Composition for the treatment of sarcopenia or disuse atrophy

Abstract

The present invention relates to substances that activate the GDF5 pathway for use in methods for the treatment of age-related sarcopenia or disuse atrophy.

Description

Technical Field

[0001] The present invention relates to a substance that activates the GDF5 pathway for use in a method for the treatment of sarcopenia or disuse atrophy.

Background Art

[0002] Sarcopenia is an age-related condition characterized by a progressive decline in skeletal muscle mass and skeletal muscle function. Muscle mass and strength begin to decline from the age of 50, and typically more than 30% of the initial muscle mass is lost by the age of 80. Sarcopenia is a major clinical problem in the public health of the elderly, and is associated with adverse outcomes such as physical disability, reduced quality of life, the need for hospitalization, and an increased risk of death. Sarcopenia can result in frailty, and several studies have shown that the risk of falls is significantly increased in subjects with reduced muscle strength. This condition raises major concerns, and it is important to prevent or delay the onset of this condition as much as possible in order to enhance survival and reduce the need for long-term care.

[0003] A very large number of studies have addressed the molecular mechanisms related to disuse atrophy and sarcopenia.

[0004] Disuse atrophy is generally characterized by an early stage in which atrogenes are rapidly activated and a late stage in which atrophy stabilizes and differential gene expression returns to basal levels. This recent finding suggests that molecular mechanisms can be activated to counteract the loss of muscle mass. Such a hypothesis is confirmed by evidence that total mRNA and protein content increase in denervated muscle (Furuno, K., Goodman, M. N., & Goldberg, A. L. Role of different proteolytic systems in the degradation of muscle proteins during denervation atrophy. J. Biol. Chem. 265, 8550-8557 (1990)) and that the protein synthesis machinery is activated (Furano Op. cit.; Sartori, R. et al. BMP signaling controls muscle mass. Nat. Genet. 45, 1309-1318 (2013)). In this compensatory response, only a few components have been identified, in particular the Gfd5 / Smad4 pathway, which is essential not only for counteracting the loss of muscle mass in denervated muscle and during starvation (Sartori Op. cit) but also for promoting reinnervation after nerve crush (Macpherson, P. C. D., Farshi, P., & Goldman, D. Dach2-Hdac9 signaling regulates reinnervation of muscle endplates. Development 142, 4038-4048 (2015)). However, surgical resection of the sciatic nerve, which mimics a very severe pathological condition that completes nerve withdrawal, can induce a sudden and irreversible molecular pathway and thus induce a compensatory response. Nevertheless, this model inadequately reflects other neuromuscular diseases that can slow down proteolysis very slowly and alter the progressive and balanced muscle loss that can change the mechanism.

[0005] Instead, aging muscle gradually loses its ability to balance muscle mass loss as a result of several molecular changes, including increased oxidation, DNA damage, inefficient autophagy, metabolic changes, immobilization and reduction of neuromuscular connections (Miljkovic, N., Lim, J.-Y., Miljkovic, I. & Frontera, W. R. Aging of skeletal muscle fibers. Ann. Rehabil. Med. 39, 155-162 (2015)). Calorie restriction aimed at enhancing autophagy and reducing oxidative DNA damage, as well as exercise or functional electrical stimulation aimed at restoring muscle activity, have so far been the best methods for maintaining the function and size of aged skeletal muscle. However, a better understanding of the mechanisms that produce compensatory responses that counteract the decline in muscle mass and function is highly significant for identifying molecular targets and improving survival and quality of life in old age.

[0006] In the present application, the inventors clearly demonstrate that CaVβ1-E plays a major role in maintaining physiological muscle mass by sustaining Gdf5 expression and signaling not only in aging muscle but also in young disused muscle. The inventors first reveal that sarcopenia is associated with a strong decrease in CaVβ1-E / GDF5, and characterize the important role of CaVβ1-E as a molecule that activates the molecular pathways necessary to balance muscle atrophy after electrical activity dysfunction in adult and aging muscle.

Prior Art Documents

Patent Documents

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Summary of the Invention

Problems to be Solved by the Invention

[0009] By clarifying the association of age-related sarcopenia with a strong decrease in CaVβ1-E / GDF5, a therapeutic solution for a major health concern is provided. As a result of the present invention, it is now possible to envision the treatment of sarcopenia by activating the GDF5 pathway.

Means for Solving the Problems

[0010] Accordingly, substances that activate the GDF5 pathway for use as a medicament are described herein. More specifically, substances that activate the GDF5 pathway are used in methods for the treatment or prevention of sarcopenia or disuse atrophy. In certain embodiments, the substance is selected from a compound that increases the activity of GDF5 or a compound that increases the expression of GDF5. Representative substances for use according to the present invention include, but are not limited to, recombinant GDF5 protein, recombinant CaVβ1-E protein, or a vector encoding GDF5 or CaVβ1-E. Other substances include substances that comprise the CaVβ1-E / GDF-5 system, such as, for example, NRSF inhibitors. Among the NRSF inhibitors, compound X5050 and valproic acid can be cited without limitation.

[0011] The substance can be administered to a subject who is 50 years old or older, particularly 55 years old or older, particularly 60 years old or older, more specifically 65 years old or older, even more specifically 70 years old or older, for example 75 years old or older, or even 80 years old or older. Moreover, the substance can be administered by oral, nasal, intravascular (e.g., intravenous or intra-arterial), intramuscular, intraperitoneal, transdermal, or subcutaneous routes. The substance can be administered periodically, for example, monthly, particularly weekly, or more specifically daily. In addition, the substance can be administered once a day or several times a day.

[0012] Treatment of sarcopenia can result in an increase in muscle mass and / or muscle function, an increase in physical ability or mobility, and / or an increase in muscle strength. Other benefits of the treatments disclosed herein will be apparent to those skilled in the art.

[0013] In another aspect, the present invention relates to a pharmaceutical composition comprising a substance that activates the GDF5 pathway, such as recombinant GDF5, particularly recombinant human GDF5, and a pharmaceutically acceptable carrier.

[0014] A further aspect relates to a substance that activates the GDF5 pathway for use in therapy, such as recombinant GDF5, particularly recombinant human GDF5.

[0015] Another aspect of the invention relates to a method for diagnosing sarcopenia in a subject, comprising the step of determining the level of GDF5 in a biological fluid of the subject.

[0016] Other objects and advantages of the invention will be readily apparent to those skilled in the art.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0018] By measuring the immediate response of skeletal muscle to changes in electrical activity in a sciatic nerve resection model, the inventors observed the appearance of a protein corresponding to the translated Cacnb1 isoform E (or isoform 5, RefSeq: NM_001282977; NP_001269906). The inventors showed that this protein, CaVβ1-E, is an embryonic variant specific to CaVβ1, thereby demonstrating that the embryonic isoform is also expressed in adult skeletal muscle lacking innervation. In mice, the inventors demonstrated that CaVβ1-E is required to balance muscle mass loss upon activation of GDF5 signaling. When CaVβ1-E is downregulated, GDF5 expression is impaired and muscle atrophy worsens after denervation. Measuring the levels of CaVβ1-E and GDF5 in aged, atrophying muscle, the inventors found that both proteins significantly decrease during the aging process. The inventors observed the same correlation for CaVβ1-E and GDF5 in human muscle biopsies from elderly healthy subjects (over 75 years old). More importantly, the inventors were able to show that high expression of CaVβ1-E by an AAV vector clearly preserves skeletal mass in treated mice. In addition, the specific force of muscles overexpressing CaVβ1-E was significantly improved. When CaVβ1-E is restored, the GDF5 pathway is rescued and then counteracts sarcopenia to abolish further decline in muscle mass. The GDF5 pathway has previously been found to play a role in the compensatory response of the denervation model. However, this application is the first report that the GDF5 pathway is involved in aging muscle. This means a significant reinforcement of the prior art and leads to a new therapeutic approach for the treatment of sarcopenia.

[0019] Accordingly, the present invention relates to the activation of the GDF5 pathway for the treatment of sarcopenia.

[0020] Growth differentiation factor 5 (GDF5; also known as BMP-14 and CDMP-1) is a member of the BMP family of TGF-beta superfamily proteins. Human GDF-5, -6, and -7 define a subgroup of the BMP family. GDF5 is synthesized as a homodimeric precursor protein consisting of a 354-amino acid N-terminal proregion and a 120-amino acid C-terminal mature peptide. Mature human GDF-5 shares 99% amino acid sequence identity with GDF5 of both mature mouse and rat. GDF5 signaling is mediated by the formation of a homodimeric complex consisting of type I (BMPR-1B) and type II (BMPR-II or activin RII) serine / threonine kinase receptors, which results in phosphorylation and activation of cytoplasmic Smad proteins (Smad 1, 5, and 8). Similar to other BMP family proteins, GDF5 signaling is attenuated by noggin. GDF5 is involved in multiple developmental processes including limb generation, chondrogenesis, joint formation, bone morphogenesis, cell survival, and neurite outgrowth. Exogenous GDF5 has been reported to promote chondrogenesis, osteogenesis, and angiogenesis in mesenchymal hepatocytes in vivo and in vitro. Inhibition of GDF5 expression or alteration of this signaling can enhance the development of osteoarthritis.

