Klotho secretory splice variants for treatment of muscle disorders

Abstract

The present invention provides a polypeptide consisting of the sequence SEQ ID NO: 1 or a variant thereof consisting of a sequence having at least 85% identity to SEQ ID NO: 1 for use in the prevention and / or treatment of muscle diseases or conditions. The invention also provides a nucleic acid sequence for coding the polypeptide, a gene construct containing the nucleic acid sequence or an expression vector containing the gene construct, which are used for the application. The polypeptides, nucleic acid sequences, gene constructs or expression vectors of the invention can be administered in the form of pharmaceutical compositions, together with at least one pharmaceutically acceptable excipient, diluent or carrier. The invention also provides a non-therapeutic method for improving muscle function and / or increasing muscle mass in a subject.

Description

[0001] This application claims priority from European patent application EP22383173.6 filed on December 2, 2022. Technical Field

[0002] The present invention relates to the field of medicine, and in particular to a medical method for preventing and / or treating muscle diseases. The compounds of the present invention are particularly suitable for treating age-related muscle diseases. Background Art

[0003] Aging represents a stage in life during which morbidity and mortality risk increase due to the accumulation of molecular alterations and tissue dysfunction, promoting the decline of the organism's protective systems.

[0004] Muscle tissue is one of the most severely impacted systems during aging, characterized by decreased strength and resistance, sarcopenia, muscle fibrosis, and diminished muscle regeneration. Skeletal muscle status is a key indicator of animal health and, in older animals, directly impacts survival by increasing the risk of falls and physical injury.

[0005] The tissue-level features of sarcopenia have been extensively described, including reduced muscle fiber size, increased intramuscular fat accumulation, preferential atrophy of type II (fast-twitch) muscle fibers, and impaired function. However, our understanding of the cellular mechanisms of sarcopenia remains inadequate, hindering the development of targeted and specific interventions.

[0006] Currently, approaches to treating and preventing sarcopenia focus primarily on prescription exercise and dietary modifications, strategies that have shown some degree of success. Furthermore, there are currently no pharmacological interventions targeting sarcopenia on the market.

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

[0008] Therefore, despite the efforts made to date, there remains a need for effective and safe treatments for muscle diseases, particularly age-related muscle diseases. Summary of the Invention

[0009] The present inventors have developed a novel therapy for preventing and / or treating muscle diseases based on the administration of s-KL, a secretory splice isoform of Klotho.

[0010] The Klotho gene exists in two major transcripts: full-length Klotho mRNA and alternatively spliced ​​Klotho mRNA. Full-length Klotho mRNA is transcribed into a single-pass transmembrane protein called m-Klotho (m-KL) with a molecular weight of 135 kDa. The extracellular domain of m-Klotho can be released from the membrane through protease-mediated shedding, producing soluble circulating processed Klotho (p-KL, 130 kDa), sometimes referred to as soluble Klotho, which has two active domains. Alternatively spliced ​​Klotho mRNA exhibits a premature stop codon and produces a secreted protein, s-KL (70 kDa), which contains only one active domain and an additional 15 amino acids at its C-terminus. Although similar abbreviations (s-KL) are sometimes used in the prior art to designate soluble Klotho and secreted Klotho, with the former being the processed version of the transmembrane full-length Klotho and the latter being the spliced ​​isoform, these two isoforms have completely different structures, sizes, and biological activities. Indeed, although full-length Klotho has been described to be involved in FGF23 receptor binding, PTH synthesis, regulation of parathyroid growth, and changes in vitamin D metabolism and calcium blood levels, the secretory splice isoform of Klotho (s-KL) has not been implicated in these processes.

[0011] The present invention is based solely on the use of the secreted splice isoform of Klotho, the biological function of which remains largely unknown, and the abbreviation s-KL used herein refers only to the secreted form of Klotho.

[0012] As shown in the following examples, the present inventors surprisingly found that administration of s-KL not only improved the physical function of elderly animals ( Figure 2 ad), and also improved the condition of elderly muscle tissue ( Figure 3 ab). In particular, administration of s-KL increased the number of muscle fibers and decreased the percentage of fibrotic tissue in muscle.

[0013] Furthermore, the present inventors found that the muscles of animals treated with s-KL exhibited a higher number of muscle stem cells and a higher proliferation capacity, particularly when treated during adult development, which provided the muscles with a higher regenerative capacity ( Figure 4 ah).

[0014] All of these effects of s-KL were highly unexpected in light of the current state of the art, as it had previously been shown that intracerebroventricular administration of s-KL had no effect on motor function in mice (Massó A. et al., "Secreted αKlothoisoform protects against age-dependent memory deficits" Mol Psychiatry, 2018, vol. 23(9), pp. 1937-1947).

[0015] The combination of significant effects that s-KL has been discovered to exert in muscle tissue clearly positions s-KL as a useful therapeutic agent for improving muscle function and treating muscle diseases, particularly age-related muscle degeneration.

[0016] Therefore, in a first aspect, the present invention provides a polypeptide or a variant thereof, the polypeptide consisting of the sequence SEQ ID NO: 1, the variant consisting of a sequence having at least 85% identity with SEQ ID NO: 1, for use in preventing and / or treating muscle diseases or disorders, in particular by improving muscle function.

[0017] In a second aspect, the present invention provides a nucleic acid sequence encoding a polypeptide as defined in the first aspect or a variant thereof, for use in preventing and / or treating muscle diseases or disorders, in particular by improving muscle function.

[0018] In a third aspect, the present invention provides a gene construct comprising a nucleic acid sequence as defined in the second aspect, operably linked to an expression promoter, for use in preventing and / or treating muscle diseases or disorders, in particular by improving muscle function.

[0019] In a fourth aspect, the present invention provides an expression vector comprising the gene construct as defined in the third aspect, for use in preventing and / or treating muscle diseases or disorders, in particular by improving muscle function.

