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Definitions

• the invention relates to a new pharmacological and/or genetic activator for its use to preserve and regenerate muscle structure and function by blocking senescence, in particular to prevent and/or treat myopathies, preferentially Duchenne muscular dystrophy (DMD) , as well as sarcopenia.
• the invention also relates to a novel adenoviral vector for its use as a medicament.
• Muscular dystrophy is a hereditary neuromuscular disorder of a progressive nature, resulting from the mutation of one or more genes intended to ensure muscular functions and structures.
• Duchenne muscular dystrophy is the most common myopathy in children. It is due to abnormalities of a protein essential to the muscles, dystrophin. It is characterized by a decline in muscle regeneration contributing to the loss of muscle mass.
• Duchenne muscular dystrophy is caused by a recessive genetic defect in the DMD gene on the X chromosome, resulting in the absence of dystrophin.
• Dystrophin is a large protein that provides the link between the contractile cytoskeleton and the extracellular matrix, through a protein complex called the dystrophin membrane complex, and plays a major role in maintaining the integrity of muscle fibers during contraction.
• Dystrophin is a protein made up of 3685 amino acids for a molecular weight of 427 kDa. It is encoded by the DMD gene which allows the synthesis of 7 main isoforms, of different sizes, thanks to the presence of 7 tissue-specific promoters.
• M, B and P promoters indicating expression in the tissues "Muscle (Muscle), “Brain (Brain)” and “Purkinje neurons of the cerebellar cortex (Purkinje cerebellar neurons)" are located upstream of the first exon and allow the synthesis of functionally equivalent full-length dystrophins.
• the other four promoters are intra-genic and allow the synthesis of isoforms of reduced size.
• Dystrophin is part of the family of filamentous proteins of the "spectrin” type which are present in skeletal and cardiac muscle tissues, the brain, the retina, glial and Purkinje cells and in very reduced quantities in the lymphocytes.
• Dystrophin is composed of four main domains which interact with several partners of the muscle cell and play an essential role during the muscle contraction-relaxation cycle: the N-terminal domain or actin binding domain (Actin Binding Domain or ABD1 ), comprising the first 246 amino acids; the central domain (or rod domain) ranging from amino acid 247 to 3045.
• ABD1 actin binding domain
• spectrin consists of 24 homologous spectrin repeats (or spectrin like, RI to R24) and 4 proline-rich hinges (or hinges, HI to H4) dividing the central domain into 3 sub-domains; the domain rich in cysteine (cysteine rich repeats or CR) ranging from amino acid 3080 to 3360; and the C-terminal domain formed from the last 325 residues.
• Gene therapies aim to obtain functional dystrophin production in the muscle.
• a first clinical trial started recently in France. It is evaluating a gene therapy product combining a shortened version of the DMD gene and an adeno-associated viral vector AAV.
• Pharmacological therapies act on certain manifestations of the disease to slow its progression.
• Several drugs are being studied to improve and/or protect muscle and/or heart function, fight against inflammation and the effects of the absence of dystrophin on muscle cells: tamoxifen, nebivolol, givinostat, rimeporide, etc.
• Patent document WO2018155913 relates to a composition comprising chemical compounds selected from pan-histone deacetylase inhibitors, ALK5 inhibitors (activin A receptor-like kinase 5 inhibitor) and activin signaling activators.
• cAMP for inducing the differentiation of somatic cells, mesenchymal stem cells or adult stem cells (excluding muscle stem cells) into skeletal muscle cells for the treatment of skeletal muscle diseases. It uses a method in which cells are cultured in a culture medium containing the composition inducing differentiation into skeletal muscle cells.
• the skeletal muscle cells thus obtained present no risk of oncogenesis after implantation due to differentiation from somatic cells, and have the advantage of being genetically stable due to the use of a mixture of compounds of low molecular weight alone, without the introduction of genetics.
• a problem which the present invention proposes to solve consists in developing a new therapy for preserving and regenerating the structure and the muscular function and in particular treating Duchenne muscular dystrophy.
• the solution to this problem has as its first object a pharmacological and/or genetic activator of the thyroid-stimulating hormone receptor (TSHR) signaling pathway in muscle stem cells (satellite cells) for its use in preserving and regenerating the structure and the muscle function by blocking senescence.
