database accession number NM_013823, version 2, dated February 15, 2015.

[0040] In another embodiment of the first aspect of the invention, the polypeptide variant consists of the sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0041] In further embodiments of the first aspect of the invention, the polypeptide has a length of 645 amino acids or less, 600 amino acids or less, or 550 amino acids or less. In even more particular embodiments, the polypeptide consists of the sequence of SEQ ID NO: 1 or a variant thereof that consists of a sequence at least 85% identical to SEQ ID NO: 1, and has a length of 645 amino acids or less, 600 amino acids or less, or 550 amino acids or less. In particular embodiments, the polypeptide consists of the sequence of SEQ ID NO: 1 or a variant thereof that consists of a sequence at least 85% identical to SEQ ID NO: 1, said variant having one of the following sequences: 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, and 600 amino acids, or a length of 545 to 600 amino acids.

[0042] In one embodiment of the first aspect of the present invention, the polypeptide is a secreted splicing isoform (s-KL) of a mammalian Klotho protein. In even more particular embodiments, the polypeptide is human s-KL. Secreted splicing isoforms (s-KL) of mammalian Klotho proteins have been disclosed in the prior art (see, for example, WO 2017085317A1). Thus, the present invention can be particularly formulated as a secreted splicing isoform (s-KL) of a mammalian Klotho protein, in particular human s-KL, or a nucleic acid sequence encoding same, for use in the prevention and / or treatment of muscle diseases or disorders.

[0043] The term "secreted spliced ​​isoform of mammalian Klotho," abbreviated as "s-KL," refers to a protein resulting from a transcript from alternative splicing, which generates a truncated form of the protein (s-KL) formed by the KL1 domain, with an estimated weight of 70 kDa, along with a specific secretion signal consisting of a 15-amino acid tail not found in the m-KL transcript. Therefore, it is also referred to as the secreted isoform of Klotho, s-KL, or secreted spliced ​​isoform of the Klotho protein. s-KL differs from other forms of soluble Klotho, namely, p-KL, p-KL1, and p-KL2. In this description, m-KL refers to the full-length transmembrane form, p-KL refers to the soluble proteolyzed Klotho resulting from cleavage of m-KL, and p-KL1 and p-KL2 refer to soluble Klotho forms consisting of the KL1 and KL2 domains of p-KL, respectively. m-KL is derived from a full-length transcript encoding a single-pass transmembrane protein (m-KL) with a molecular weight of approximately 130 kDa. The protein contains three domains: a short C-terminal transmembrane domain, an extracellular domain consisting of two internal repeats of approximately 550 amino acids, designated KL1 and KL2, respectively, and a very short intracellular domain of 10 amino acids. The transmembrane extracellular domain can be cleaved by the metalloproteinases ADAM10 and ADAM17 to yield another form of soluble Klotho of approximately 130 kDa (abbreviated as p-KL for proteolyzed membrane isoform). In addition, there is a second recognition site for the proteases ADAM10 and 17 located between the KL1 and KL2 domains, which generates two new 70 kDa isoforms, one contained only in the KL1 domain (e.g., resulting from alternative splicing but without the specific amino acid tail) and the other contained in the KL2 domain. However, proteolysis of p-KL into p-KL1 and p-KL2 has not been demonstrated in vivo.

[0044] In another embodiment of the invention, the polypeptide is for use in the prevention and / or treatment of a muscle disease or disorder by improving muscle function, by increasing muscle mass, by increasing the number of muscle fibers, by reducing muscle fibrosis, by increasing muscle regenerative capacity, and / or by improving physical condition or performance.

[0045] In another embodiment of the invention, the polypeptide is for use in the prevention and / or treatment of a muscle disease or disorder by directly improving muscle function.

[0046] In another embodiment of the invention, the prevention and / or treatment of muscle diseases or disorders comprises improving muscle function, increasing muscle mass, increasing the number of muscle fibers, reducing muscle fibrosis, increasing muscle regenerative capacity, and / or improving physical condition or performance. In more particular embodiments, the prevention and / or treatment of muscle diseases or disorders comprises improving muscle function, increasing muscle mass, increasing the number of muscle fibers, reducing muscle fibrosis, increasing muscle regenerative capacity, and / or improving physical condition or performance by a direct effect on muscle (i.e., muscle cells).

[0047] As used herein, "muscle disease or disorder" refers to a disease, disorder, or condition in a muscle-containing animal characterized by deterioration or weakness or degeneration of skeletal and / or smooth muscle, such that normal muscle function is impaired. Typically, this results in a worsened physical condition or performance of the animal. Muscle function can be measured, for example, by rotarod, horizontal, or grip strength tests, as described in the Examples below.