[0020] In a fifth aspect, the present invention provides a host cell transformed or transfected with the nucleic acid sequence defined in the second aspect, the gene construct defined in the third aspect or the expression vector defined in the fourth aspect, for preventing and / or treating muscle diseases or disorders, in particular by improving muscle function.

[0021] In a sixth aspect, the present invention provides a polypeptide or a variant thereof, or a nucleic acid sequence encoding the polypeptide or a variant thereof, wherein the polypeptide consists of the sequence SEQ ID NO: 1, and the variant consists of a sequence having at least 85% identity with SEQ ID NO: 1, for 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, the method comprising administering to the subject a polypeptide or a variant thereof, or a nucleic acid sequence encoding the polypeptide or a variant thereof, wherein the polypeptide consists of the sequence SEQ ID NO: 1 and the variant consists of a sequence having at least 85% identity with SEQ ID NO: 1.

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

[0024] In a ninth aspect, the present invention provides a non-therapeutic method for improving a subject's physical condition or performance, the method comprising administering to the subject a polypeptide or a variant thereof, or a nucleic acid sequence encoding the polypeptide or a variant thereof, the polypeptide consisting of the sequence SEQ ID NO: 1, and the variant consisting of a sequence having at least 85% identity with SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Treatment with s-KL effectively increased s-KL protein concentrations. (a) Schematic diagram of the experimental design. (b) Analysis of s-KL gene expression in male (left) and female (right) livers. (c) Quantification of total s-KL protein concentrations in male (left) and female (right) serum. Analysis was performed on samples from a subset of animals sacrificed at 24 months of age. Data in (b) are presented as fold change in expression relative to untreated animals. Mean ± standard error of the mean (SEM), n = 4; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001.

[0026] Figure 2 Treatment with s-KL improved the physical performance of aged animals in a fitness test. (a) Results of the accelerating rotarod test are shown as the maximum speed achieved by the animals. (bc) Results of the horizontal rod test are shown, showing the time it took for male (b) and female (c) animals to fall from the rod. (d) Results of the grip strength test. Results represent the average force achieved by the animals across three trials. Data are expressed as mean ± standard error of the mean (SEM), n = 8-11; *p < 0.05; **p < 0.01; ***p < 0.001.

[0027] Figure 3Treatment with s-KL reduces age-related muscle fibrosis in mice. (a) Quantification of soleus muscle fibers. (b) Quantification of the percentage of fibrotic area in soleus muscle. Data are expressed as mean ± standard error of the mean (SEM), n = 4; *p < 0.05; **p < 0.01.

[0028] Figure 4 Aged muscles from treated animals exhibited enhanced regenerative capacity. (A, B) Quantitative analysis of transplanted muscle fibers, including average fiber size (A) and frequency of different fiber sizes (B). (C, E) Quantitative analysis of Pax7, Ki67, and cells expressing both Pax7 and Ki67 in transplanted tissue. (F, H) Quantitative analysis of MyoD, Ki67, and cells expressing both MyoD and Ki67 in transplanted tissue. Data are expressed as mean ± standard error of the mean (SEM), n = 8-11; *p < 0.05; **p < 0.01; ***p < 0.001. DETAILED DESCRIPTION

[0029] Unless otherwise specified, all terms used in this application should be understood to have the ordinary meaning known in the art. Other more specific definitions of certain terms in this application are as follows, which are intended to be uniformly applied throughout the specification and claims, unless otherwise expressly provided.

[0030] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more". Unless otherwise indicated, definite articles such as "the" as used herein also include plural forms of nouns.

[0031] As described above, in the first aspect, the present invention provides a polypeptide or a variant thereof for preventing and / or treating muscle diseases or disorders, in particular by improving muscle function, wherein the polypeptide consists of the sequence SEQ ID NO: 1, and the variant consists of a sequence having at least 85% identity with SEQ ID NO: 1.

[0032] This aspect can also be expressed as the use of the polypeptide described above in the preparation of a medicament for preventing and / or treating a muscle disease or condition. The present invention also relates to a method for treating and / or preventing a muscle disease or condition, comprising administering a therapeutically effective amount of the polypeptide described above and a pharmaceutically acceptable excipient or carrier to a subject (including a human) in need thereof.

[0033] In a more specific embodiment of the first aspect of the invention, the polypeptide consists of the sequence of SEQ ID NO: 1 and a variant thereof 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 a more specific embodiment, the polypeptide consists of the sequence of SEQ ID NO: 1 and a variant thereof consists of a sequence that is at least 88% or 98% identical to SEQ ID NO: 1.

[0034] In a more specific embodiment of the first aspect of the invention, the polypeptide consists of the sequence of SEQ ID NO: 1 and a variant thereof consists of a sequence having 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% identity to SEQ ID NO: 1, wherein the variant substantially maintains or enhances the muscle therapeutic effect of SEQ ID NO: 1.

[0035] In another embodiment of the first aspect of the invention, the polypeptide consists of the sequence SEQ ID NO: 1 or SEQ ID NO: 2.

[0036] For those skilled in the art, variants of proteins and polypeptides are well understood and may involve modifications of the amino acid sequence. For example, modifications of the amino acid sequence may generally be classified into one or more of the following categories: substitution, insertion, or deletion variants.

[0037] In the present invention, the term "identity" refers to the percentage of identical residues in a sequence when two sequences are optimally aligned. In the optimal alignment, if the amino acid residue at a position in the first sequence is the same as the corresponding position in the second sequence, then the sequences exhibit identity with respect to that position. The percentage of identity determines the number of identical residues within a defined length in a given alignment. Therefore, the level of identity between two sequences or ("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., percentage sequence identity = (number of identical positions / total number of compared positions) × 100). Gaps, i.e., positions where there is a residue in one sequence but not in the other, are considered to have non-identical residues and are counted as compared positions.

[0038] By way of illustration, if an amino acid sequence is at least (for example) 95% identical to the reference amino acid sequence of SEQ ID NO: 1, 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 per 100 amino acids in the reference amino acid sequence of SEQ ID NO: 1. In other words, to obtain a polypeptide having an amino acid sequence 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 up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the amino- or carboxyl-terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, and may be interspersed individually among the residues in the reference sequence or in one or more contiguous groups.