• TSHR thyroid-stimulating hormone receptor
• TSHR signaling pharmacologically and/or genetically preserves the proliferation and differentiation capacities of muscle stem cells by blocking senescence.
• the activation of TSHR signaling in muscle stem cells increases the regenerative properties of muscles affected in particular by Duchenne muscular dystrophy and preserves muscle structure and function, by preventing the rapid muscle wasting that occurs with the progression of the disease. pathology.
• a subject of the invention is also an adenoviral vector characterized in that it comprises a protein sequence of the thyroid stimulating hormone receptor (TSHR) of SEQ ID NO: 1 or a functional variant having a protein sequence which is at least 60% identical to the sequence SEQ ID NO:1, or a nucleotide sequence of the TSHR of SEQ ID NO:2 or a functional variant having a nucleotide sequence that is at least 60% identical to the sequence SEQ ID NO:2 for its use as a medicament.
• TSHR thyroid stimulating hormone receptor
• the Applicant has in particular been able to develop a pharmacological and/or genetic activator of the thyroid-stimulating hormone receptor (TSHR) signaling pathway in muscle stem cells, making it possible to increase muscle regeneration by preventing the loss of muscle mass and substitution by fatty and fibrous tissues. This approach will help maintain muscle functionality and slow the progression of Duchenne muscular dystrophy.
• TSHR thyroid-stimulating hormone receptor
• FIG. 1 illustrates the generation of R-DMDdel52 rats as detailed in example 1 in which: IA) is an observation of Dystrophin and
• 3-Dystroglycan (in gray) by immunofluorescence in the anterior tibia of WT rats and R-DMDdel52 rats at 3 weeks and 12 months of age. (Scale bar at 20
• 1B is an analysis of the level of dystrophin expression in the anterior tibia of a WT rat and two R-DMDdel52 rats (DMD #1, DMD#2) at 16 weeks of age by Western Blot . (3-Tubulin is used as a housekeeping gene.
• I C represents a survival curve of WT rats and DMD rats by the Kaplan Meier estimator.
• 1 G corresponds to the percentage distribution of the sizes of the muscle fibers of the transverse section at the level of the anterior tibia in WT and R-DMDdel52 rats of the age of 12 months.
• Mean ⁇ standard deviation (n 3), ANOVA test.
• the p values are calculated by Sidak's post test.
• 1 J corresponds to the percentage of central nucleus myofibrils in the extraocular muscles of WT and R-DMDdel52 rats at the age of 12 months.
• Mean ⁇ standard deviation (n 3), Mann-Whitney test.
• FIG. 2 illustrates the continuous muscular regeneration of progressive extraocular muscles in the rat DMD model as detailed in example 2 in which:
• 2A is an observation of the embryonic myosin light chain (eMHC - gray cluster in the lower left panel) and laminin (as a network) in the anterior tibia of WT and R-D rats Ddel52 aged 3 weeks,
• 2D corresponds to the quantifications of the number of eMHC per mm2 in the extraocular muscles of WT and R-DMDdel52 rats aged 3 weeks, 6 months and 12 months.
• Mean ⁇ standard deviation (n 3), Mann-Whitney test.
• 2G represents the quantification of the percentage of quiescent (Pax7+/Ki67-) MSCs (muscle stem cells) and proliferative MSCs (Pax7+/Ki67+) on AT isolated from WT and R-DMDdel52 rats aged 3 weeks, 6 months and 12 month.
• Mean ⁇ standard deviation (n 3), Mann-Whitney test.
• FIG. 3 illustrates the myogenic potential of MSCs from AT R-DMDdel52 rats in vitro in which:
• 3A-B correspond to representative co-immunofluorescence staining of Ki67 (round), MyoD (square) and nuclei (Hoechst, star) of myoblasts isolated from the anterior tibia (A) and extraocular muscles (B ) 3-week-old WT and R-DMDdel52 rats. Arrows indicate proliferating myoblasts co-labeled with Ki67 and MyoD. Scale bar 20 ⁇ m.
• 3C corresponds to the quantifications of MyoD+/KI67+ and MyoD+/KI67- myoblasts from the anterior tibia and extraocular muscles of 3-week-old WT and R-DMDdel52 rats.
• Mean ⁇ standard deviation (n 3), Mann-Whitney test.