[0048] In one embodiment of the first aspect of the invention, the muscle disease or disorder is a skeletal muscle disease or disorder.

[0049] In one embodiment of the present invention, the muscle disease or disorder is muscle degeneration and / or sarcopenia. In more particular embodiments, the muscle disease or disorder is not associated with cognitive and / or behavioral dysfunction and / or neurodegenerative and / or neuropathological disorders. In even more particular embodiments, the muscle degeneration and / or sarcopenia is not associated with cognitive and / or behavioral dysfunction and / or neurodegenerative and / or neuropathological disorders.

[0050] As used herein, "muscle degeneration" refers to any condition in which the structural integrity of muscle, particularly skeletal muscle, is altered. As used herein, the term "sarcopenia" refers to any condition in which muscle mass, particularly skeletal muscle mass, is reduced.

[0051] In one embodiment of the first aspect of the invention, the muscle disease or disorder is age-related muscle degeneration and / or sarcopenia.

[0052] In one embodiment of the first aspect of the present invention, the muscle disease or disorder is selected from the group consisting of sarcopenia, muscular dystrophy, muscle atrophy, muscle wasting syndrome, cachexia, and combinations thereof. The term "sarcopenia" refers to the age-related loss of skeletal muscle mass and function.

[0053] In another embodiment of the first aspect of the invention, optionally in combination with any one of the embodiments provided below, the muscle disease or disorder is selected from the group consisting of achondroplasia, cleidocranial dysplasia, enchondromatosis, fibrous dysplasia, Gaucher disease, hypophosphatemic rickets, Marfan syndrome, hereditary multiple exotoses, neurofibromatosis, osteogenesis imperfecta, bone poikilosis, sclerotic lesions, pseudoarthrosis, pyogenic osteomyelitis, periodontal disease, antiepileptic drug-induced sarcopenia, primary and secondary hyperparathyroidism, familial hyperparathyroidism syndrome, weightlessness-induced sarcopenia, male osteoporosis, menopause Post-operative sarcopenia, osteoarthritis, nephrogenic gastrointestinal disease, muscle infiltration disorders, oral sarcopenia, osteonecrosis of the jaw, juvenile Paget's disease, melorheostosis, metabolic muscle disease, mastocytosis, sickle cell anemia / disease, organ transplant-associated sarcopenia, kidney transplant-associated sarcopenia, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritis rash, thalassemia, mucopolysaccharidoses, Fabry disease, Turner syndrome, Down syndrome, Klinefelter syndrome, leprosy, Perthes disease, adolescent idiopathic scoliosis, childhood-onset multisystem inflammatory disease, Winchester syndrome, Menkes disease, Wilson's disease, ischemic muscle Diseases (such as Legg-Calve-Perthes disease and regional migratory osteoporosis), anemic conditions, steroid-induced conditions, glucocorticoid-induced sarcopenia, heparin-induced sarcopenia, muscle marrow disorders, scurvy, malnutrition, calcium deficiency, idiopathic osteopenia or osteoporosis, congenital osteopenia or osteoporosis, alcoholism, chronic liver disease, postmenopausal conditions, chronic inflammatory conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus, hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, Acromegaly, hypogonadism, immobility or disuse, recurrent sympathetic dystrophy syndrome, regional osteoporosis, osteomalacia, sarcopenia associated with joint replacement, HIV-associated sarcopenia, sarcopenia associated with loss of growth hormone, sarcopenia associated with cystic fibrosis, fibrous dysplasia, chemotherapy-associated sarcopenia, tumor-induced sarcopenia, cancer-associated sarcopenia, hormone-ablation sarcopenia, multiple myeloma, drug-induced sarcopenia, anorexia nervosa, disorders associated with facial sarcopenia, disorders associated with cranial sarcopenia, disorders associated with jaw sarcopenia, disorders associated with skull sarcopenia,or caused by muscle loss associated with space travel.

[0054] In certain embodiments of the first aspect, optionally in combination with any of the embodiments provided above and below, the polypeptide is linked to a heterologous moiety.

[0055] As used herein, a "heterologous moiety" refers to any molecule that is coupled to a polypeptide via either a covalent or non-covalent bond. In certain embodiments, the heterologous moiety is located at either the N-terminus or the C-terminus of the polypeptide. In certain embodiments, the heterologous moiety is located at both the N-terminus and the C-terminus of the polypeptide.

[0056] The heterologous moiety can be, for example, a molecule that facilitates purification of the polypeptide. In particular embodiments, the heterologous moiety is a peptide. In even more particular embodiments, the heterologous moiety is a polyhistidine track. As will be appreciated by those skilled in the art, small peptides that aid in the purification of a protein can be maintained in the final compound without affecting its functionality.