[0039] Many mathematical algorithms for rapidly obtaining optimal alignments of two or more sequences and calculating the identity between them are known and are incorporated into many available software programs. For purposes of the present invention, it is preferred to use an algorithm based on a global alignment to determine the sequence identity between two amino acid sequences, such as the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), preferably 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, v. 215, pages 403-410) using the default settings. When the compared sequences are substantially the same length, a local alignment may also be used.

[0040] Polypeptides having at least 88% identity to either SEQ ID NO: 1 or SEQ ID NO: 2 include s-KL from mammals other than mouse and human.

[0041] SEQ ID NO:1 is the amino acid sequence of an alternatively spliced ​​transcript of the human α-klotho gene. It contains the KL1 domain, has a molecular weight of approximately 70 kDa, and possesses a specific 15-amino acid secretion signal sequence, which is absent from the m-KL transcript. The human α-klotho gene is located on chromosome 13, NC_000013.11 (33016063..33066145), in the human genome GRCh38 (2013.12.24) maintained by the Genome Reference Consortium. SEQ ID NO:1 is derived from the corresponding cDNA of SEQ ID NO:3, which was derived from the mRNA sequence of an alternatively spliced ​​transcript. This cDNA is GenBank accession number NM_004795, 5012 base pairs, version 3, March 3, 2014.

[0042] SEQ ID NO:2 is the amino acid sequence of an alternatively spliced ​​transcript from the mouse α-klotho gene. It contains the KL1 domain, has a molecular weight of approximately 70 kDa, and possesses a specific 15-amino acid secretion signal sequence that is absent from the m-KL transcript. The mouse α-klotho gene is located on chromosome 5 (150952607-150993809) of the July 2007 (NCBI37 / mm9) assembly of the mouse genome available on the UCSC Mouse Genome Browser. SEQ ID NO:2 is derived from the cDNA corresponding to SEQ ID NO:4, an alternatively spliced ​​transcript derived from the mRNA sequence, GenBank database accession number NM_013823, 5124 base pairs, version 2, February 15, 2015.

[0043] In another embodiment of the first aspect of the invention, the polypeptide variant consists of the sequence SEQ ID NO:5 or SEQ ID NO:6.

[0044] In another embodiment of the first aspect of the invention, the polypeptide is equal to or less than 645 amino acids, 600 amino acids, or 550 amino acids in length. In a more specific embodiment, the polypeptide consists of the sequence of SEQ ID NO: 1, a variant thereof consists of a sequence having at least 85% identity to SEQ ID NO: 1, and is equal to or less than 645 amino acids, 600 amino acids, or 550 amino acids in length. In a specific embodiment, the polypeptide consists of the sequence of SEQ ID NO: 1, a variant thereof consists of a sequence having at least 85% identity to SEQ ID NO: 1, and the length of the variant is selected from the group consisting of 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 584, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599 and 600 amino acids, or a group consisting of 545 to 600 amino acids in length.

[0045] In one embodiment of the first aspect of the present invention, the polypeptide is a secreted splice isoform of the mammalian Klotho protein (s-KL). In a more specific embodiment, the polypeptide is human s-KL. Secreted splice isoforms of the mammalian Klotho protein (s-KL) have been disclosed in the prior art (e.g., see WO2017085317A1). Therefore, the present invention can be specifically formulated as a secreted splice isoform of the mammalian Klotho protein (s-KL), particularly human s-KL, or a nucleic acid sequence encoding the same, for use in preventing and / or treating muscle diseases or disorders.

[0046] The term "secreted splice isoform of mammalian Klotho," abbreviated as "s-KL," refers to a protein produced from an alternatively spliced ​​transcript that yields a truncated form of the protein (s-KL) consisting of the KL1 domain, with a molecular weight of approximately 70 kDa and a specific 15-amino acid secretion signal sequence. This sequence is absent from the m-KL transcript and is therefore also referred to as the secreted isoform of Klotho, s-KL, or the secreted splice isoform of the Klotho protein. s-KL is distinct from other soluble forms of klotho, namely p-KL, p-KL1, and p-KL2. Throughout this specification, m-KL represents the full-length transmembrane form; p-KL represents the soluble proteolytic form of klotho, produced by cleavage of m-KL; and p-KL1 and p-KL2 represent soluble forms of klotho composed 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. This protein comprises three domains: a short transmembrane domain at the C-terminus, an extracellular domain consisting of two internal repeats of approximately 550 amino acids, designated KL1 and KL2, and a very short intracellular domain consisting of 10 amino acids. The transmembrane form of the extracellular domain can be cleaved by the metalloproteases ADAM10 and ADAM17, generating a soluble, approximately 130 kDa form of Klotho (the proteolytic membrane isoform is abbreviated as p-KL). Furthermore, a second recognition site for the proteases ADAM10 and ADAM17 exists between the KL1 and KL2 domains, generating two new 70 kDa isoforms: one containing only the KL1 domain (similar to the form produced by alternative splicing, but lacking the specific amino acid tail) and the other containing the KL2 domain. However, proteolysis of p-KL into p-KL1 and p-KL2 has not been demonstrated in vivo.

[0047] In another embodiment of the present invention, the polypeptide is used to prevent and / or treat muscle diseases or disorders by improving muscle function, increasing muscle mass, increasing the number of muscle fibers, reducing muscle fibrosis, improving muscle regeneration capacity and / or improving physical condition or performance.

[0048] In another embodiment of the invention, the polypeptides are used to prevent and / or treat muscle diseases or disorders by directly improving muscle function.

[0049] In another embodiment of the present invention, the prevention and / or treatment of a muscle disease or condition comprises improving muscle function, increasing muscle mass, increasing the number of muscle fibers, reducing muscle fibrosis, enhancing muscle regeneration capacity, and / or improving physical condition or performance. In a more specific embodiment, the prevention and / or treatment of a muscle disease or condition comprises improving muscle function, increasing muscle mass, increasing the number of muscle fibers, reducing muscle fibrosis, enhancing muscle regeneration capacity, and / or improving physical condition or function by directly acting on muscle (i.e., muscle cells).