[0021] The relevance of the GDF5 / SMAD4 pathway in the maintenance of skeletal muscle after atrophic stimuli (nerve injury, fasting) was clearly shown in a 2013 publication by Sartori et al., which demonstrated that muscles from SMAD4 knockout mice lacked a compensatory response to denervation. They showed that in wild-type mice, SMAD4 was activated by GDF5 (also known as BMP14), a paracrine factor that is strongly upregulated upon denervation. GDF5, which is expressed after nerve resection, acts on BMP receptor 1, which stimulates phosphorylation of the Smad1 / 5 / 8 complex. In this change, this complex binds to SMAD4 and mediates its translocation to the nucleus, where it limits atrophy by regulating gene transcription and inhibiting the activation of the ubiquitin ligase MUSA1 (Fbox32). This publication defined an essential pathway required for the counteraction of excessive muscle loss after nerve withdrawal. Moreover, it was also shown that GDF5 governs GDF8 (commonly known as myostatin) signaling, and that muscle hypertrophy induced after myostatin inhibition is due to the spread of the GDF5 pathway. Although a few other studies have confirmed the essential role of GDF5 / SMAD4 in muscle mass homeostasis (Winbanks et al. 2013, Macpherson et al. 2015), no study has elucidated the upstream signaling that causes the induction of GDF5. A recent paper has shown that DNA methylation has an important role in the activation of the GDF5 promoter (Reynard et al., 2014 - Hum Genet (2014) 133:1059 - 1073), and that the NFKB-TAK1 pathway is involved in SMAD4 signaling in skeletal muscle (Sadejah et al., JCI Insight. 2018;3(3):e98441), only discussing the possibility of GDF5 involvement.

[0022] The present invention provides a substance for activating the GDF5 pathway for use in a method for the treatment or prevention of sarcopenia.

[0023] In certain embodiments, the substance that activates the GDF5 pathway is a GDF5 peptide, particularly a synthetic or recombinant GDF5, more particularly a recombinant GDF5, such as recombinant human GDF5. Untreated wild-type human GDF-5 (Uniprot accession number P43026) has the following sequence.

[0024]

Chemical formula

[0025] (SEQ ID NO: 1)

[0026] SEQ ID NO: 1 includes a single peptide at amino acid positions 1 to 27, a propeptide at amino acid positions 28 to 381, and the portion corresponding to the mature peptide, which is underlined in the above sequence, at amino acid positions 382 to 501.

[0027] Therefore, the mature peptide has the sequence shown in SEQ ID NO: 2 below. APLATRQGKRPSKNLKARCSRKALHVNFKDMGWDDWIIAPLEYEAFHCEGLCEFPLRSHLEPTNHAVIQTLMNSMDPESTPPTCCVPTRLSPISILFIDSANNVVYKQYEDMVVESCGCR (SEQ ID NO: 2)

[0028] Other recombinant human GDF5 is commercially available and is, for example, a protein having the sequence shown in SEQ ID NO: 3, which is available from Thermo Fischer (catalog number RP-8663). APSATRQGKRPSKNLKARCSRKALHVNFKDMGWDDWIIAPLEYEAFHCEGLCEFPLRSHLEPTNHAVIQTLMNSMDPESTPPTCCVPTRLSPISILFIDSANNVVYKQYEDMVVESCGCR (SEQ ID NO: 3)

[0029] In the context of the present invention, the peptides shown in SEQ ID NO: 2 or SEQ ID NO: 3 may be referred to as "reference recombinant human GDF5".

[0030] According to another specific embodiment, the substance that activates the GDF5 pathway is a functional derivative of the GDF5 peptide. The functional derivative according to the present invention is a peptide having at least one, particularly all, of the activities of the reference peptide. In the context of the present invention, a functional variant of the GDF5 peptide may have the ability to induce the production of alkaline phosphatase by ATDC5 mouse chondrogenic cells with an ED50 of 0.01 to 10 μg / mL, for example, 0.2 to 4 μg / mL, for example, 0.2 to 1.2 μg / mL (Nakamura, K. et al. (1999) Exp. Cell Res. 250:351). In particular, a functional variant of the GDF5 peptide is a peptide that can treat or prevent sarcopenia in an animal model of the symptoms shown in the experimental part of the present application or in a human subject. Also, GDF5 signaling can be evaluated by measuring the phosphorylation of SMAD1 / 5 / 8, the nuclear translocation of SMAD 4, and the transcription of Id-1 as shown in the following experimental part. The activity of the functional variant can be at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 100% of the activity of the reference GDF5 peptide. In a specific embodiment, the functional peptide has an activity superior to that of the reference GDF5 peptide, for example, at least 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% of the activity of the reference GDF5 peptide or at least 150% of the activity. In addition, according to the present invention, a functional variant of the GDF5 peptide has at least 80% sequence identity to the reference GDF5 amino acid sequence, particularly at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the reference human recombinant GDF5.For example, a functional variant of the GDF5 peptide can include 1 to 20 amino acid modifications (i.e., amino acid addition, deletion, or substitution) compared to the reference recombinant human GDF5, for example, 1 to 15 amino acid modifications compared to the reference recombinant human GDF5, particularly 1 to 10 amino acid modifications, more particularly 1 to 6 amino acid modifications, and even more particularly 1, 2, 3, 4, 5, or 6 amino acid modifications. Such functional variants of recombinant human GDF5 can be natural variants of GDF5. In certain embodiments, the functional variant is an optimized GDF5 peptide. Optimization can include various changes in the peptide, such as the amino acid modifications, glycosylation, acetylation, phosphorylation, etc., described above, or the incorporation of at least one D-amino acid, for example, at least 2, at least 3, at least 4, or at least 5 D-amino acids. In another embodiment, the GDF5 peptide includes at least one non-natural amino acid incorporated by insertion, addition, or substitution of another amino acid of the GDF5 sequence. In yet another embodiment, the recombinant GDF5 can be fused to another moiety, for example, another peptide moiety. Such other moieties can, for example, stabilize the peptide.

[0031] In another specific embodiment, the substance corresponds to a GDF5-related protein described in WO 2013 / 08649, and is a functional variant of a GDF5 peptide with increased affinity for BMP receptor IB (BMPR-IB) and / or decreased affinity for BMP receptor IA (BMPR-IA). In certain embodiments, the protein is derived from human wild-type GDF5. In certain embodiments, the GDF5-related protein is preferably obtained by substituting at least one amino acid residue associated with the BMPR-IB and / or BMPR-IA binding site within the amino acid sequence of the GDF-5 peptide by genetic modification techniques. In a further embodiment, at least one hydrophobic amino acid at the BMPR-IB and / or BMPR-IA binding site of the GDF5 peptide is substituted with a hydrophilic or polar amino acid residue selected from the group consisting of, for example, aspartic acid, glutamic acid, lysine, arginine, histidine, serine and threonine. In an alternative embodiment, at least one hydrophilic or polar amino acid at the BMPR-IB and / or BMPR-IA binding site of the GDF5 peptide is substituted with a hydrophobic amino acid selected from the group consisting of, for example, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, valine. In another alternative embodiment, the GDF5-related protein comprises a conservative substitution of at least one amino acid at the BMPR-IB and / or BMPR-IA binding site of the GDF peptide, in particular, a hydrophobic amino acid is substituted with a small or large hydrophobic amino acid, or a hydrophobic or polar amino acid is substituted with a small or large hydrophilic or polar amino acid. The region of the GDF-5-related protein involved in binding to the BMPR-IA and / or BMPR-IB binding site is well known in the art or can be readily determined using methods within the scope of general knowledge. With reference to SEQ ID NO: 1, which is the untreated full-length amino acid sequence of wild-type human GDF5, in certain embodiments, one or more substitutions of the following amino acids with any different amino acid are provided. R399; Any one of F409 to W417, particularly M412, G413, W414 and / or W417; Any one of E434 to M456, particularly F435, P436, L437, R438, S439, H440, P443, N445, V448, I449, L452, M453, S455 and / or M456; S475; I476; F478; Any one of K488 to M493, particularly K488, Y490 and / or D492.