[0057] The heterologous moiety can also be any vehicle to facilitate absorption, transport, and delivery of the polypeptide.

[0058] These polypeptides, in particular those derived from KL proteins such as s-KL, may be used directly in protein form or may be expressed in target cells of tissues of interest by gene therapy. To this end, in a second aspect, the present invention also provides a nucleic acid sequence encoding a polypeptide as defined in the first aspect or a variant thereof for use in the prevention and / or treatment of muscle diseases or disorders.

[0059] The term "nucleic acid sequence encoding a polypeptide" is to be understood in particular as meaning an mRNA encoding said polypeptide or a cDNA sequence resulting from reverse transcription (RT-PCR) of an mRNA encoding said polypeptide.

[0060] This aspect can also be formulated as the use of a nucleic acid sequence as defined above for the manufacture of a medicament for the prevention and / or treatment of a muscular disease or disorder. The present invention also relates to a method for the treatment and / or prevention of a muscular disease or disorder, comprising administering a therapeutically effective amount of a nucleic acid sequence as defined above, together with a pharmaceutically acceptable excipient or carrier, to a subject, including a human, in need thereof.

[0061] In particular embodiments of the second aspect, the nucleic acid sequence comprises SEQ ID NO: 3 or SEQ ID NO: 4. In even more particular embodiments, the nucleic acid sequence consists of SEQ ID NO: 3 or SEQ ID NO: 4.

[0062] In more particular embodiments of the second aspect of the invention, the nucleic acid sequence consists of SEQ ID NO:3 or SEQ ID NO:4 or a variant thereof consisting of a sequence that is at least 85%, 86%, 87%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to SEQ ID NO:3 or SEQ ID NO:4.

[0063] In more particular embodiments of the second aspect of the invention, the nucleic acid sequence consists of SEQ ID NO:3 or SEQ ID NO:4 or a variant thereof consisting of a sequence that is at least 85%, 86%, 87%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to SEQ ID NO:3 or SEQ ID NO:4, which variant substantially maintains or improves the muscle treating effect of SEQ ID NO:3 or SEQ ID NO:4.

[0064] In a third aspect, the present invention provides a genetic construct comprising a nucleic acid sequence as defined in the second aspect operably linked to an expression promoter, for use in the prevention and / or treatment of a muscle disease or disorder.

[0065] This aspect can also be formulated as the use of the genetic construct defined above for the manufacture of a medicament for the prevention and / or treatment of a muscle disease or disorder. The present invention also relates to a method for the treatment and / or prevention of a muscle disease or disorder, comprising administering a therapeutically effective amount of the genetic construct defined above, together with a pharmaceutically acceptable excipient or carrier, to a subject, including a human, in need thereof.

[0066] In a specific embodiment of the third aspect, the operably linked expression promoter is selected from the group consisting of a constitutive expression promoter, an inducible promoter, a muscle-specific expression promoter, and a neuron-specific expression promoter. In a more specific embodiment, the genetic construct according to the present invention comprises a cytomegalovirus intermediate-early (CMV IE) promoter, a sequence encoding s-KL (mouse or human s-KL cDNA), and a polyadenylation sequence (polyA). In another specific embodiment, the genetic construct according to the present invention comprises a CAG promoter, a sequence encoding s-KL (mouse or human s-KL cDNA), and a polyadenylation sequence (polyA).

[0067] In particular embodiments of the third aspect, optionally in combination with any of the embodiments provided above and below, the genetic construct comprises or consists of SEQ ID NO:7 or SEQ ID NO:8.

[0068] All these genetic constructs are capable of expressing the protein of interest once in the cell. To facilitate administration of the construct, the present invention also provides in a fourth aspect an expression vector comprising a genetic construct as defined in the third aspect for use in the prevention and / or treatment of a muscle disease or disorder, and thus comprising a nucleic acid sequence of the second aspect encoding the polypeptide of the first aspect operably linked to an expression promoter, in particular a constitutive expression promoter.

[0069] This aspect can also be formulated as the use of an expression vector as defined above for the manufacture of a medicament for the prevention and / or treatment of a muscle disease or disorder. The present invention also relates to a method for the treatment and / or prevention of a muscle disease or disorder, comprising administering a therapeutically effective amount of an expression vector as defined above, together with a pharmaceutically acceptable excipient or carrier, to a subject, including a human, in need thereof.

[0070] In certain embodiments of the fourth aspect, optionally in combination with any of the embodiments provided above and below, the expression vector is a viral vector.