• 3D-E represent the immunofluorescence labeling of sarcomeric myosins (MF20 in the form of filaments) and nuclei (Hoechst,) of differentiated myotubes at the level of the anterior tibia (D) and extraocular muscles (E) of WT rats and R-DMDdel52 aged 3 weeks.
• H-1) correspond to co-immunofluorescence staining of KI67, MyoD and nuclei (Hoechst) of myoblasts isolated from TA (H) and MEO (I) of 6-month-old WT and R-DMDdel52 rats. Arrows indicate proliferating myoblasts co-labeled with Ki67 and MyoD, while stars highlight MyoD+/KI67- myoblasts, 20 ⁇ m scale bar.
• 3K-L correspond to immunofluorescence staining of sarcomeric myosins (MF20, green) and nuclei (Hoechst, blue) of differentiated myotubes from TA (K) and MEO (L) of 6-year-old WT and R-DMDdel52 rats month. 20m scale bar.
• 3P is the percentage of cell type (Pax7+/MyoD+ and Pax7+/MyoD-) to assess cell purity after FACS sorting.
• 3R is an immunofluorescence staining of sarcomeric myosins (MF20, as a broad band (WT) and a thick line (DMD)) and nuclei (Hoechst, in circles) on myotubes differentiated from FACS-sorted myoblasts of 6 month old WT and R-DMDdel52 rats. Scale bar 50 m.
• 3S corresponds to the quantification of the fusion index per field for the experiment carried out in (R).
• FIG. 4 illustrates the expression of the senescence markers in the satellite cells of the Tibialis anterior (TA) muscle R-DMDdel52 as detailed in Example 4, in which:. 4A) is a plot plotted by the t-SNE algorithm on the clustering of muscle-resident cell populations revealed by scRNA-seq analysis on the AT of 12-month-old WT and R-DMDdel52 rats.
• 4B is a t-SNE plot split between WT and R-DMDdel52.
• 4C is a t-SNE plot showing expression of Cdkn1a and Cdkn2a in muscle stem cells (MSCs) of WT and R-DMDdel52 samples.
• 4D depicts violin diagrams showing the gene expression distribution of Pax7 and Myf5 as CSM-specific genes and Cdkn 1a and Cdkn2a as senescence markers.
• 4E-F-G correspond to staining of TA by co-immunofluorescence, using antibodies against Pax7, Cdkn2a (also called p16; star, E) or Cdknl a (also called p21; star, F) or yH2AX (star, G), laminin (in the form of a network) and Hoechst for the nuclei.
• Triangles highlight PAX7+ CSMs. Scale bar 10pm.
• 4J corresponds to the quantification of G showing the percentage of MSC (Pax7+) co-expressing yH2AX at the age of 3 weeks, 6 months and 12 months at the level of the AT of the WT and R-DMDdel52 rats.
• Means ⁇ standard deviation (n 3), Mann-Whitney test.
• FIG. 5 illustrates the expression of senescence markers in the MSCs of human patients with DMD as detailed in example 5, in which:
• 5A depicts immunofluorescence for embryonic myosin heavy chain (eMHC, large filled gray circle) and laminin (as a lattice) on human control and DMD biopsies at ages 1-2, 3-4, 5-6, 7-8 years old. Scale bar 20pm.
• 5C is co-immunofluorescence staining of DMD and control muscle biopsies, using antibodies against Pax7, Cdkn2a (red) and Hoechst. Yellow triangles highlight PAX7+ CSMs. Scale bar 10 m.
• 5F is a representative co-immunofluorescence staining of DMD and control muscle biopsies, using antibodies against Pax7, yH2AX (star), and Hoechst. Triangles highlight PAX7+ CSMs. lOpm scale bar.
• FIG. 6 illustrates the expression of a specific signature of MSCs at the MEC level distinct from that at the TA level as detailed in Example 6, in which:
• 6A is a graph plotted by the t-SNE algorithm of clustering of muscle-resident cell populations revealed by scRNA-seq at the TA (left panels) and MEOs (right panels) of WT rats and 12-month-old R-DMDdel52 showing expression of Pax7, Pitx2, Cdkn la, Cdkn2a and Tshr. The squares indicate the CSM group.