[0032] In certain embodiments, with reference to SEQ ID NO: 1 which is an untreated full-length amino acid sequence, one or more of the following amino acids are substituted with specific amino acids. - R399 is substituted with V, L, I, M, F, Y, W, E or D, - M412 is substituted with V, L, I, F, Y, W, H, K or R, - W414 is substituted with R, K, F, Y, H, E or D, - W417 is substituted with R, K, F, Y, H, E or D, - F435 is substituted with V, L, I, M, P, Y, W, H, K or R, - P436 is substituted with V, L, I, M, F, Y or W, - L437 is substituted with D or E, - R438 is substituted with K, D, H, N, M, E, Q, S, T, Y or W, - S439 is substituted with K, D, E, H, R, M, T, N, Q, Y or W, - H440 is substituted with V, I, M, F, Y, W, E or D, - P443 is substituted with V, L, I, M, F, Y, W, A or S, - N445 is substituted with D, Q, H, F, L, R, K, M, S, Y or W, - V448 is substituted with F, L, I, M, P, Y or W, - I449 is substituted with F, L, V, M, P, Y or W, - L452 is substituted by F, I, V, M, P, Y or W, - M456 is substituted by F, I, L, P, Y, W, S, T, N, Q, K or D, - S475 is substituted by M, T, N, Q, Y or W, - K488 is substituted by R, M, S, T, N, Q, Y or W, - Y490 is substituted by E, H, K, R, Q, F, T, M, S, N, Q or W, - D492 is substituted by G, E, M, S, T, N, Q, Y, W, H, K or R, - I476 is substituted by G, A, V, L, M, F, Y or W, - F478 is substituted by G, A, V, L, I, Y or W.

[0033] In another specific embodiment, with reference to SEQ ID NO: 1 which is an untreated full-length amino acid sequence, one or more of the following amino acids are substituted by specific amino acids. R399 is substituted by M or E, W414 is substituted by R, W417 is substituted by R or F, R438 is substituted by K, S439 is substituted by K or E, I449 is substituted by V.

[0034] The corresponding positions in the mature peptide (e.g., SEQ ID NO: 2 or SEQ ID NO: 3) can be readily obtained from the above information regarding the untreated full-length wild-type human GDF-5.

[0035] In a specific embodiment of the present invention, the substance is a GDF5 peptide consisting of amino acids SEQ ID NO: 2 or SEQ ID NO: 3. In another specific embodiment, the substance is a GDF5 peptide consisting of an amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 3 with a methionine residue added at the N-terminus. In another embodiment, the substance is a GDF5 peptide consisting of amino acids SEQ ID NO: 2 or SEQ ID NO: 3 with the first alanine residue substituted by a methionine residue.

[0036] In a more specific embodiment, the substance that activates the GDF5 pathway is a substance that includes the CaVβ1-E / GDF5 system. In this embodiment, the variant includes the use of small chemical molecules. In a non-limiting variant of this embodiment, the substance that activates the GDF5 pathway is an inhibitor of NRSF (neuron-specific silencer factor; also called REST or RE1 transcription silencer factor).

[0037] In a specific embodiment, the substance that activates the GDF5 pathway and is an NRSF inhibitor is valproic acid. In a more specific embodiment, the substance that activates the GDF5 pathway is an NRSF inhibitor disclosed by Charbord et al., Stem Cells. 2013 Sep;31(9):1816-28, particularly 2-(2-hydroxy-phenyl)-1H-benzimidazole-5-carboxylic acid allyloxyamide (X5050), 2-thiophen-2-yl-1H-benzimidazole-5-carboxylic acid (2-ethyl-hexyl)-amide (X5917), 3-[1-(3-bromo-phenyl)-3,5-dimethyl-1H-pyrazol-4-yl]-1-{4-[5-(morpholine-4-carbonyl)-pyridin-2-yl]-2-phenyl-piperazin-1-yl}-propan-1-one (X38210) or 3-[1-(2,5-difluoro-phenyl)-3,5-dimethyl-1H-pyrazol-4-yl]-1-{4-[5-(morpholine-4-carbonyl)-pyridin-2-yl]-2-phenyl-piperazin-1-yl}-propan-1-one (X38207) molecules disclosed therein, more particularly selected from the X5050 molecule disclosed therein.

[0038] In yet another embodiment, the substance that activates the GDF5 pathway is a vector containing a nucleic acid encoding GDF5, such as human GDF5 or a functional variant thereof. In certain embodiments, the vector is a plasmid or a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. Accordingly, the present invention also relates to a vector, such as a viral vector, such as the retroviral, lentiviral, adenoviral, or AAV vector described above, containing a sequence encoding GDF5. According to certain embodiments, the viral vector is suitable for transducing muscle and / or nerve cells. In a more specific embodiment, such a viral vector suitable for transducing muscle and / or nerve cells is an AAV vector, such as an AAV vector having an AAV2 / 2, AAV2 / 6, AAV2 / 8, AAV2 / 9, or AAV2 / 10 capsid. In a further specific embodiment, the sequence encoding GDF5 may be under the control of regulatory sequences, such as a promoter, an enhancer, a repressor, and a polyadenylation signal. In certain embodiments, the vector contains an expression cassette containing a promoter, a sequence encoding GDF5, and a polyadenylation signal in this order. The promoter may be ubiquitous or tissue-specific. In certain embodiments, the promoter is the native promoter of the GDF5 gene, such as the promoter of the human GDF5 gene.

[0039] In another specific embodiment, the substance that activates the GDF5 pathway is a substance that increases the activity or expression of GDF5. In a more specific embodiment, the substance is recombinant CaVβ1-E, such as recombinant human CaVβ1-E.

[0040] In a further embodiment, the substance that activates the GDF5 pathway is CaVβ1-E, for example, a vector comprising a nucleic acid encoding human CaVβ1-E. In certain embodiments, the vector is a plasmid or a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector. Accordingly, the invention also relates to a vector, such as a viral vector, such as the above retroviral, lentiviral, adenoviral or AAV vector, comprising a sequence encoding CaVβ1-E. The sequence encoding CaVβ1-E may be under the control of regulatory sequences, such as a promoter, an enhancer, a repressor and a polyadenylation signal. In certain embodiments, the vector comprises an expression cassette comprising a promoter, a sequence encoding CaVβ1-E and a polyadenylation signal in this order. The promoter may be ubiquitous or tissue-specific. In certain embodiments, the promoter is the native promoter of the CaVβ1-E gene, such as the promoter of the human CaVβ1-E gene.

[0041] In a preferred embodiment, the substance that activates the GDF5 pathway is the above recombinant GDF5, such as recombinant human GDF5 or a functional variant thereof.

[0042] In certain embodiments, the substance is administered to a subject who is 50 years of age or older. In certain embodiments, the subject is 55 years of age or older, particularly 60 years of age or older, more particularly 65 years of age or older, even more particularly 70 years of age or older, for example 75 years of age or older, or even 80 years of age or older. In certain embodiments, the subject exhibits progressive muscle mass loss. The subject can be male or female, particularly a postmenopausal female. In certain embodiments, the subject's motor neurons are intact or substantially intact, meaning that denervation has not occurred in the subject's body.

[0043] The subject can be screened by any method known to those skilled in the art as having or potentially having sarcopenia. Such methods include evaluation by calculating the skeletal muscle mass index by dual-energy X-ray absorptiometry (DEXA) and / or by calculating the body mass index of the subject. Other methods for identifying subjects who would benefit from the present invention include measurement of muscle function parameters.

[0044] Furthermore, the present invention can be beneficial to a subject of the above age when the subject is subject to partial or complete physical movement restriction, for example, restriction of movement of a limb, for example, restriction of movement of the arm, leg, shoulder, hip joint, particularly, after fracture of the above-mentioned limbs, and most particularly, after fracture of the hip joint. For example, the movement restriction can be the result of a fracture, for example, a fracture of the humerus, femur, or a fracture of the hip joint. Also, the movement restriction can be associated with replacement of a body part with an artificial part, for example, an orthosis, a partial or complete artificial knee joint, a partial or complete artificial hip joint, and a partial or complete shoulder joint. In certain embodiments, the substance is administered to an elderly subject having a hip fracture. Accordingly, the present invention relates to a substance that activates the GDF5 pathway for use in a method for the treatment or prevention of age-related muscle mass loss in a subject having partial or complete physical movement restriction. Particularly, the present invention relates to a substance that activates the GDF5 pathway for use in a method for the treatment or prevention of age-related muscle mass loss in a subject having a hip fracture.