[0071] In a particular embodiment of the third aspect, optionally in combination with any of the embodiments provided above and below, the expression vector consists of the sequence of SEQ ID NO:9.

[0072] In certain embodiments of the fourth aspect, optionally in combination with any of the embodiments provided above and below, the viral vector is an adeno-associated virus. In certain embodiments, it is an adeno-associated virus of a serotype selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, PHPeB, and 9P31. In more particular embodiments, it is an adeno-associated virus of the serotype AAV9.

[0073] In another embodiment of the first, second, third and fourth aspect, optionally in combination with any of the embodiments provided above and below, the polypeptide for use according to the first aspect, the nucleic acid sequence for use according to the second aspect, the genetic construct for use according to the third aspect or the expression vector for use according to the fourth aspect is administered together with at least one pharmaceutically acceptable excipient, diluent or carrier in the form of a pharmaceutical composition.

[0074] The expression "pharmaceutical composition" encompasses compositions intended for both humans and non-human animals. Those skilled in the art will understand that a pharmaceutical composition must contain a therapeutically effective amount of a compound. As used herein, the expression "therapeutically effective amount" refers to an amount of a polypeptide, nucleic acid sequence, gene construct, or expression vector that, when administered, is sufficient to prevent the occurrence of, or alleviate to some extent, one or more symptoms of the disease being addressed. The specific dosage of a compound to be administered in accordance with the present invention will, of course, be determined by the specific circumstances surrounding the case, including the compound to be administered, the route of administration, the specific condition being treated, and similar considerations.

[0075] The expression "pharmaceutically acceptable excipient, diluent, or carrier" refers to a pharmaceutically acceptable material, composition, or vehicle. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissues or organs of humans and non-human animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problem or complication commensurate with a reasonable benefit-to-risk ratio.

[0076] Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, tonicity adjusting agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc. Use of any conventional excipient medium is contemplated within the scope of the present invention so long as it is not incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition.

[0077] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition of the present invention will vary depending on the identity, size, and / or condition of the subject being treated, and further depending on the route by which the composition is intended to be administered.

[0078] Pharmaceutically acceptable excipients for use in preparing pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Excipients such as coloring agents, coating agents, sweetening agents, and flavoring agents can be present in the composition, according to the judgment of the formulator.

[0079] Pharmaceutical compositions containing the proteins or nucleic acids of the invention can be in any dosage form, e.g., solid or liquid, and can be administered by any suitable route, e.g., oral, parenteral, rectal, topical, intranasal, intraocular, intraperitoneal, or sublingual, and therefore, they contain pharmaceutically acceptable excipients necessary for formulation into the desired dosage form, e.g., topical formulations (ointments, creams, lipogels, hydrogels, etc.), eye drops, aerosol sprays, injectable hydrogels, injectable solutions, osmotic pumps, etc.

[0080] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and combinations thereof.

[0081] Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and combinations thereof.

[0082] Exemplary binders include, but are not limited to, starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), natural and synthetic gums (e.g., acacia, sodium alginate, Irish moss extract, panwar gum, ghatti gum, isapol husk mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone), magnesium aluminum silicate (veegum), and larch arabinogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and combinations thereof.

[0083] Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, ascorbyl stearate, ascorbyl oleate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, edetate disodium, edetate dipotassium, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and edetate trisodium.

[0084] Exemplary buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and combinations thereof.

[0085] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0086] In another embodiment of the first, second, third, and fourth aspects, optionally in combination with any of the embodiments provided above and below, the pharmaceutical composition is for administration to a patient mucosally (e.g., nasally, sublingually, vaginally, buccally, or rectally), parenterally (e.g., subcutaneously, intravenously, intramuscularly, or intraarterially, either by bolus or infusion), orally, transdermally, or via inhalation, e.g., by aerosol. In certain embodiments, the pharmaceutical composition is for systemic administration. Suitable formulations for parenteral administration, e.g., by intra-articular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Injection solutions and suspensions can also be prepared from sterile powders, granules, and tablets. In some embodiments, the compositions are administered, for example, by subcutaneous, intraperitoneal, intravesical, intravenous, or intracerebroventricular injection, by infusion, for example, using a reservoir or osmotic minipump, or by intramuscular infusion. The formulations can be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials. In even more particular embodiments, the pharmaceutical compositions are for intracerebroventricular or intravenous administration, more particularly for systemic intravenous administration.

[0087] In another embodiment of the first, second, third and fourth aspects, optionally in combination with any of the embodiments provided above and below, the polypeptide for use according to the first aspect, the nucleic acid sequence for use according to the second aspect, the genetic construct for use according to the third aspect or the expression vector for use according to the fourth aspect is administered in combination with another active agent. Suitable active agents for administration in combination with the compounds of the invention include, but are not limited to, vitamin D, an ACE inhibitor, a myostatin inhibitor, growth hormone, testosterone, metformin or creatine.