• 6B-C depict Violin plots showing the gene expression distribution of Fax/, Cdkn la, Cdkn2a in B and Myf5, Pitx2 and Tshr in C from scRNAseq analysis performed at TA and MEO 12-month-old WT and R-DMDdel52 rats.
• 6E-G correspond to co-immunofluorescence staining of MEOs, using antibodies against Pax7, Cdkn2a (E) or Cdknl a (F) or yH2AX (, G), laminin (as array) and Hoechst.
• Triangles highlight CSMs (PAX7+) .
• FIG. 7 illustrates the regulation of senescence in myoblasts isolated from MEOs in R-DMDdel2 rats, in which:
• 7A is immunofluorescence for TSHR (stars) and Pax7/MyoD on myoblasts from 3 month old rats isolated from MEO (left) and limb muscle (right). Nuclei were stained with Hoechst. 20m scale bar.
• 7B is co-immunofluorescence staining of Edll (middle panels), Pax7/MyoD (left panels) and nuclei (Hoechst, right panels) of vehicle-treated (DMSO) OME-derived myoblasts ) or ML224. 20m scale bar.
• 7D is an immunofluorescence staining of sarcomeric myosins (MF20, as a band) and nuclei (Hoechst) of differentiated myotubes from MEO cells treated with vehicle (DMSO) or ML224. 20pm scale bar.
• FIG. 7G depicts representative co-immunofluorescence staining of Pax7/MyoD (left panels), EdU (middle panels), and nuclei (Hoechst, right panels) on myoblasts isolated from WT and R limb muscles -DMDdel52 treated with vehicle (DMSO) or with the adenylate cyclase activator Forskolin. 20m scale bar.
• 7K shows immunofluorescence staining of sarcomeric myosins (MF20, as bands) and nuclei (Hoechst, round) of differentiated myotubes from WT and R-DMDdel52 limb cells treated with vehicle (DMSO) or Forskolin. 20m scale bar.
• the invention relates to an activator of the thyroid stimulating hormone receptor (TSH R) signaling pathway in muscle stem cells (satellite cells) for its use in preserving and regenerating muscle structure and function by blocking senescence, preferentially in ' Man and animal, even more preferentially in Man.
• TSH R thyroid stimulating hormone receptor
• the invention relates to a pharmacological and/or genetic activator of the thyroid-stimulating hormone receptor (TSH R) signaling pathway in muscle stem cells (satellite cells) for its use in preserving and regenerating muscle structure and function by blocking senescence, preferentially in humans and animals, even more preferentially in humans.
• TSH R thyroid-stimulating hormone receptor
• the Applicant has thus been able to identify new pharmacological and/or genetic activators of the thyroid-stimulating hormone receptor (TSHR) signaling pathway, taken alone or in combination, for their use specifically at the level of muscle stem cells (cells satellites) to block their senescence.
• TSHR thyroid-stimulating hormone receptor
• TSHR belongs to the family of glycoprotein rhodopsin receptors which, when activated, stimulate adenylate cyclase, activating the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway (Morshed et al. , “Characterization of thyrotropin receptor antibody-induced signaling cascades.” Endocrinology vol. 150.1 (2009): 51-29: Rowe et al., "Tezacaftor-lvacaftor in Residual-Function Heterozygotes with Cystic Fibrosis.” The New England journal of medicine vol 377.21 (2017): 2024-2035).
• cAMP cyclic adenosine monophosphate
• PKA protein kinase A pathway
• DMD Duchenne muscular dystrophy
• preserving and regenerating muscle structure and function by blocking the senescence of muscle stem cells makes it possible to prevent and/or treat myopathies, more preferably DMD.
• the pharmacological and/or genetic activators of the TSHR signaling pathway in muscle stem cells (satellite cells) used according to the invention make it possible to prevent and/or treat sarcopenia.
• sarcopenia we mean a geriatric syndrome characterized initially by a decrease in muscle capacity due to age and which, as it gets worse, will be the cause of a deterioration in muscle strength and physical performance.
• the sarcopenia observed in the elderly is attributable to the aging process but can be accelerated by pathological and behavioral factors such as malnutrition and a sedentary lifestyle. From the age of 30, muscle degeneration of 3 to 8% per decade is observed. Muscle tissue is replaced by fat mass and this loss accelerates from the age of 50. Indeed, muscle mass declines approximately 1 to 2% per year after the age of 50, while strength declines on average by 1.5% per year between 50 and 60 years (-15%), then at the rate 3% per year, i.e.