[0045] Furthermore, the present invention can provide benefits to a subject suffering from progeria. Progeria, also known as Hutchinson-Gilford syndrome, is a very rare progressive genetic disorder that causes rapid aging in children and onset occurs at 2 years of age. Children with progeria appear genetically normal at birth. During the first year of life, signs and symptoms such as growth retardation and hair loss are seen. Also, a decrease in muscle mass is observed in children suffering from this disease. Therefore, the present invention can be beneficial to a subject suffering from progeria by at least reducing one of the symptoms of the disease. For this reason, the present invention relates to a substance that activates the GDF5 pathway for use in a method for treating or preventing a decrease in muscle mass associated with progeria, increasing or stabilizing muscle mass and / or muscle function, or increasing or stabilizing the physical ability or mobility of a subject suffering from progeria.

[0046] According to another embodiment, the subject is identified by determining the level of GDF5 in a biological sample of the subject. In certain embodiments, the biological sample is a biofluid sample, such as blood, plasma, serum, urine, or saliva. Accordingly, another aspect of the invention relates to a method for diagnosing sarcopenia in a subject, comprising the step of determining the level of GDF5 in a biological sample of the subject, such as a biofluid. In certain embodiments, the biofluid is blood, plasma, or serum. In a further particular embodiment, the level of GDF5 in the subject sample is compared to the level of GDF5 in a reference sample. The reference sample can be a sample from a young non-sarcopenic subject that has been processed in the same manner as the subject's sample. Also, the level of GDF5 in the reference sample can be a published predetermined criterion, such as a criterion obtained from the measurement of the average level of GDF5 in young non-sarcopenic subjects. For example, the reference sample can be a sample from a young subject (e.g., a subject 45 years old or younger, 40 years old or younger, 35 years old or younger, or 30 years old or younger). In this case, when the level of GDF5 in the test sample is lower than the level of GDF5 in the reference sample, sarcopenia can be suspected. The reference sample can correspond to an age and / or gender-specific subject. In certain embodiments, the subject is a human male, and the level of GDF5 in the reference sample is the level of GDF5 in a reference sample from a young human male subject. In another particular embodiment, the subject is a human female, and the level of GDF5 in the reference sample is the level of GDF5 in a reference sample from a young human female subject. For example, if the subject is a human male, the reference sample can be the level of GDF5 in a reference sample from a human male aged 30 - 40 years. In certain embodiments, if the subject is a human male, the reference sample level is the average value of the levels of GDF5 measured in non-sarcopenic human male subjects aged 30 - 40 years. In another embodiment, if the subject is a human female, the reference sample can be the level of GDF5 in a reference sample from a human female aged 40 - 50 years. In certain embodiments, if the subject is a human female, the reference sample level is the average value of the levels of GDF5 measured in non-sarcopenic human female subjects aged 40 - 50 years. For clarity, such average values are for a plurality of young human male subjects aged 30 - 40 years or 40 - 50 Obtained from levels measured in a plurality of young women of a certain age, and such values are averaged. In the definition of the reference level, attention should be paid to the state of the subject for obtaining the GDF5 level. For example, in the case of human female reference, it may be noted to exclude pregnant women.

[0047] Also disclosed herein is a pharmaceutical composition comprising a substance that activates the GDF5 pathway or the above-mentioned vector in a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise other additives, such as preservatives, buffers and / or solvents. Suitable carriers include, but are not limited to, water or physiological saline. Also, carrier proteins, such as serum albumin, may be included in the pharmaceutical composition. The finally formulated pharmaceutical composition prepared according to the present invention may be stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid or dehydrated or lyophilized powder. Such a formulation may be stored either in a form ready for use or in a form that requires reconstitution before administration, for example, in the case of a lyophilized powder. The above and further suitable pharmaceutical formulations are known in the art and are described, for example, in Gus Remington's Pharmaceutical Sciences (18th Ed., Mack Publishing Co., Eastern, Pa., 1990, pages 1435 - 1712). Such formulations may affect the physical state, stability, in vivo release rate and in vivo clearance rate of the pharmaceutically active compound.

[0048] Other effective administration forms include parenteral sustained release, i.e., delayed formulations, inhalation mists, orally active formulations. For example, the sustained release formulation may include particulate preparations of high molecular weight compounds (such as polylactic acid, polyglycolic acid, etc.) or proteins bound to or incorporated into liposomes.

[0049] Also, the pharmaceutical composition according to the present invention may be formulated for parenteral administration, for example, by infusion or injection, and may include sustained release or continuously circulating formulations.

[0050] The substance can be administered enterally or parenterally via various routes such as oral, rectal, nasal, intravascular (e.g., intravenous or intra - arterial), intramuscular and intraperitoneal, transdermal and subcutaneous routes. The pharmaceutical composition is adapted to the particular route of administration finally selected.

[0051] In certain embodiments, the substance is a recombinant protein, e.g., recombinant human GDF5, which is administered via the transdermal or intravascular route, more particularly, the intravenous route.

[0052] In certain aspects, the substance is contained in liposomes, nanoparticles (e.g., lipid - containing nanoparticles) or lipid - based carriers. In other aspects, the substance is contained in a skin patch so as to act for transdermal delivery. In another aspect, the substance is contained in an implantable device, e.g., a device for subcutaneous implantation. In certain embodiments, the implantable device includes a pump for delivering the substance gradually and / or continuously. Such an implantable device may include a refill system.

[0053] The substance is administered in a therapeutically effective amount, i.e., an amount that results in the recovery of at least one symptom of sarcopenia. The therapeutically effective amount can be readily determined by one of ordinary skill in the art based on the substance to be administered, the subject to be treated, the stage of sarcopenia, the route of administration, etc.

[0054] In certain embodiments, the substance is administered in a single dose. For example, the substance may be a gene therapy vector, e.g., a vector encoding GDF5 or CaVβ1 - E, which is administered in a single dose for the sustained expression of the encoded gene.

[0055] In another particular embodiment, the substance is administered periodically, e.g., monthly, especially weekly, or more particularly, daily. In addition, the substance can be administered once a day or several times a day. In a further particular embodiment, the substance is administered for the lifetime of the aging subject.

[0056] Also disclosed herein is a pharmaceutical composition comprising a substance that activates the GDF5 pathway and a pharmaceutically acceptable carrier.

[0057] Another aspect relates to a substance that activates the GDF5 pathway for use as a medicament.

[0058] The substance of the present invention can be used in a method for treating or preventing sarcopenia, i.e., for treating or preventing age-related muscle mass loss in a subject. Non-limiting benefits of the present invention can include an increase or stabilization of muscle mass and / or muscle function, an increase or stabilization of physical ability or mobility, a reduction in hospital stay, an increase in autonomy, prevention of the risk of death associated with sarcopenia, prevention of cancer mortality, and / or treatment or prevention of frailty in a subject.

[0059] In another aspect, the present invention also relates to a substance that activates the GDF5 pathway for use in a method for treating muscle weakness in a subject suffering from myopathy or neuromuscular disorder. Weakness is one of the main clinical symptoms of myopathy and neuromuscular diseases and strongly affects the daily life, prognosis, and outcome of affected patients. One of the main treatment goals in subjects suffering from such diseases is to fully recover muscle weakness. Various treatment options are possible and include physical therapy, electrotherapy, diet, drugs, avoidance or discontinuation of muscle toxins and weakness-inducing substances, detoxification, hepatocyte therapy, plasma exchange, respiratory therapy, or surgery. As a result of the present invention, muscle weakness can be treated or prevented by administration of a substance that activates the GDF5 pathway to a subject in need thereof. The substance that activates the GDF5 pathway can be administered alone or in combination with a treatment for myopathy or neuromuscular disease. Accordingly, the present invention also relates to a substance that activates the GDF5 pathway for use in combination with a treatment for myopathy or neuromuscular disease, for example, in combination with another pharmaceutically active substance suitable for the treatment of the above symptoms. As a result of the present invention, in the context of such treatment for myopathy or neuromuscular disease, a great benefit may be obtained, such as an increase or stabilization of muscle mass and / or muscle function, or an increase or stabilization of physical ability or mobility, thereby synergistically increasing the therapeutic efficiency of the above treatment for myopathy or neuromuscular disease. Further, the present invention further relates to a substance that activates the GDF5 pathway for use in increasing or stabilizing muscle mass and / or muscle function or increasing or stabilizing physical ability or mobility in a subject undergoing treatment for myopathy or neuromuscular disease, for example, a subject suffering from myopathy, such as centronuclear myopathy and dystrophinopathy (e.g., Duchenne muscular dystrophy or Becker muscular dystrophy) or neuromuscular disease, such as spinal muscular atrophy and amyotrophic lateral sclerosis. Preferably, the subject's motor neurons are intact or substantially intact.