[0088] All embodiments of the first aspect are intended to apply to the second to sixth aspects of the invention as well.

[0089] The positive effects that s-KL exerts on muscle can also be applied to healthy subjects, such as athletes, to improve muscle function or overall physical condition.

[0090] Thus, as noted above, in the seventh, eighth, and ninth aspects, the present invention provides non-therapeutic methods based on the administration of s-KL to increase muscle regenerative capacity, improve muscle function, and / or increase muscle mass, as well as methods for improving the physical condition or performance of a subject. Embodiments of the first and second aspects, particularly those relating to the sequences of the polypeptides or variants thereof, or nucleic acid sequences, are intended to apply to the seventh, eighth, and ninth aspects of the invention.

[0091] As used herein, the term "subject" is meant to include human and non-human animals.

[0092] In certain embodiments of the seventh aspect of the invention, improving muscle function and / or increasing muscle mass comprises increasing the number and / or size of muscle fibers.

[0093] In an embodiment of the seventh, eighth and ninth aspects of the invention, the subject is a healthy subject.

[0094] In further embodiments of the seventh, eighth and ninth aspects of the invention, the nucleic acid sequence is comprised in a genetic construct operably linked to an expression promoter. In more particular embodiments, the genetic construct is comprised in an expression vector. It is intended that the embodiments of the second, third and fourth aspects relating to nucleic acid sequences, expression constructs and vectors also apply to the seventh, eighth and ninth aspects of the invention.

[0095] In another embodiment of the above aspect, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered for a specific period of time or for a chronic treatment period, which is an extended period including the entire life of the subject. Within the treatment period, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered on a specific time schedule. In further embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered one, two, three, or four times per day. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered once per day. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered twice per day. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered in the morning and evening. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered one, two, three, or four times per week. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered once per week. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered 1, 2, 3, or 4 times per month. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered once per month. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered for at least 3 months every year. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered for 1 month every 6 months.

[0096] Throughout the specification and claims, the word "comprise" and variations of this word are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" also includes the example of "consisting of." Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the present invention. The following examples and drawings are provided by way of illustration and are not intended to be limiting of the present invention. Reference signs placed in parentheses in connection with the drawings and in the claims are intended solely to enhance the comprehension of the claims and should not be construed as limiting the scope of the claims. Furthermore, the present invention covers all possible combinations of the specific and preferred embodiments described herein.

[0097] For reasons of completeness, the various aspects of the invention are set out in the following numbered clauses.

[0098] 1. A polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence which is at least 85% identical to SEQ ID NO: 1, for use in the prevention and / or treatment of a muscle disease or disorder.

[0099] 2. The polypeptide for use according to claim 1, wherein the muscle disease or disorder is muscle degeneration and / or sarcopenia.

[0100] 3. The polypeptide for use according to claim 1 or 2, wherein said muscle disease or disorder is age-related muscle degeneration and / or muscle loss.

[0101] 4. The polypeptide for use according to any one of items 1 to 3, wherein the muscle disease or disorder is selected from the group consisting of sarcopenia, muscular dystrophy, muscle atrophy, muscle wasting syndrome, cachexia, and combinations thereof.

[0102] 5. A polypeptide for use according to any one of clauses 1 to 4, consisting of the sequence SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 88% identical to SEQ ID NO: 1.

[0103] 6. A polypeptide for use according to any one of clauses 1 to 5, consisting of the sequence SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 98% identical to SEQ ID NO: 1.

[0104] 7. A polypeptide for use according to any one of items 1 to 5, consisting of SEQ ID NO: 1 or SEQ ID NO: 2.

[0105] 8. A nucleic acid sequence encoding the polypeptide or variant thereof according to any one of paragraphs 1 to 7, for use in the prevention and / or treatment of a muscular disease or disorder.

[0106] 9. A genetic construct comprising the nucleic acid sequence according to paragraph 8, operably linked to an expression promoter, for use in the prevention and / or treatment of a muscle disease or disorder.

[0107] 10. An expression vector comprising the genetic construct according to paragraph 9, for use in the prevention and / or treatment of a muscle disease or disorder.

[0108] 11. An expression vector for use according to paragraph 10, which is a viral vector.

[0109] 12. An expression vector for use according to item 11, which is an adeno-associated virus of a serotype selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, PHPeB, and 9P31.