[0050] As used herein, "muscle disease or disorder" refers to a disease, disorder, or condition in a muscle-containing animal characterized by deterioration, weakening, or loss of skeletal and / or smooth muscle, resulting in a decrease in normal muscle function. Typically, this results in a deterioration in the animal's physical condition or performance. Muscle function can be measured, for example, by rotating rod, horizontal rod, or handgrip strength tests, as described in the examples below.

[0051] In one embodiment of the first aspect of the invention, the muscle disease or disorder is a skeletal muscle disease or disorder.

[0052] In one embodiment of the invention, the muscle disease or disorder is muscle degeneration and / or muscle loss. In a more specific embodiment, the muscle disease or disorder is not associated with cognitive and / or behavioral disorders and / or neurodegenerative and / or neuropathological diseases. In a more specific embodiment, the muscle degeneration and / or muscle loss is not associated with cognitive and / or behavioral disorders and / or neurodegenerative and / or neuropathological diseases.

[0053] 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 "muscle loss" refers to any condition in which muscle mass, particularly skeletal muscle mass, is reduced.

[0054] In one embodiment of the first aspect of the invention, the muscle disease or disorder is age-related muscle degeneration and / or muscle loss.

[0055] In one embodiment of the first aspect of the 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.

[0056] In another embodiment of the first aspect of the invention, optionally in combination with any of the examples provided below, the muscle disease or disorder is caused by: achondroplasia, cleidocranial dysplasia, enchondromatosis, fibrous dysplasia, Gaucher's disease, hypophosphatemic rickets, Marfan syndrome, multiple hereditary exostoses, neurofibromatosis, osteogenesis imperfecta, osteophytes, sclerotic lesions, pseudoarthritis, suppurative osteomyelitis, periodontal disease, antiepileptic drug-induced muscle loss, primary and secondary hyperparathyroidism, familial hyperparathyroidism, muscle loss caused by weightlessness, male osteoporosis, postmenopausal muscle loss, osteoarthritis , renal osteodystrophy, infiltrative muscle disease, oral muscle loss, osteonecrosis of the jaw, juvenile Paget's disease, hyperostosis, metabolic muscle disease, mastocytosis, sickle cell anemia / disease, organ transplant-related muscle loss, kidney transplant-related muscle loss, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritis, thalassemia, mucopolysaccharidoses, Fabry disease, Turner syndrome, Down syndrome, Klinefelter syndrome, leprosy, avascular necrosis of the femoral head (Perthes' Disease), juvenile idiopathic scoliosis, infantile-onset multisystem inflammatory disease, Winchester syndrome, Menkes disease, Wilson's Disease), ischemic muscle disease (eg, Legg-Calve-Perthes disease, regional migratory osteoporosis), anemic states, steroid-induced disease, glucocorticoid-induced muscle loss, heparin-induced muscle loss, muscle bone marrow disease, scurvy, malnutrition, calcium deficiency, idiopathic osteopenia or osteoporosis, congenital osteopenia or osteoporosis, chronic alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory disease, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus, hyperthyroidism, thyroid disease, parathyroid disease, Cushing's disease, limb Hypertrophy, hypogonadism, immobilization or disuse states, reflex sympathetic dystrophy syndrome, regional osteoporosis, osteomalacia, muscle loss associated with joint replacement, muscle loss associated with HIV, muscle loss associated with loss of growth hormone, muscle loss associated with cystic fibrosis, fibrous dysplasia, chemotherapy-related muscle loss, tumor-induced muscle loss, cancer-related muscle loss, hormone-dependent muscle loss, multiple myeloma, drug-induced muscle loss, anorexia nervosa, disease-related facial muscle loss, disease-related cranial muscle loss, disease-related jaw muscle loss, disease-related cranial muscle loss, or muscle loss associated with spaceflight weightlessness.

[0057] In a specific embodiment of the first aspect, optionally in combination with any of the above or below embodiments, the polypeptide is linked to a heterologous moiety.

[0058] As used herein, a "heterologous group" refers to any molecule coupled to a polypeptide by a covalent or non-covalent bond. In a specific embodiment, the heterologous group is located at the N-terminus or C-terminus of the polypeptide. In a specific embodiment, the heterologous group is located at the N-terminus and C-terminus of the polypeptide.

[0059] The heterologous group can be, for example, a molecule that facilitates the purification of a polypeptide. In a specific embodiment, the heterologous group is a peptide. In a more specific embodiment, the heterologous group is a polyhistidine tag. Those skilled in the art will appreciate that small peptides that aid in protein purification can be retained in the final compound without affecting its function.

[0060] The heterologous group can also be any excipient to facilitate absorption, transport, and delivery of the polypeptide.

[0061] These polypeptides produced by KL proteins, in particular s-KL, can be used directly in the form of proteins, or they can be expressed in target cells of target tissues through gene therapy. To this end, in a second aspect, the present invention also provides a nucleic acid sequence encoding a polypeptide or a variant thereof as defined in the first aspect, which is used to prevent and / or treat muscle diseases or disorders.

[0062] 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 generated by reverse transcription (RT-PCR) of an mRNA encoding said peptide.

[0063] This aspect can also be expressed as the use of the nucleic acid sequence described above in the preparation of a medicament for preventing and / or treating a muscle disease or condition. The present invention also relates to a method for treating and / or preventing a muscle disease or condition, comprising administering a therapeutically effective amount of the nucleic acid sequence described above and a pharmaceutically acceptable excipient or carrier to a subject (including a human) in need thereof.

[0064] In a specific embodiment of the second aspect, the nucleic acid sequence comprises SEQ ID NO: 3 or SEQ ID NO: 4. In a more specific embodiment, the nucleic acid sequence consists of SEQ ID NO: 3 or SEQ ID NO: 4.