[0060] Another aspect of the present invention relates to the treatment of muscle diseases mediated by motor neuron disorders. In fact, it is shown herein that activation of the GDF5 pathway results in activation of compensatory pathways in atrophic or hypoplastic muscles. Thus, it is possible to compensate for the lack or disorder of innervation by a substance that activates the GDF5 pathway. Muscle diseases that can be treated by the present invention include, but are not limited to, myopathies such as centronuclear myopathy and dystrophinopathies (e.g., Duchenne muscular dystrophy or Becker muscular dystrophy), neuromuscular diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis, and congenital and traumatic spinal cord injuries.

[0061] Although the present invention mainly focuses on the treatment of age-related decline in muscle mass and / or muscle function, other benefits of administering a substance that activates the GDF5 pathway can be envisioned based on the finding that the CaVβ1-E / GDF-5 system is involved in the homeostasis of muscle mass and muscle function. As described above, the substance can be advantageously administered to young progeria subjects. In addition, young subjects receiving treatment for symptoms selected from the above myopathies and neuromuscular diseases can benefit from treatment with a substance that activates the GDF5 pathway, either alone or in combination with another pharmaceutically active substance suitable for the treatment of the above symptoms.

[0062] In another aspect, a substance that activates the GDF5 pathway can also be administered to treat or prevent disuse atrophy in a subject in need thereof. In this aspect, the subject can be either young or old. For example, disuse atrophy can be the result or future result of partial or complete physical movement inhibition, such as movement inhibition of the limbs, such as movement inhibition of the arms, legs, shoulders, or hip joints. For example, movement inhibition can be the result of a fracture, such as a fracture of the arm bone, leg bone, or hip bone. Also, movement inhibition can be associated with the replacement of a body part with an artificial part, such as an orthosis, a partial or complete artificial knee joint, a partial or complete artificial hip joint, and a partial or complete shoulder joint. Also, movement inhibition can be the result when the subject is in a coma state. Thus, the present invention can be beneficial in that it can treat or prevent muscle loss observed during the onset of coma. For example, the present invention can be beneficial for a subject in a coma state for increasing or stabilizing muscle mass and / or muscle function, increasing or stabilizing physical ability or mobility, or preventing or treating frailty associated with coma.

[0063] In another specific aspect, the present invention relates to the use of a substance that activates the GDF5 pathway for non-therapeutically increasing muscle mass and / or muscle strength. For example, such an increase in muscle mass and / or muscle strength can be desired in the context of sports practice or exercise. In this embodiment, the subject can be a young subject, such as a teenager or young adult, such as between 11 and 50 years old, particularly between 15 and 40 years old, such as between 18 and 30 years old.

[0064] Of course, the present invention may also have applications and benefits in the veterinary field. In particular, any of the above embodiments may be practiced in non-human mammals, such as pets and livestock. In particular, a substance that activates the GDF5 pathway can be administered to a pet for treating or preventing sarcopenia in the pet or livestock, particularly in a pet. In particular, a substance that activates the GDF5 pathway can be used in the pet to increase or stabilize muscle mass and / or muscle function, increase or stabilize physical ability or mobility, reduce the length of hospital stay in a veterinary hospital, increase autonomy, prevent the risk of death associated with sarcopenia, prevent cancer mortality, and / or treat or prevent frailty. According to certain embodiments, a pet includes, but is not limited to, any non-human mammal living with humans. Such pets include, but are not limited to, cats, dogs, rabbits, mice, hamsters, guinea pigs, ferrets, horses, and pigs. In certain embodiments, the pet is a cat or a dog.

[0065] In another aspect, the present invention relates to a non-therapeutic method for increasing muscle mass and / or muscle function in a non-human animal, particularly a livestock, comprising the step of administering to the animal an amount of a substance that activates the GDF5 pathway effective to induce and increase muscle mass and / or muscle function. In the context of the present invention, livestock are domesticated animals raised in an agricultural environment to provide labor and / or commodities such as meat, eggs, milk, fur, leather, and wool. The term includes, but is not limited to, cows, pigs, sheep, goats, and horses.

[0066] Of course, in all of the above non-human mammalian embodiments, the substance that specifically activates the GDF5 pathway to be used is selected according to the specific non-human mammal that ingests the substance. For example, when the substance is a GDF5 peptide, a CaVβ1-E protein, or a vector encoding a GDF5 peptide or a CaVβ1-E protein, it may be more suitable to use a peptide, a protein, or a vector encoding a peptide or a protein derived from the above human mammal. By way of illustration, the GDF5 peptides, CaVβ1-E proteins, or vectors encoding the above of cats, dogs, rabbits, rats, mice, hamsters, guinea pigs, ferrets, horses, pigs, cows, sheep and goats may preferably be used respectively in the subjects of cats, dogs, rabbits, rats, mice, hamsters, guinea pigs, ferrets, horses, pigs, cows, sheep and goats.

Example

[0067] (Example 1) Materials and methods Generation of plasmids and AAVs. AAV-sh CaVβ1-Ex2 (sh CaVβ1-E) (Individual: TRC mouse Cacnb1 shRNA clone Id: TRCN000006951, Dharmacon) was generated by cloning pALK0.1shCaVβ1-Ex2 by PCR insertion of BglII and HindIII sites under the control of the H1 promoter in pSUPER. Subsequently, the H1 cassette was introduced between the two ITRs in an AAV1-based vector using the BamHI and SalI sites of the pSMD2-sh AAV2 vector backbone (Vassillopoulos et al., J. Cell Biol. 205, 377-393 (2014)). pSUPER retro puro Scr shRNA (SCRA) was a gift from John Gurdon (Addgene plasmid # 30520) (Pasque et al., EMBO J. 30, 2373-2387 (2011)). The BamHI site was inserted by PCR, and the H1-SCRA cassette was cloned into pSMD2-sh using BamHI and SalI sites. AAV2 / 1 pseudotyped vectors were prepared by transfection in 293 cells as previously described by the AAV production facility of the Center of Research in Myology (Riviere et al., Gene Ther. 13, 1300-1308 (2006)).

[0068] AAV-CaVβ1-E was generated by directly cloning the Cacnb-E ORF (NM_001282977) adjacent to the EcoRI and NheI sites under the CMV promoter in the pSMD2 AAV2 vector (GeneArt string; ThermoFisher). The final virus preparation was maintained at -80 °C in PBS solution. The particle titer (number of viral genomes) was determined by quantitative PCR. All AAV2 / 1 were used at a final titer of 1×10 12 vector genomes (vg) / TA.

[0069] In addition, shCacnb-Ex2 and sh-SCRA were cloned into pCDNA3, and a luciferase assay was performed.

[0070] The Gdf5 promoter region was designed by obtaining the sequence from -312 to the Gdf5 TSS using the public domain http: / / epd.vital-it.ch. This sequence adjacent to the EcoRI and NheI sites was synthesized (GeneArt string; ThermoFisher), and the upstream firefly luciferase gene was cloned into the HSVTK-Luc3' modified plasmid to perform a luciferase assay.

[0071] Gene transfer in vivo The experiments were performed on adult C57 / BL6 mice at 6 - 8 or 78 - 80 weeks of age. Anesthesia was performed using isoflurane, and analgesia was provided by buprenorphine (vetergesic). Intramuscular injection (40 μl / TA) was performed once in both TA muscles. As a control, C57 / BL6 mice at 6 - 8 weeks or 78 - 80 weeks of age were injected using the same procedure as the SCRA AAV vector. The mice were sacrificed 3 months after injection.

[0072] Denervation experiment Ten weeks after injection of AAV or the control into the mice, under general anesthesia (isoflurane), the sciatic nerve was denervated (resection of a 5-mm sciatic nerve segment). The mice were sacrificed 2 weeks after denervation. After the TA muscles were dissected and weighed, they were frozen with pre-cooled isopentane in liquid nitrogen and stored at -80°C until histological examination or molecular analysis.

[0073] Gene expression analysis Total RNA was prepared from 600-μm tibialis anterior (TA) muscle cryosections using TRIzol (Life Technologies) according to the manufacturer's instructions. Complementary DNA was generated using Invitrogen Superscript II reverse transcriptase (Invitrogen), and analyzed by performing real-time qPCR using Power SyberGreen PCR MasterMix (Applied Biosystems) on a StepOne Plus Real-Time PCR System (Applied Biosystems). All data were normalized to the PO expression level. The primers used are listed in the following table.