[0110] 13. A polypeptide for use according to any one of clauses 1 to 7, a nucleic acid sequence for use according to clause 8, a genetic construct for use according to clause 9, or an expression vector for use according to any one of clauses 10 to 12, administered in the form of a pharmaceutical composition together with at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0111] 14. A polypeptide for use according to any one of paragraphs 1 to 7 and 13, a nucleic acid sequence for use according to paragraphs 8 and 13, a genetic construct for use according to paragraphs 9 and 13, or an expression vector for use according to any one of paragraphs 10 to 13, administered in combination with another active agent.

[0112] 15. A non-therapeutic method for improving muscle function and / or increasing muscle mass in a subject, comprising the step of administering to the subject a polypeptide consisting of the sequence of SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, or a nucleic acid sequence encoding the polypeptide or variant thereof.

[0113] [Example] Materials and Methods Animal housing C57BL / 6J male (n = 48) and female mice (n = 48) were purchased from Charles River. The animals were randomly divided into three groups per sex. Two of these were treated with a null control vector (SEQ ID NO: 10) (n = 16) or an s-KL expression vector (SEQ ID NO: 9) (n = 16) when they were 7 months old. The third group was treated with an s-KL expression vector (SEQ ID NO: 9) (n = 16) when they were 12 months old. The majority of these animals were followed throughout their lifespan to study health and viability, and a second subset of four animals / group was randomly selected and euthanized when they were 24 months old to study viral vector function.

[0114] Mice were allowed free access to food and water and maintained under standard temperature conditions (22 ± 2°C) and a 12-h light / dark cycle (300 lux / 0 lux). Mice were checked periodically for overall health until natural death or euthanasia. The euthanasia protocol was cervical dislocation, administered by a blinded veterinarian at the animal housing facility when the animal was deemed to have reached the endpoint criteria.

[0115] Generation and Administration of Treatment Gene therapy treatment consisted of an expression cassette under the control of the CAG promoter containing either a control null sequence or the secreted isoform of the mouse α-KL gene (SEQ ID NO: 2).

[0116] Two adeno-associated virus vectors, serotype 9 (AAV9), independently containing these constructs were generated according to the triple transfection method described in Piedra JX et al., 2015. To transduce as many mouse tissues as possible, animals were simultaneously administered by intracerebroventricular and intravenous injection. Intracerebroventricular stereotactic injection of AAV vectors was performed as previously described (Masso A. et al., supra). Briefly, treatments were administered into the right hemisphere at coordinates of -0.2 mm anterior-posterior, -2 mm dorsoventral, and +1 mm medial-lateral from the bregma. The vector dose was 1 x 10 in 6 μl. 11 Intravenous injections were performed using 4 × 10 viral genomes / animal, diluted with NaCl 0.9% to a final volume of 200 μL, using an ultramicropump (World Precision Instruments) at a rate of 0.5 μl / min. 11 The dose consisted of 100 viral genomes / animal and was manually injected into the lateral tail vein of the mice using a syringe.

[0117] Serum biochemical analysis Blood samples were obtained by decapitation of deeply anesthetized animals using SST serum collection tubes (BD microtainer). Blood was allowed to stand at room temperature for 5 minutes and then placed on ice. Serum was isolated by centrifugation of the tubes at 3000 rpm for 10 minutes for 15 minutes, and finally aliquoted and kept frozen at -80°C. KL serum levels were measured using a mouse KL-specific ELISA kit (IBL) according to the manufacturer's instructions.

[0118] rotarod Prior to the rotarod experiment, animals underwent three consecutive days of training. Training consisted of two accelerating rotarod trials for each animal to reduce stress, improve coordination with novel tasks, and allow for the detection of differences due to physical condition. On the test day, animals were carefully placed into a pre-cleaned rotarod apparatus, and the latency to fall was quantified. Tests were performed twice for each animal, and the results presented are the average of both trials per animal.

[0119] Horizontal bar A circular wooden bar with a diameter of 1 cm was placed horizontally 40 cm above a soft floor made of expanded polystyrene. Mice were carefully hung from the bar by their upper limbs, and the maximum test length was 40 seconds. The time spent on the bar (tolerance) and the distance walked along the bar (coordination) were recorded. Two trials were performed per animal, and the best performance was selected.

[0120] Grip strength The animals were given a force platform with their front legs and slowly pulled backward by the tail until they reached the edge of the platform, allowing them to demonstrate resistance. This procedure was repeated three times, and data are presented as the average force for each group for each trial to assess fatigue effects in the animals.