[0065] In a more specific embodiment of the second aspect of the invention, the nucleic acid sequence consists of the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a variant thereof, said variant 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.

[0066] In a more specific embodiment of the second aspect of the invention, the nucleic acid sequence consists of the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a variant thereof, said variant consisting of a sequence having 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% identity to SEQ ID NO: 3 or SEQ ID NO: 4, wherein the variant thereof substantially maintains or improves the muscle therapeutic effect of SEQ ID NO: 3 or SEQ ID NO: 4.

[0067] In a third aspect, the present invention provides a gene construct comprising a nucleic acid sequence as defined in the second aspect operably linked to an expression promoter, for use in preventing and / or treating a muscle disease or disorder.

[0068] This aspect can also be expressed as the use of the gene construct described above in the preparation of a medicament for preventing and / or treating a muscle disease or condition. The present invention also relates to a method for treating and / or preventing a muscle disease or condition, comprising administering a therapeutically effective amount of the gene construct described above and a pharmaceutically acceptable excipient or carrier to a subject (including a human) in need thereof.

[0069] 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 early-mid (CMV IE) promoter, a sequence encoding s-KL (mouse or human s-KL cDNA), and a polyadenylation chain (poly A). 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 chain (poly A).

[0070] In a specific embodiment 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.

[0071] All of these gene constructs are capable of expressing the target protein in cells. To facilitate administration of the construct, in a fourth aspect, the present invention further provides an expression vector comprising the gene construct as defined in the third aspect, thereby comprising the nucleic acid sequence encoding the polypeptide of the first aspect in the second aspect, the nucleic acid sequence being operably linked to an expression promoter, particularly a constitutive expression promoter, and the expression vector being used to prevent and / or treat muscle diseases or conditions.

[0072] This aspect can also be expressed as the use of the expression vector described above for preparing a medicament for preventing and / or treating a muscle disease or condition. The present invention also relates to a method for treating and / or preventing a muscle disease or condition, comprising administering a therapeutically effective amount of the expression vector described above and a pharmaceutically acceptable excipient or carrier to a subject (including a human) in need thereof.

[0073] In a specific embodiment of the fourth aspect, optionally in combination with any of the embodiments provided above and below, the expression vector is a viral vector.

[0074] In a specific embodiment of the third aspect, optionally in combination with any of the embodiments provided above and below, the expression vector consists of the sequence SEQ ID NO:9.

[0075] In a specific embodiment 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 a specific embodiment, it is selected from the group consisting of the following serotypes of adeno-associated viruses: AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, PHPeB and 9P31. In a more specific embodiment, it is an adeno-associated virus of serotype AAV9.

[0076] 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 used according to the first aspect, the nucleic acid sequence used according to the second aspect, the gene construct used according to the third aspect or the expression vector used according to the fourth aspect can be administered in the form of a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient, diluent or carrier.

[0077] The term "pharmaceutical composition" includes both compositions for use in humans and compositions for use in non-human animals. It will be understood by those skilled in the art 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 development of one or more symptoms of the disease being treated, or to alleviate these symptoms of the disease being treated to some extent. The specific dosage of the compound administered in accordance with the present invention will, of course, be determined by the specific circumstances surrounding the case, including the compound being administered, the route of administration, the specific condition being treated, and similar considerations.

[0078] A "pharmaceutically acceptable excipient, diluent, or carrier" means 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 human and non-human animal tissues or organs without producing excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications disproportionate to a reasonable benefit / risk ratio.

[0079] Examples of suitable pharmaceutically acceptable excipients include solvents, dispersion media, diluents or other liquid carriers, dispersion or suspension aids, surfactants, isotonicity agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. Unless any conventional excipient medium is incompatible with the substance or its derivatives, for example, produces any adverse biological effect or interacts in other deleterious ways with any other components of the pharmaceutical composition, its use is considered to be within the scope of the present invention.

[0080] The relative amounts of the active ingredient, pharmaceutically acceptable excipients and / or any other ingredients in the pharmaceutical compositions of the present invention will vary depending on the characteristics, size and / or health of the subject being treated and the route of administration of the composition.

[0081] Pharmaceutically acceptable excipients for the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients such as coloring agents, coating agents, sweeteners and flavorings may be present in the composition at the discretion of the formulator.

[0082] Pharmaceutical compositions containing proteins or nucleic acids of the present invention can be in any dosage form, such as solid or liquid, and can be administered by any suitable route, such as oral, parenteral, rectal, topical, nasal, ophthalmic, intraperitoneal or sublingual, for which purpose these compositions will include pharmaceutically acceptable excipients for formulating the desired dosage form, such as topical preparations (ointments, creams, lipogels, hydrogels, etc.), eye drops, aerosol sprays, injectable hydrogels, injectable solutions, osmotic pumps, etc.

[0083] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dibasic calcium phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and combinations thereof.

[0084] Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinyl pyrrolidone (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, 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.

[0085] Exemplary binders include, but are not limited to, starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, levulose, dextrin, molasses, lactose, lactitol, mannitol), natural and synthetic gums (e.g., acacia, sodium alginate, Irish moss extract, Panwar gum, Ghatti gum, isapol shell mucilage, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, polyvinyl pyrrolidone), magnesium aluminum silicate (Veegum) and larch acacia polysaccharides, alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, ethanol, and combinations thereof.

[0086] Exemplary preservatives may include antioxidants, chelating agents, antibacterial 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, disodium EDTA, dipotassium EDTA, EDTA, fumaric acid, malic acid, phosphoric acid, sodium EDTA, tartaric acid, and trisodium EDTA.

[0087] Exemplary buffers include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium glucoheptonate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium levulinate, valeric acid, dibasic calcium phosphate, phosphoric acid, calcium phosphate, calcium hydroxyphosphate, 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, disodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and combinations thereof.