[0074]

Table 1A

[0075]

Table 1B

[0076] Antibodies for immunoblotting The following antibodies were used, obtained from Cell Signaling Technology: rabbit polyclonal antibody against phosphorylated Smad1 / 5 (Ser463 / 465) / Smad8 (Ser426 / 438); rabbit monoclonal antibody against phosphorylated Smad3 (Ser423 / 425), mouse polyclonal antibody against Smad4, mouse monoclonal antibody against Cav3. A rabbit polyclonal antibody against the Cavβ1 C-terminus (AP16144b) was purchased from AbGent, while a rabbit polyclonal antibody against the Cavβ1 internal region (currently unavailable: sc-25689) and a luminescent mouse monoclonal antibody against GDF5 were obtained from Santa Cruz Biotechnologies. A mouse monoclonal antibody against actin (A4700) was purchased from Sigma. A rat polyclonal AchR antibody was obtained from Covance; a mouse anti-actin EA53 antibody was obtained from Sigma.

[0077] Immunoblotting Frozen TA muscle or frozen sections derived from liquid nitrogen-frozen human muscle biopsies were homogenized in a lysis buffer containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5% NP40, and a Halt protease and phosphatase inhibitor cocktail (Pierce) using a Dounce-type homogenizer. The samples were then centrifuged at 5000 g for 5 minutes and denatured with Laemmli buffer for 30 minutes at room temperature. Protein concentration was determined by Bradford assay (Pierce). Proteins were separated by electrophoresis (Nu-PAGE 4-12% Bis-Tris gel; Life Technologies), transferred to a nitrocellulose membrane (GE Healthcare), and labeled with a primary antibody and a secondary antibody conjugated to horseradish peroxidase. Signals were visualized with SuperSignal West Pico Chemiluminescent substrate (Pierce). Images were acquired with a LAS4000 camera (GE Healthcare). Western blot image analysis was performed using the public domain software Fiji ImageJ (gel analysis tool) (Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nature Methods (2012). doi:10.1038 / nmeth.2089). Blots were stripped using Restore Western Blotting Stripping Buffer (Thermo) according to the manufacturer's instructions and reprobed as needed.

[0078] Immunolabeling experiment In the immunolabeling procedure, 10-μm tissue sections were generated on a cryostat (Leica), fixed on glass slides, and stored at -80°C. The slides were rehydrated in phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich), and blocked in PBS / 4% bovine serum albumin / 0.1% Triton X-100 for 1 hour. The sections were incubated overnight at 4°C with the primary antibody in PBS / 2% BSA / 0.1% Triton X-100, washed with PBS, incubated with the secondary antibody for 1 hour, thoroughly washed with PBS, incubated with 4',6'-diamidino-2-phenylindole for 5 minutes for nuclear staining, and mounted with Fluoromount (Southern Biotech). Images were acquired with a Leica SPE confocal microscope.

[0079] Cell culture, transfection, and luciferase assay The C2C12 myoblast cell line was purchased from ATCC and cultured in IMDM (Gibco-Life Technologies) supplemented with 15% FBS and 1% penicillin-streptomycin mixture at 37°C and 5% CO2 until the cells reached confluence. Differentiation was induced by changing the medium to IMDM supplemented with 2% HS and 1% penicillin-streptomycin mixture. Cells were transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. The cell lines used in the experiments were verified and tested for mycoplasma contamination. Gdf5 promoter-luciferase was co-transfected into C2C12 cells using Lipofectamine 2000 diluted in Optimem reduced serum medium with either 25 pg of pCDNA3-sh CaVβ1-E or pCDNA3-sh Scra alone. Plasmid CMV Renilla luciferase (0.25 ng) was also transfected as a reference for each condition.

[0080] Five hours after transfection, the Optimem medium was replaced with IMDM supplemented with 2% HS. Cells were analyzed at 24 and 48 hours after medium replacement.

[0081] The luminescence of firefly and Renilla luciferase was quantified on a Flexstation3 microplate reader (according to the manufacturer's instructions) using the Dual-Glo Luciferase Assay System. Firefly luciferase activity was normalized to Renilla luciferase activity.

[0082] Results Embryonic CaVβ1-E expression is controlled in adult muscle by innervation An appropriate model for measuring the skeletal muscle response to changes in activity is sciatic nerve resection. In this model, the inventors probed for Cacnb1 mRNA with primers for exons 2 and 3. The inventors observed a time-dependent increase in amplification of this region in the denervated tibialis anterior (TA) muscle (Figure 1A). Western blot of CaVβ1 protein using an antibody that recognizes CaVβ1 central peptide 18 under the same conditions revealed the appearance of an additional 70 kDa band that increased over time after denervation. In contrast, the intensity of the band at 53 kDa, the molecular weight of the expected muscle-specific CaVβ1A (or isoform 1 of Cacnb1 NM_031173; NP_112450), remained unchanged (Figure 1B).

[0083] The inventors speculated that the 70 kDa band could be a longer CaVβ1 isoform not previously described for muscle. The Cacnb1 gene (GSMG0007319) has 14 exons, which could give rise to 6 alternatively spliced transcript variants (NM_031173; NM_145121; NM_001159319; NM_001159320; NM_001282977; NM_001282978) (Figure S1H).

[0084] To identify potential splicing events of Cacnb1 occurring in neuromuscular tissues, the inventors performed whole-genome transcriptome analysis at the exon level on RNA extracted from innervated or denervated mouse TA muscles. The inventors found 1,022 differentially regulated alternative splicing events (from 706 different genes), and this redistribution showed a predominance of the first exon splicing event. Among these, Cacnb1 showed first exon splicing, and this transcript initiated in the putative non-coding sequence 5' of exon 3 in innervated muscle samples. In denervated muscle samples, it was found that another transcript starting at exon 1 was upregulated in addition to the first Cacnb1 mRNA (Figure 1C and Figure S2), which means the transcription of two different splicing isoforms. By RT-PCR, the Cacnb1 open reading frame (ORF) in innervated TA muscle was present 5' of exon 3 (ATG1), while two Cacnb1 transcripts in denervated muscle were expressed, one starting at the level of exon 1 (ATG2) and the other starting at the level of ATG1, which was confirmed (Figure 1D). By searching the mRNA sequence of Cacnb1 starting from exon 3 immediately upstream in the Blast or NCBI database, the inventors concluded that the specific CaVβ1 isoform expressed in adult mouse skeletal muscle was CaVβ1D (NP_001152792.1) (Figure S1H, Figure S2A, Figure S2B). On the other hand, the size of the additional CaVβ1 band appearing in the protein extract from denervated muscle was reliable by the translation of Cacnb1-E (or isoform 5, NM_001282977; NP_001269906). To confirm this hypothesis, the inventors designed specific primers that match at the level of exon 13 for Cacnb1-D and at the level of exon 14 for Cacnb1-E at the 3' of the two sequences (Figure S2A, the primer regions are underlined), and the inventors confirmed by RT-PCR that only the Cacnb1-E transcript increased in adult denervated muscle, consistent with the protein expression data (Figure 1E).

[0085] Denervation induces several embryonic proteins, such as troponin T, myosin, and acetylcholine receptor subunits. The inventors suspected whether this applied to CaVβ1-E. By real-time PCR and qPCR, it was revealed that Cacnb1-E is a variant specific to embryonic and neonatal muscle (E12.5, E16, P0) (Figure S1A, Figure S1B, Figure S1C), has an ORF in exon 1 (Figure S1D, Figure S1E), and is identical to Cacnb1-E expressed in adult muscle after denervation.

[0086] The Cacnb1-B variant, shown to be specific to nerve terminals, shares the same 3'-terminal sequence as Cacnb1-E (Figure S2C). Amplification of the region between exons 5 and 9 showed abundant mRNA expression of Cacnb1-E (381bp) in embryonic and neonatal muscle, and almost undetectable expression of Cacnb1-B (246bp) only in E12.5 muscle (presumably due to the presence of mixed progenitor cells at this embryonic stage) (Figure S1I). Moreover, probing the region between exon 7A and exon 14, excluding the Cacnb1-B and Cacnb1-C variants, the inventors confirmed that Cacnb1-E is the only CaVβ1 isoform upregulated after denervation (Figure S1J). As further confirmation, Western blot was performed using an antibody specific to mouse CaVβ1E and embryonic muscle protein extracts, staining only denervated adult mice (Figure S1G). Immunofluorescence of nerve-innervated and denervated muscle slides and isolated fibers with either CaVβ1 (central peptide) or CaVβ1E antibody showed that the CaVβ1 staining intensity and triple localization rather reflected the main expression of CaVβ1D. Indeed, the specific CaVβ1-E staining that appeared increased in denervated fibers and slides, and most was distributed at the level of the Z-line and localized to the nucleus, consistent with the expression of the NLS predicted by the free software cNLM mapper (http: / / nlsmapper.iab.keio.ac.jp / cgibin / NLS_Mapper_form.cgi).