[0121] Muscle histology and immunohistochemistry Mice were euthanized, and the indicated muscles were dissected, embedded in OCT solution (TissueTek), frozen in liquid nitrogen-cooled isopentane, and stored at -80°C until analysis, or fixed in 2% PFA solution in PBS. Ten-micrometer muscle cryosections were taken and stained for hematoxylin / eosin (H / E) or Sirius red (Sigma-Aldrich). The following primary antibodies were used for immunohistochemistry: anti-MyoD (Dako, M3512), anti-Pax7 (DSHB), and anti-Ki67 (Abcam, ab15580). Digital images were acquired using a Leica DMR600B microscope equipped with a DFC300FX camera. Image J software was used to quantify fiber type distribution, CSA, and the percentage of muscle area positive for Sirius red staining, according to the manufacturer's instructions.

[0122] Extensor digitorum longus (EDL) muscle transplant Xenotransplantation experiments were performed by removing the extensor digitorum longus muscle from its anatomical bed and transplanting it onto the surface of the tibialis anterior muscle of wild-type (WT) recipient mice. Seven days after transplantation, the muscle grafts were harvested for analysis.

[0123] Gene expression Total RNA isolation was performed using TRIsure™ Reagent according to the manufacturer's instructions (Bioline Reagent). The tissue used for RNA extraction was liver. Samples were homogenized using a TissueLyzer LT sample disruption device (QIAGEN). RNA quantity and purity were measured using a NanoDrop™ 1000 spectrophotometer (Thermo Scientific). RNA reverse transcription was performed using the iScript™ Advanced cDNA Synthesis Kit (Bio-Rad). Gene expression was analyzed by real-time quantitative PCR (RT-qPCR) using a Bio-Rad CFX-384 PCR instrument at the Analysis and Photodocumentation Service of the Universitat Autónoma de Barcelona according to the manufacturer's instructions. Each reaction contained 25 ng of cDNA, 7.5 μL of iTaq™ Universal SYBR Green Supermix (Bio-Rad), and a primer concentration of 0.2 nM, with a final reaction volume of 15 μL. The primers used are listed in Table 1.

[0124] [Table 1]

[0125] statistical analysis Statistical analysis and graphical presentation were performed using GraphPad Prism version 8 (GraphPad Software). Statistical differences between groups were analyzed using a two-tailed unpaired Student's t-test when comparing two groups, or one-way analysis of variance (ANOVA), followed by Tukey's as a post-hoc analysis for comparisons of all treatment groups. Data were expressed as the mean ± standard error of the mean (SEM). Statistical differences were accepted when p values ​​were ≤ 0.05.

[0126] result s-KL treatment efficiently increased s-KL protein concentrations Viral vector administration was performed by continuous intravenous and intracerebroventricular injection (Fig. 1a). Of the 96 treated animals, one died immediately after the intervention. To evaluate viral vector function, a randomly selected subset of four animals from each group was euthanized at 24 months of age. s-KL gene expression was studied in the liver, as this organ is transfected after AAV9 serotype injection and is the main secretory organ in adult animals (Fig. 1b). s-KL cDNA expression was significantly increased in all KL-treated groups, higher in males than in females, and higher in the 12MO (12-month-old) group compared with 6MO (6-month-old)-treated animals. Furthermore, efficient protein production and secretion into the bloodstream was confirmed by ELISA (Fig. 1c). At both 6 and 12 months of age, significantly higher KL protein levels were detected in serum in treated mice compared with animals treated with null-containing AAV9. Again, concentrations were much higher in males than in females. In males, this was also significantly increased in 12MO males compared to the 6MO group, showing twice as high s-KL concentrations.

[0127] s-KL treatment improved the performance of aged animals in physical tests Various behavioral tests were performed to assess the physical condition of aging animals. The accelerating rotarod test provides information on the animal's coordination and tolerance status. As can be seen in Figure 2a, females treated with s-KL showed improved performance in the accelerating rotarod test at both administration points. Furthermore, both males and females showed improved horizontal bar performance, reaching statistical significance in s-KL-treated animals at 12 months of age (Figure 2b, c). Horizontal bar performance parameters also show increased tolerance compared to null-treated animals. Finally, grip strength was measured, and improvements in grip strength parameters were observed in s-KL-treated males (Figure 2d).

[0128] s-KL treatment reduced age-related muscle fibrosis in male mice To confirm the state of aging muscle tissue, two muscles from the animals were analyzed. The parameters analyzed were the number of muscle fibers measured using hematoxylin and eosin staining, and the level of muscle fibrosis, which quantified the percentage of connective tissue in Sirius Red staining. As can be seen in Figure 3a, there was a trend toward an increased number of fibers in the soleus muscle of aging male mice treated with s-KL for 6 mo. Importantly, there was a significant and consistent decrease in the percentage of fibrous tissue in the muscles of male mice treated with s-KL for 12 mo (Figure 3b).