[0088] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0089] 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 by mucosal (e.g., nasal, sublingual, vaginal, oral or rectal), parenteral (e.g., subcutaneous, intravenous, intramuscular or intraarterial injection, whether bolus or infusion), oral, transdermal or by inhalation such as an aerosol. In a specific embodiment, the pharmaceutical composition is for systemic administration. Formulations suitable for parenteral administration, such as by intraarticular, 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, as well as aqueous and non-aqueous sterile suspensions, which may include 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 composition is administered by injection, e.g., subcutaneously, intraperitoneally, intravesically, intravenously, intracerebroventricularly, by infusion, such as using a reservoir or osmotic minipump, or intramuscularly. The formulation can be packaged in unit-dose or multi-dose sealed containers, such as ampoules and vials. In a more specific embodiment, the pharmaceutical composition is for intracerebroventricular administration or intravenous administration; even more particularly, systemic intravenous administration.

[0090] In another embodiment of the first, second, third and fourth aspects, optionally in combination with any of the embodiments provided above or below, the polypeptide used according to the first aspect, the nucleic acid sequence used according to the second aspect, the gene construct used according to the third aspect, or the expression vector used according to the fourth aspect is administered in combination with another active agent. Suitable active agents for co-administration with the compounds of the invention include, but are not limited to, vitamin D, ACE inhibitors, myostatin inhibitors, growth hormone, testosterone, metformin, or creatine.

[0091] All embodiments of the first aspect are also intended to apply to the second to sixth aspects of the present invention.

[0092] The positive effects of s-KL on muscle could also be applied in healthy subjects to improve muscle function or general physical condition, such as in athletes.

[0093] Thus, as described above, the present invention provides, in its seventh, eighth, and ninth aspects, non-therapeutic methods based on the administration of s-KL for improving muscle function and / or increasing muscle mass, enhancing muscle regeneration capacity, and improving the physical condition or performance of a subject. The embodiments of the first and second aspects, particularly those relating to the sequence or nucleic acid sequence of the polypeptide or variant thereof, also apply to the seventh, eighth, and ninth aspects of the present invention.

[0094] As used herein, the term "subject" is meant to include human and non-human animals.

[0095] In a specific embodiment 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.

[0096] In embodiments of the seventh, eighth and ninth aspects of the invention, the subject is a healthy subject.

[0097] In another embodiment of the seventh, eighth and ninth aspects of the present invention, the nucleotide sequence is contained in a gene construct operably linked to an expression promoter. In a more specific embodiment, the gene construct is contained in an expression vector. The embodiment of the second, third and fourth aspects relevant to the nucleotide sequence, expression construct and vector is also applicable to the seventh, eighth and ninth aspects of the present invention.

[0098] In another embodiment of the above aspects, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered over a specific time period or chronic treatment period, which refers to a longer time period, including the entire life cycle of the subject. During the treatment period, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered at specific times. In further embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered once, twice, three times, or four times daily. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered once daily. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered twice daily. 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 once, twice, three times, or four times weekly. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered once weekly. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered once, twice, three times, or four times monthly. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered once monthly. In some embodiments, the polypeptide, nucleic acid sequence, gene construct or expression vector is administered for at least three months per year. In some embodiments, the polypeptide, nucleic acid sequence, gene construct or expression vector is administered for one month out of every six months.

[0099] Throughout the description and claims, the word "comprise" and its variations are not intended to exclude other technical features, additives, components or steps.

[0100] In addition, the word "comprising" includes the case of "consisting of...". Other objects, advantages and features of the present invention will become clear to those skilled in the art after reading the specification or may be learned by practicing the invention. The following examples and drawings are for illustrative purposes only and are not intended to limit the present invention. Reference symbols in brackets in the claims relative to the drawings are only used to increase the intelligibility of the claims and should not be construed as limiting the scope of the claims. In addition, the present invention covers all possible combinations of the specific and preferred embodiments described herein.

[0101] For reasons of completeness, various aspects of the invention are listed in the following numbered clauses: 1. A polypeptide consisting of the sequence SEQ ID NO: 1, or a variant consisting of a sequence having at least 85% identity with SEQ ID NO: 1, for use in preventing and / or treating muscle diseases or disorders.

[0102] 2. The polypeptide according to clause 1, wherein the muscle disease or disorder is muscle degeneration and / or muscle loss.

[0103] 3. The polypeptide according to any one of clauses 1-2, wherein the muscle disease or disorder is age-related muscle degeneration and / or muscle loss.

[0104] 4. The polypeptide according to any one of clauses 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.

[0105] 5. A polypeptide according to any one of clauses 1 to 4, wherein the polypeptide consists of the sequence of SEQ ID NO: 1 and a variant thereof consists of a sequence having at least 88% identity to SEQ ID NO: 1.

[0106] 6. A polypeptide according to any one of clauses 1 to 5, wherein the polypeptide consists of the sequence of SEQ ID NO: 1, and a variant thereof consists of a sequence having at least 98% identity with SEQ ID NO: 1.

[0107] 7. The polypeptide according to any one of clauses 1 to 5, wherein the polypeptide consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0108] 8. A nucleic acid sequence encoding a polypeptide as defined in any one of clauses 1 to 7 or a variant thereof for use in preventing and / or treating a muscle disease or disorder.

[0109] 9. A gene construct comprising the nucleic acid sequence defined in clause 8, operably linked to an expression promoter, for use in preventing and / or treating a muscle disease or disorder.

[0110] 10. An expression vector comprising the gene construct defined in clause 9 for use in preventing and / or treating a muscle disease or disorder.

[0111] 11. The expression vector for use according to item 10, which is a viral vector.

[0112] 12. The expression vector according to item 11, which is selected from the group consisting of adeno-associated virus serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, PHPeB and 9P31.

[0113] 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 gene 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.

[0114] 14. A polypeptide for use according to any one of items 1 to 7 and 13, a nucleic acid sequence for use according to items 8 and 13, a gene construct for use according to items 9 and 13, or an expression vector for use according to any one of items 10 to 13, administered in combination with another active agent.