[0087] Overall, such data demonstrate that skeletal muscle expresses various innervation-dependent CaVβ1 isoforms. Alternative first exon splicing occurs at the onset of differential expression of adult and embryonic Cacnb1 variants. CaVβ1D, rather than CaVβ1A, is the isoform expressed in innervated adult skeletal muscle, while only CaVβ1E is expressed in embryonic muscle. In addition, lack of innervation specifically induces the expression of CaVβ1E, which is almost undetectable in innervated adult muscle. Moreover, CaVβ1D and CaVβ1E exhibit various intracellular localizations in adult skeletal muscle fibers.

[0088] CaVβ1-E is required for the activation of GDF5 signaling after denervation To understand whether CaVβ1-E may have a role in disuse atrophy, the inventors generated a tool to ablate CaVβ1-E expression by targeting a specific sequence in the Cacnb1 exon (shCaVβ1 Ex2). The shCaVβ1-E construct (AAV-shCaVβ1-E) transported by the AAV2 / 1 vector was injected into the mouse TA muscle.

[0089] CaVβ1-E expression induced after denervation disappeared 2 months after AAV-shCaVβ1-E injection (FIGS. 2A, 2C, 2D, 2E). No decrease in CaVβ1D expression was observed (FIGS. 2C, 2D, 2E). The lack of induction of CaVβ1-E suggested a protective role for this protein in increasing atrophy and preventing muscle mass loss from disuse after denervation (FIG. 2F).

[0090] Among the molecular pathways involved in muscle mass homeostasis, GDF5 signaling has been shown to be essential for limiting muscle loss in atrophic conditions (Sartori Op. cit).

[0091] The absence of CaVβ1-E significantly affected the increase in Gdf5 after denervation (Figure 2G), suggesting a positive control of CaVβ1-E on the GDF5 pathway. Indeed, the phosphorylation of Smad 1 / 5 / 8, the nuclear translocation of Smad 4, and the transcription of Id-1 were inhibited under CaVβ1-E knockdown conditions (Figure 2H, Figure 2I).

[0092] As CaVβ1 has been described as a transcription factor in muscle progenitor cells (Taylor et al. J. Cell Biol. 205, 829 - 846 (2014)), the inventors asked whether CaVβ1E could have transcriptional activity on GDF5 expression. For this purpose, the inventors used C2C12 cells. First, the inventors confirmed whether such cells express CaVβ1E during differentiation. The inventors' data showed that Cacnb1-E is expressed and increased during differentiation in C2C12 (Figure S3A), and that CaVβ1E is the main isoform expressed in this myoblast cell line (Figure S3B). Moreover, the expression of Gdf5 was also increased in differentiating C2C12. Consistently, inhibition of Cacnb1-E expression by transfection of a plasmid transporting shCaVβ1-E (pCDNA3-shCaVβ1-E) (Figure S3D) blocked the expression of Gdf5 in differentiating C2C12 (Figure S4C), mimicking this effect in vivo. Such data confirmed C2C12 cells as an appropriate in vitro tool for measuring CaVβ1-E transcriptional activity.

[0093] Previous studies have shown that several standard and non-standard DNA E-Box sequences (CANNTG and CANNNTG) in the promoter regions (Taylor et al., J. Cell Biol. 205, 829-846 (2014)) can be targeted by CaVβ1. Therefore, the sequence from -312 to the Gdf5 TSS containing two CANNNTG E-Boxes and one CANNTG E-Box was cloned into the firefly luciferase upstream of the HSVTK-Luc3' modified plasmid and transfected into C2C12 cells (Figure 2J). The firefly / sea pansy signal increased during cell differentiation, which reflects the activation of the Gdf5 promoter, and disappeared due to the downregulation of CaVβ1-E induced by co-transfection with the shCaVβ1-E plasmid. Such data strongly suggest that CaVβ1-E can target the Gdf5 promoter, and it is confirmed that this effect was observed in vivo.

[0094] Aging muscle: The key role of CaVβ1-E Surgical resection of the sciatic nerve mimics a very severe condition, and nerve withdrawal induces molecular pathways that avoid complete muscle loss. However, the inventors questioned the role of CaVβ1 in the physiological processes when the compensatory response to muscular atrophy is impaired. This scenario is represented by sarcopenia.

[0095] Aging is a complex physiological state that involves many tissues and organs. Aging skeletal muscle exhibits denervation-like symptoms, becomes sarcopenic, and gradually loses its ability to counteract muscle mass loss. In aging muscle, there is some debate about CaVβ1 expression and function (Taylor et al. Aging Cell 8, 584-594 (2009)). In the TA of C57b1 / 6 mice, muscle loss becomes significant at approximately 78 weeks of age and highly significant at 92 weeks of age (26.3 ± 8.9% and 38.7 ± 6.4% of sarcopenia, respectively, compared to 12-week-old mice) (Figure 3A). The inventors observed a slight decrease in Cacnb1-D in the TA of 95-100 weeks of age compared to adult TA muscle of 12 weeks of age (Figures 3C and S4A), but in contrast, a significant decrease in the basal level of Cacnb1-E was observed (Figures 3B, S4B). Nothing is known about Gdf5 levels in aged muscle.

[0096] In addition, the inventors measured CaVβ1-E expression in innervated and denervated TA muscle during aging. The inventors found that since 52 weeks of age, the response of CaVβ1-E to denervation did not increase, the increase in Gdf5 was attenuated (Figures 3D, 3E, 3F, 3G), and it affected the phosphorylation of Smad 1 / 5 / 8 (Figures S4C, S4D). No significant change in CaVβ1D expression was measured (Figures 3H, 3I).

[0097] The inventors wondered whether this mechanism was conserved in humans as well. Only three human CACNB1 variants have been identified to date (NM_000723.4, NM_199247.2, NM_199248.2), corresponding to mouse isoforms A, B, and C (Figure S1H). The inventors probed exons 5-9, exon 13, and exon 14 of human CACNB1 mRNA extracted from the muscles of healthy subjects aged 30-89 years. Amplification of the region between exon 5 and exon 9 showed that all muscles expressed the expected CACNB-A (380 bp) variant and an unexpected CACNB-B (245 bp) variant, which was confirmed by amplification of a specific region in exon 13. Surprisingly, amplification of a specific region in exon 14 revealed that human muscles expressed a novel, unidentified variant that the inventors named CACNB1-E, and that this transcript was strongly downregulated in muscles over 75 years old (aged) (Figure 3J). The inventors measured the GDF5 transcript in the same samples and found a highly significant decrease in this level in aged muscles (Figure 3K). Such results demonstrate that human CACNB1-E has actually been elucidated and strongly suggest that the CaVβ1-GDF5 system is conserved in humans as well.

[0098] To understand whether CaVβ1-E could improve sarcopenia, the inventors highly expressed CaVβ1-E by injecting AAV-CaVβ1-E into the TA of 78- to 80-week-old mice. High expression of CaVβ1-E was very efficient 3 months later (Figure 4A) without affecting the transcription of Cacnb1-D (Figure 4B). High expression of CaVβ1-E rescued the transcription of Gdf5 (Figure 5C) and the GDF-5 signaling measured by phosphorylation of Smad 1 / 5 / 8, nuclear translocation of Smad 4, and transcription of Id-1 (Figures 4D, 4E, 4F).

[0099] The rescue of GDF5 signaling by CaVβ1-E was evidenced by a marked preservation of the amount of aged skeletal muscle (Figure 4G) and an improvement in specific force compared to scramble (Figure 4H). Overall, such data demonstrated that CaVβ1-E plays a highly essential role in maintaining muscle mass in age-related sarcopenia.

[0100] Conclusion Proteins and mechanisms that link the sensing of skeletal muscle activity to translation into gene expression have not been found to date.

[0101] Here, the inventors showed a molecular pathway triggered by a voltage sensor subunit that sustains muscle flexibility and is strictly required for muscle maintenance, independent of E-C coupling. Such evidence suggests that CaVβ1-E-dependent signaling may also be central in neuromuscular disorders and could uncover potential therapeutic targets that preserve muscle and nerve degeneration in such pathologies.

[0102] Most importantly, the inventors established that CaVβ1-E is essential for activating the Gdf5 promoter. In adult skeletal muscle, the expression of CaVβ1-E rather than CaVβ1D is required to sustain the GDF5 pathway. Importantly, the inventors questioned GDF5 signaling and the protein CaVβ1-E that elicits it when the ability of skeletal muscle to maintain skeletal muscle mass is lost, as occurs in age-related sarcopenia. To date, the correlation between progressive muscle loss and GDF5 pathway deficiency has not been reported. Here, the inventors show that the levels of Gdf5 are decreased in age-related sarcopenia. Overall, the inventors' data indicate that the GDF5 pathway plays a key role during this process. Thus, the inventors propose a potent therapeutic strategy based on the administration of GDF5 to subjects suffering from a decrease in muscle mass and / or muscle function, in either the context of sarcopenia or disuse atrophy.