• the activator of the TSHR signaling pathway in muscle stem cells (satellite cells) used according to the invention is an activator chosen from a TSHR receptor agonist or an activator of the adenylate cyclase, taken alone or in combination.
• agonist of the TSHR receptor used according to the invention is meant a compound capable of activating the TSHR receptor directly by binding at the level of the TSHR receptor or indirectly via other compound(s).
• TSHR receptor agonist By way of example of a TSHR receptor agonist, mention may be made of recombinant human TSH (rhTSH) Thyrogen®, an antibody stimulating the TSHR (such as M22), NCGC00161870 known as C2 and consisting of the El and E2 enantiomers, NCGC00379308, and MSql.
• rhTSH recombinant human TSH
• Thyrogen® an antibody stimulating the TSHR (such as M22)
• NCGC00161870 known as C2 and consisting of the El and E2 enantiomers
• NCGC00379308 NCGC00379308
• MSql MSql
• the pharmacological activator used according to the invention is preferably an adenylate cyclase activator chosen from NKH 477 (6-[3-(dimethylamino)propionyl]forskolin), cw008, DMAPD (6-[3-(dimethylamino)propionyl] -14,15-dihydro-forskolin), PACAP-27, PACAP-38, Glucagon, Isoproterenol, Octopamine, PACAP 27 Amine, Prostaglandin D2/E1/E2/I2, Vasopressina, Forskolin and its analogues like 6-Acetyl-7- deacetyl-Forskolin, 7-Deacetyl-7-O- 4hemisuccinyl-Forskolin, 7-Deacetyl-7-(O-N- methylpiperazino)-y-butryl-Dihydrochlonde- Forskolin, 7-deacetyl-
• the activator of the TSHR signaling pathway in muscle stem cells (satellite cells) used according to the invention is a genetic activator.
• the genetic activator used according to the invention consists of a vector chosen from a liposome comprising a protein sequence of the thyroid stimulating hormone receptor (TSHR) of SEQ ID NO: 1 or a functional variant having a protein sequence identical to at least 60 % to the sequence SEQ ID NO: 1, an adenoviral vector comprising the protein sequence of SEQ ID NO: 1 or a functional variant having a protein sequence identical to at least 60% to the sequence SEQ ID NO: 1, or a nucleotide sequence of the TSHR of SEQ ID NO: 2 or a functional variant having a nucleotide sequence that is at least 60% identical to the sequence SEQ ID NO: 2, and an adenoviral vector comprising a CRISPR-Cas9 sequence fused with a transactivator domain preferentially chosen from VP16 , VP64, p65, NCO1A1, FOXO1A to activate TSHR expression.
• TSHR thyroid stimulating hormone receptor
• nucleotide any polyribonucleotide or polydeoxyribonucleotide, which may be an unmodified RNA or DNA or a modified RNA or DNA.
• Polynucleotides include, without limitation, single and double stranded DNA, DNA that is a mixture of single and double stranded regions or single and triple stranded regions, single and double stranded RNA, and RNA which is a mixture of single and double stranded regions, hybrid molecules comprising DNA and RNA which may be single stranded or, more typically, double stranded, or regions triple stranded, or a mixture of single and double stranded regions.
• polynucleotide(s) also includes DNAs or RNAs described above which contain one or more modified bases.
• DNAs or RNAs whose backbones are modified for reasons of stability or for other reasons are “polynucleotides” in the sense that this term is understood here.
• polynucleotide(s) as used herein encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells.
• polynucleotide(s) also encompasses short polynucleotides often referred to as oligonucleotides.
• polypeptide(s) designates both short chains, commonly called peptides, oligopeptides and oligomers, and longer chains generally called proteins. Polypeptides may contain amino acids other than the 20 amino acids encoded by the gene. "Polypeptides” include those that are modified by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in the scientific literature and are well known to those skilled in the art. It will be appreciated that the same type of modification may be present to the same degree or to different degrees at several sites in a given polypeptide.