[0115] 15. A non-therapeutic method for improving muscle function and / or increasing muscle mass in a subject, the method comprising administering to the subject a polypeptide consisting of the sequence SEQ ID NO: 1 or a variant thereof, wherein the variant consists of a sequence having at least 85% identity with SEQ ID NO: 1, and a nucleic acid sequence encoding the polypeptide or the variant thereof.

[0116] Example Materials and Methods C57BL / 6J male (n=48) and female (n=48) mice were purchased from Charles River. The animals were randomly divided into three groups by sex. Two of the groups were treated with either a blank control vector (SEQ ID NO: 10) (n=16) or an s-KL expression vector (SEQ ID NO: 9) (n=16) at 7 months of age. The third group was treated with an s-KL expression vector (SEQ ID NO: 9) (n=16) at 12 months of age. The majority of these animals were followed throughout their lifespan to investigate their health and survival, but a second subgroup of four animals per group was randomly selected and euthanized at 24 months of age to investigate viral vector function.

[0117] Mice were housed under standard temperature conditions (22 ± 2°C) and a 12-h light / dark cycle (300 lux / 0 lux) with free access to food and water. Mice were regularly checked for general health until natural death or euthanasia. Euthanasia was performed by cervical dislocation when a blinded veterinarian at the animal shelter determined that an animal had reached endpoint criteria.

[0118] Preparation and administration of treatment regimens The gene therapy comprises an expression cassette containing either a control empty sequence or a secreted isoform of the mouse α-KL gene (SEQ ID NO: 2) under the control of the CAG promoter.

[0119] Two adeno-associated viral vectors (AAV9) containing the above constructs, serotype 9, were prepared according to the triple transfection method published in Piedra J. X et al., 2015. Animals were administered simultaneously via intracerebroventricular and intravenous injections to transduce as many mouse tissues as possible. AAV vectors were injected stereotaxically into the lateral ventricle as previously described (Massó A. et al., supra). Briefly, the therapeutic drug was injected into the right cerebral hemisphere at the following coordinates: -0.2 mm in the anterior-posterior direction, -2 mm in the dorsal-ventral direction, and +1 mm in the medial-lateral direction from the bregma. The vector dose per animal was 1×10 11 The viral genomes were dissolved in 6 μL and administered via an ultra-micropump (World Precision Instruments) at a rate of 0.5 μL per minute. The intravenous dose was 4 × 10 11 The viral genomes were diluted to a final volume of 200 μL with 0.9% sodium chloride solution and manually injected into the tail vein of the mouse with a syringe.

[0120] Serum biochemical analysis Blood samples were obtained by decapitation from deeply anesthetized animals using SST serum collection tubes (BD Microtainer). The blood was allowed to stand at room temperature for 5 minutes and then placed on ice. Serum was separated by centrifugation at 3000 rpm for 15 minutes and then for an additional 10 minutes. The serum was aliquoted and stored at −80°C. Serum levels of mouse Klotho (KL) were measured using an ELISA kit (IBL) specific for Klotho (KL) according to the manufacturer's instructions.

[0121] Rotating rod Prior to the rotarod test, animals were trained for three consecutive days. Training consisted of two accelerating rotarod trials per animal to reduce stress, improve coordination for the novel task, and enable recognition of differences due to physical state. On the day of the test, the animals were carefully placed in the pre-cleaned rotarod apparatus, and the time to fall was quantified. Each animal was tested twice, and the results presented are the average of the two trials per animal.

[0122] Horizontal rod A 1-cm-diameter, circular wooden pole was placed horizontally 40 cm above a soft, expanded polystyrene floor. Mice were carefully suspended from the pole by their upper limbs for a maximum test duration of 40 seconds. The time the mouse remained on the pole (endurance) and the distance it traveled along the pole (coordination) were recorded. Each mouse underwent two trials, and the best performance was selected.

[0123] grip strength The animal was asked to grasp the grip strength test platform from the upper limbs and then slowly pulled backward from the tail, allowing the animal to generate resistance until it reached the end of the platform. This procedure was repeated three times, and the data were expressed as the average strength per trial per group to observe the effect of animal fatigue.

[0124] Muscle histology and immunohistochemistry Mice were euthanized, and the indicated muscles were dissected and embedded in OCT solution (TissueTek), then frozen in liquid nitrogen-cooled isopentane and stored at −80°C until analysis or fixed in PBS containing 2% PFA. Muscles were frozen and stained with hematoxylin / eosin (H / E) or Sirius red (Sigma-Aldrich) at 10 μm. Immunohistochemistry was performed using the following primary antibodies: 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. Fiber type distribution, CSA, and the percentage of muscle area positive for Sirius red staining were quantified using Image J software according to the manufacturer's instructions.

[0125] Extensor digitorum longus (EDL) transplant Xenograft experiments were performed by removing the extensor digitorum longus muscle from its anatomical location and transplanting it onto the surface of the tibialis anterior muscle in wild-type (WT) recipient mice. Muscle graft samples were collected for analysis 7 days after transplantation.

[0126] Gene expression Total RNA was isolated using TRIsure™ reagent according to the manufacturer's instructions (Bioline Reagent). Liver tissue was used for RNA extraction. Samples were homogenized using a TissueLyser LT sample grinder (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. Experiments were performed at the Center for Analysis and Image Recording of the Autonomous University of Barcelona according to the manufacturer's instructions. Each reaction contained 25 ng of cDNA, 7.5 µL of iTaqt™ 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.

[0127] Table 1

[0128] Data Analysis Statistical analysis and graphical presentation were performed using GraphPad Prism version 8 (GraphPad Software). When comparing two groups, statistical differences between groups were analyzed using a two-tailed unpaired Student's t-test. For comparisons of all treatment groups, one-way analysis of variance (ANOVA) was performed, followed by a Tukey test as a post hoc analysis. Data are presented as mean ± standard error of the mean (SEM). Statistical differences were considered significant when the p-value was ≤ 0.05.