[0129] Aged muscles of treated animals exhibit higher regenerative capacity The regenerative capacity of aged muscle was assessed by transplanting extensor digitorum lengus (EDL) muscles into young recipient mice as described above. Fibers in the muscle grafts were quantified, and their quantity and size were observed to increase. Animals treated with s-KL for 6 mo showed a significant increase in the number of fibers, while animals treated with s-KL for 12 mo showed an increase in both the quantity and percentage of thicker fibers (Figure 4a-b). To assess regenerative capacity, various cell proliferation markers were analyzed. The amount of satellite cells was quantified using the paired box 7 (Pax7) marker. As can be seen in Figure 4c-e, the amount of these cells increased in grafts from treated animals. These satellite cells also exhibited increased proliferative capacity, as measured by the number of cells that also expressed the cell proliferation marker Ki67. Myoblast determination protein 1, a marker of myogenic commitment of muscle satellite cells, was increased in explants from animals treated with s-KL and reached statistical difference when colocalized with stem cell markers (Fig. 4f–h).

[0130] All these results suggest that administration of s-KL may provide a useful therapeutic approach for the prevention and / or treatment of muscle disorders, particularly age-related muscle disorders.

[0131] List of References Altschul et al., “Basic local alignment search tool”, 1990, J. Mol. Biol, vol. 215, pp. 403-410. International Publication No. WO 2017 / 085317 Masso A. et al., “Secreted αKlotho isoform protects against age-dependent memory deficits” Mol Psychiatry, 2018, vol. 23(9), pp. 1937-1947 Piedra, J. et al., “Development of a rapid, robust, and universal picogreen-based method to titer adeno-associated vectors”, 2015, Human Gene Therapy Methods, vol. 26(1), pp. 35-42

Claims

1. A polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, for use in the prevention and / or treatment of muscle diseases or disorders by improving muscle function.

2. 2. The polypeptide for use according to claim 1, wherein the muscle disease or disorder is muscle degeneration and / or muscle loss.

3. 3. The polypeptide for use according to claim 1 or 2, wherein the muscle disease or disorder is age-related muscle degeneration and / or muscle loss.

4. 4. The polypeptide for use according to any one of claims 1 to 3, wherein the muscle disease or disorder is selected from the group consisting of sarcopenia, muscular dystrophy, muscle atrophy, muscle wasting syndrome, cachexia, and combinations thereof.

5. 5. The polypeptide for use according to any one of claims 1 to 4, wherein the muscle disease or disorder is not associated with cognitive and / or behavioral dysfunction, and / or a neurodegenerative and / or neuropathological disease.

6. A polypeptide for use according to any one of claims 1 to 5, consisting of the sequence SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 88% identical to SEQ ID NO:

1.

7. A polypeptide for use according to any one of claims 1 to 6, consisting of the sequence SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 98% identical to SEQ ID NO:

1.

8. A polypeptide for use according to any one of claims 1 to 7, consisting of SEQ ID NO: 1 or SEQ ID NO:

2.

9. 9. A nucleic acid sequence encoding a polypeptide or variant thereof as defined in any one of claims 1 to 8, wherein said nucleic acid is for use in the prevention and / or treatment of a muscle disease or disorder by improving muscle function.

10. 10. A genetic construct comprising a nucleic acid sequence as defined in claim 9, wherein the genetic construct is for use in the prevention and / or treatment of a muscle disease or disorder by improving muscle function, and is operably linked to an expression promoter.

11. 11. An expression vector comprising the gene construct defined in claim 10, wherein the expression vector is for use in the prevention and / or treatment of a muscle disease or disorder by improving muscle function.

12. 12. The expression vector for use according to claim 11, which is a viral vector.

13. 13. The expression vector for use according to claim 12, which is an adeno-associated virus of a serotype selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, PHPeB, and 9P31.

14. 14. A polypeptide for use according to any one of claims 1 to 8, a nucleic acid sequence for use according to claim 9, a genetic construct for use according to claim 10, or an expression vector for use according to any one of claims 11 to 13, administered in the form of a pharmaceutical composition together with at least one pharmaceutically acceptable excipient, diluent, or carrier.

15. A polypeptide for use according to any one of claims 1 to 8 and 14, a nucleic acid sequence for use according to claims 9 and 14, a genetic construct for use according to claims 10 and 14 or an expression vector for use according to any one of claims 11 to 14, administered in combination with another active agent.

16. A non-therapeutic method for improving muscle function and / or increasing muscle mass in a subject, comprising the step of administering to the subject a polypeptide consisting of the sequence of SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, or a nucleic acid sequence encoding said polypeptide or said variant thereof.

Images