[0103] Finally, but most importantly, CaVβ1-E may be a critical component of the pathway that results in muscle reinnervation after reversible nerve injury. The significance of CaVβ1-E should be a central factor mediating the restoration of nerve connection as a result of crosstalk with GDF5 signaling.

[0104] (Example 2) Human muscle: A new CaVβ1 isoform associated with aging of skeletal muscle Given the apparent importance of CaVβ1-E in mouse skeletal muscle, the inventors wondered whether a similar mechanism might be conserved in humans. To date, only three human CACNB1 variants have been identified, corresponding to mouse isoforms A, B, and C (Figure 5A). Human mRNA extracted from biopsies of one quadriceps and two vastus lateralis muscles of healthy adult subjects was probed for exons 13 and 5-9, together with human mRNA extracted from cervical spinal cord as a positive control for hCACNB1-B (Table 1).

[0105] [Table 2]

[0106] Amplification of the sequences in exon 13 showed that both muscle types expressed hCACNB1-A and / or hCACNB1-C. As in mouse muscle, amplification of the region between exons 5 and 9 demonstrated that hCACNB1-B (245 bp) was expressed only in human spinal cord (SC), but not in muscle, where only 380 bp corresponding to hCACNB1-A or hCACNB1-E appeared. Moreover, the expression of hCACNB1-C was excluded in muscle because the amplified sequence was 245 bp (Figure 5B). Amplification of the region in exon 14 revealed that human muscle expressed a previously uncharacterized variant, hCACNB1-E (Figure 5C). This isoform corresponded to the predicted XM_006722072.2 variant and had an initiation codon (ATG2) upstream of exon 3 (Figure 5A, Figure 5C). This expression was confirmed by Western blot experiments in two different human vastus lateralis muscle biopsies (Figure 5D).

[0107] Since the inventors found that changes in the CaVβ1-E / GDF5 system were associated with muscle loss during aging in mice, the inventors compared muscle characteristics indicating a decrease in muscle mass (percentage of fat-free mass) and function (strength) in cohorts of healthy young (20 - 42 years) and elderly (70 - 81 years) volunteers and included them in previous tests (Table 2 (Table 3)).

[0108]

Table 3

[0109] Parameters were examined in young (Y) participants aged 20 - 42 years and elderly (O) participants aged 70 - 81 years, with gender being male (M) and female (F). Fat-free mass (%) was evaluated by dual-energy X-ray absorptiometry. The force exerted on a force platform was expressed as watts per kilogram (W / kg), and ND represents undetermined.

[0110] The inventors found that the elderly group had significantly lower lean muscle mass and strength than the young group (Figure 6A). The inventors then measured the hCACNB1-E transcript and found a significant decrease in this expression in the elderly group, while the hCACNB1-A level did not change between the groups (Figure 6B). Significantly lower hCACNB1-E expression was also associated with a lower percentage of lean muscle mass (Figure 6C). hGDF5 was detected at very low levels in muscle biopsies. However, in routine muscle samples, the inventors were able to associate low expression of both hCACNB1-E and hGDF5 with a lower percentage of lean muscle mass. In addition, participants with a higher percentage of lean muscle mass exhibited high levels of both hCACNB1-E and hGDF5 (Figure 6D).

[0111] Overall, the inventors discovered CaVβ1-E as a new factor in the GDF5-regulated compensatory response that counteracts muscle loss in young and old muscle. In particular, the results obtained in this study indicate that the association between age-related muscle loss and the CaVβ1-E / GDF5 system is conserved between mice and humans, strongly suggesting an essential role for this protein in maintaining mammalian muscle mass.

[0112] Systemic administration of rGdf5 counteracts age-related sarcopenia Recombinant mouse (Rm) Gdf5 was diluted to 0.2 mg / kg in 0.1% PBS / BSA and administered intraperitoneally (I.P.) twice a week for 10 weeks to four C57b1 / 6 mice (90 weeks old). As a control, only 0.1% PBS / BSA was injected. Body composition measurements (RMN) and grip strength tests were performed every two weeks (Figure 7A).

[0113] Analysis of the grip strength test showed no differences between the groups, probably due to a decrease in the sensitivity of this assay. The percentages of lean muscle mass and fat mass did not change in mice treated with the vehicle (Figure 7B). However, the introduction of RmGdf5 induced a significant increase in the percentage of lean muscle mass and a decrease in the percentage of fat mass (Figure 7C).

[0114] The muscle / body weight ratios of TA and QUAD were increased in 100-week-old C57B1 / 6 mice treated with Rm-GDF5 (Gdf5) for 10 weeks compared to untreated or vehicle-treated 92-week-old CD57B1 / 6 mice (Figure 7D), suggesting that introduction of Rm-Gdf5 results in a significant increase in muscle mass. Collectively, such data suggest that systemic treatment with Rm-Gdf5 effectively counteracts sarcopenia.

Claims

**Claim 1** A pharmaceutical composition for the treatment or prevention of sarcopenia or disuse atrophy, comprising a substance that activates the GDF5 pathway selected from a synthetic or recombinant GDF5 protein, a functional GDF5 peptide having at least 90% sequence identity with reference human GDF5 having the amino acid sequence shown in SEQ ID NO: 2 or 3, a vector encoding GDF5, a recombinant CaVβ1-E protein, and a vector encoding human CaVβ1-E. **Claim 2** A pharmaceutical composition for the treatment or prevention of muscle weakness in myopathy or neuromuscular disease, comprising a substance that activates the GDF5 pathway selected from a synthetic or recombinant GDF5 protein, a functional GDF5 peptide having at least 90% sequence identity with reference human GDF5 having the amino acid sequence shown in SEQ ID NO: 2 or 3, a vector encoding GDF5, a recombinant CaVβ1-E protein, and a vector encoding human CaVβ1-E, and used alone or in combination with the treatment of myopathy or neuromuscular disease. **Claim 3** The pharmaceutical composition according to claim 1 or 2, wherein the substance is selected from a compound that increases the activity of GDF5 or a compound that increases the expression of GDF5. **Claim 4** The pharmaceutical composition according to any one of claims 1 to 3, wherein the substance is recombinant human GDF5. **Claim 5** The pharmaceutical composition according to any one of claims 1 to 3, wherein the substance is recombinant human CaVβ1-E or a vector encoding human CaVβ1-E. **Claim 6** The pharmaceutical composition according to any one of claims 1 to 5, which is administered to a subject aged 50 years or older, 55 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, or 80 years or older. **Claim 7** The pharmaceutical composition according to any one of claims 1 to 6, which is administered by oral, nasal, intravascular, intramuscular, intraperitoneal, transdermal or subcutaneous routes. **Claim 8** The pharmaceutical composition according to any one of claims 1 to 7, which is administered periodically, or monthly, or weekly, or daily. **Claim 9** The pharmaceutical composition according to any one of claims 1 to 8, wherein treatment of sarcopenia results in an increase in muscle mass and / or muscle function, an increase in physical ability or mobility, and / or an increase in muscle strength. **Claim 10** A pharmaceutical composition for the treatment or prevention of sarcopenia or disuse atrophy, or for the treatment or prevention of muscle weakness in myopathy or neuromuscular disease, which comprises a substance that activates the GDF5 pathway selected from a synthetic or recombinant GDF5 protein, a functional GDF5 peptide having at least 90% sequence identity with the reference human GDF5 having the amino acid sequence shown in SEQ ID NO: 2 or 3, a vector encoding GDF5, a recombinant CaVβ1-E protein, and a vector encoding human CaVβ1-E, and a pharmaceutically acceptable carrier, and is used alone or in combination with the treatment of myopathy or neuromuscular disease.

11. The pharmaceutical composition according to claim 10, wherein the substance is synthetic GDF5 or recombinant human GDF5.

12. Use of a substance that activates the GDF5 pathway in the manufacture of a medicament for the treatment or prevention of sarcopenia or disuse atrophy, or for the treatment or prevention of muscle weakness in myopathy or neuromuscular disease, which is used alone or in combination with the treatment of myopathy or neuromuscular disease, wherein the substance is selected from a synthetic or recombinant GDF5 protein, a functional GDF5 peptide having at least 90% sequence identity with the reference human GDF5 having the amino acid sequence shown in SEQ ID NO: 2 or 3, a vector encoding GDF5, a recombinant CaVβ1-E protein, and a vector encoding human CaVβ1-E.

13. Use of the substance that activates the GDF5 pathway according to claim 12, wherein the substance is synthetic GDF5 or recombinant human GDF5.

14. A method for providing data for the diagnosis of sarcopenia in a subject, which comprises the step of determining the level of GDF5 in a biological sample of the subject.

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