[0129] result s-KL treatment effectively increased s-KL protein concentration Viral vectors were administered by continuous intravenous and intracerebroventricular injection ( Figure 1 a). Of the 96 treated animals, one died shortly after the intervention. At 24 months of age, four animals in each group were randomly selected for euthanasia to evaluate viral vector function. Because the liver is transfected after injection of AAV9 serotypes and is the major secretory organ in adult animals, s-KL gene expression was studied in the liver ( Figure 1b). s-KL cDNA expression was significantly increased in all KL-treated groups, with males showing higher expression than females and the 12MO (12-month-old) group showing higher expression than the 6MO (6-month-old) group. Furthermore, ELISA confirmed efficient protein production and secretion into the blood ( Figure 1 c). Significantly higher levels of KL protein were detected in the serum of mice administered at 6 and 12 months of age compared to animals treated with AAV9 containing a null control (Null). Similarly, concentrations were significantly higher in males than in females. In males, s-KL concentrations were also significantly increased in males receiving 12 MO compared to the 6 MO group, exceeding double the s-KL concentration in the 6 MO group.

[0130] s-KL treatment improves performance in physical fitness tests in aged animals Different behavioral tests were performed to assess the physical state of aged animals. The accelerating rotarod test provides information on the coordination and endurance of the animals. Figure 2 As shown in a, s-KL-treated females showed improved performance in the test at both administration time points. In addition, both males and females improved their performance on the horizontal bar, with statistical significance being reached in animals treated with s-KL at 12 months of age ( Figure 2 b, c). This parameter also showed an increase in endurance compared to animals in the blank control group. Finally, grip strength was measured and an improvement in this parameter was observed in s-KL treated males ( Figure 2 d).

[0131] s-KL treatment reduces age-related muscle fibrosis in male mice Two muscles of the animals were analyzed to examine the state of aging muscle tissue. The parameters analyzed were the number of muscle fibers measured by hematoxylin and eosin staining and the percentage of connective tissue quantified by picrosirius red staining to assess the level of muscle fibrosis. Figure 3 As shown in a, there is a trend toward increased fiber number in the soleus muscle of aged male mice treated with s-KL for 6 MO. Importantly, at 12 MO, the percentage of fibrotic tissue in the muscles of male mice treated with s-KL was significantly and consistently reduced ( Figure 3 b).

[0132] Aging muscles in treated animals showed greater regenerative capacity As described above, the regenerative capacity of aged muscles was assessed by transplanting the extensor digitorum longus (EDL) muscle into young mouse recipients. Fiber quantification of the muscle grafts revealed an increase in both the number and size of these fibers. Animals treated with s-KL at 6 MO significantly increased the number of fibers, and animals treated with s-KL at 12 MO increased the number and percentage of thicker fibers ( Figure 4ab). To assess regenerative capacity, different cell proliferation markers were analyzed. Paired Box 7 (Pax7) marker was used to quantify the number of satellite cells. Figure 4 As shown in Figure ce, the number of these cells increased in the grafts of treated animals. The proliferation capacity of these satellite cells was enhanced, as was the increase in the number of cells expressing the cell proliferation marker Ki67. Myoblast-determining protein 1, a marker of myogenic commitment of muscle satellite cells, was increased in the grafts of s-KL-treated animals, reaching statistical significance when colocalized with stem cell markers ( Figure 4 fh).

[0133] All these results suggest that s-KL administration may provide a useful therapeutic approach for preventing and / or treating muscle diseases, particularly age-related muscle diseases.

[0134] References Altschul et al., "Basic local alignment search tool", 1990, J. Mol.Biol, vol. 215, pp. 403-410. WO2017085317A1 Massó 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 universalpicogreen-based method to titer adeno-associated vectors", 2015, Human GeneTherapy Methods, vol. 26(1), pp. 35–42

Claims

1. A polypeptide or a variant thereof for preventing and / or treating a muscle disease or disorder by improving muscle function, wherein the polypeptide consists of the sequence SEQ ID NO: 1, and the variant consists of a sequence having at least 85% identity with SEQ ID NO:

1.

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

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

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

6. The polypeptide for use according to any one of claims 1 to 5, wherein The polypeptide consists of the sequence of SEQ ID NO: 1, or the variant consists of a sequence having at least 88% identity to SEQ ID NO:

1.

7. The polypeptide for use according to any one of claims 1 to 6, wherein The polypeptide consists of the sequence of SEQ ID NO: 1, or the variant consists of a sequence having at least 98% identity to SEQ ID NO:

1.

8. The polypeptide for use according to any one of claims 1 to 7, wherein The polypeptide consists of SEQ ID NO: 1 or SEQ ID NO:

2.

9. A nucleic acid sequence encoding a polypeptide as defined in any one of claims 1 to 8 or a variant thereof, wherein: The nucleic acid sequence is used to prevent and / or treat muscle diseases or disorders by improving muscle function.

10. A gene construct comprising a nucleic acid sequence as defined in claim 9 operably linked to an expression promoter, wherein: The gene construct is used to prevent and / or treat muscle diseases or disorders by improving muscle function.

11. An expression vector comprising the gene construct according to claim 10, wherein The expression vector is used to prevent and / or treat muscle diseases or disorders by improving muscle function. The expression vector according to claim 11 , which is a viral vector.

13. The expression vector according to claim 12, which is selected from the group consisting of adeno-associated virus serotypes: AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, PHPeB and 9P31.

14. The polypeptide for use according to any one of claims 1 to 8, the nucleic acid sequence for use according to claim 9, the gene construct for use according to claim 10, or the 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. The polypeptide for use according to any one of claims 1 to 8 and 14, the nucleic acid sequence for use according to claims 9 and 14, the gene construct for use according to claims 10 and 14 or the 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, the method comprising administering to the subject a polypeptide or a variant thereof, or a nucleic acid sequence encoding the polypeptide or a variant thereof, wherein the polypeptide consists of the sequence SEQ ID NO: 1 and the variant consists of a sequence having at least 85% identity with SEQ ID NO: 1.

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