• a given polypeptide may contain many types of modification. Modification can occur anywhere in a polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme fragment, covalent attachment of a nucleotide, or of a nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cysteine formation, pyroglutamate formation , formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycos
• the protein acid sequence of TSH R is at least 60% identical to the sequence SEQ ID NO: 1, for example at 65%, 70%, 75%, 80%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, more preferably identical at least 95% to the sequence SEQ ID NO: 1, for example 96%, 97%, 98%, 99%, even more preferably is identical to the sequence SEQ ID NO: 1.
• the nucleic acid sequence of TSH R is at least 60% identical to the sequence SEQ ID NO: 2, for example at 65%, 70%, 75%, 80%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, more preferably identical at least 95% to the sequence SEQ ID NO: 2, for example 96%, 97%, 98%, 99%, even more preferably is identical to the sequence SEQ ID NO: 2.
• identity is meant a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparison of the sequences.
• identity also means the degree of sequence relatedness between the polypeptide or polynucleotide sequences, as the case may be, as determined by the correspondence between the chains of these sequences.
• Identity and similarity can be readily calculated by known methods, including but not limited to those described in the following references (Computational Molecular Biology, Lesk A.M., ed travers Oxford University Press, New York, 1988; Biocomputing: Informatics and genome Projects, Smith D.W., ed therapeutic Academic Press, New York.
• variant is meant a polynucleotide or a polypeptide which differs from a reference polynucleotide or polypeptide, respectively, but which retains essential properties.
• a typical variant of a polynucleotide differs in its nucleotide sequence from another reference polynucleotide. Alterations to the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes can lead to substitutions, additions, deletions, fusions and/or truncations of amino acids in the polypeptide encoded by the reference sequence, as discussed below.
• a typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide.
• a variant polypeptide and a reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and/or deletions in any combination.
• a substituted or inserted amino acid residue may or may not be a residue encoded by the genetic code.
• the present invention also includes variants of each of the polypeptides of the invention, i.e. polypeptides which vary from the references by conservative amino acid substitutions, whereby one residue is replaced by another having characteristics similar.
• Typical conservative amino acid substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acid residues, Asp and Glu; among Asn and Gin; and from the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
• conservative mutations include mutations that swap one amino acid for another in one of the following groups:
• Aromatic residues Phe, Tyr and Trp.
• variants in which several, for example 5-10, 1-5, 1-3, 1-2 or 1 amino acid are substituted, deleted or added in any combination.
• a variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to be naturally occurring.
• Unnatural polynucleotide and polypeptide variants can be obtained by mutagenesis techniques, by direct synthesis and by other recombinant methods known to those skilled in the art.
• sequence optimization may also include decreasing the number of CpG islands in the sequence and/or decreasing the number of splice donor and acceptor sites.
• sequence optimization is a balance between all these parameters, which means that a sequence can be considered optimized if at least one of the above parameters is enhanced while one or more of the other parameters are not, so long as the optimized sequence leads to an enhancement of the transgene, such as enhanced expression and/or decreased immune response to the transgene in vivo .
• the transgene of interest codes for a human TSHR
• the nucleic acid sequence coding for the human TSHR protein comprises an optimized sequence identical to the sequence SEQ ID NO: 2.
• liposome an artificial vesicle formed by concentric lipid bilayers, trapping aqueous compartments between them. It is obtained from a wide variety of amphiphilic lipids, most often phospholipids. When such compounds are placed in the presence of an excess of aqueous solution, they organize themselves in such a way as to minimize the interactions between their hydrocarbon chains and water.
• the genetic activator preferentially used according to the invention is an adenoviral vector, more preferentially an adeno-associated virus (AAV), even more preferentially an AAV8 or an AAV9.
• AAV adeno-associated virus
• Another subject of the invention relates to a combination of a pharmacological activator of the TSHR signaling pathway in muscle stem cells (satellite cells) and a genetic activator of the TSHR signaling pathway in muscle stem cells ( satellite cells) for its use in preserving and regenerating the muscle structure and function by blocking senescence, preferentially to prevent and/or treat myopathies, more preferentially DMD or even to prevent and/or treat sarcopenia.
• the pharmacological activator is Forskolin and the genetic activator is an adeno-associated virus (AAV) comprising a protein sequence of the receptor of thyroid stimulating hormone (TSH R) of SEQ ID NO: 1 or a functional variant having a protein sequence that is at least 60% identical to the sequence SEQ ID NO: 1, or a nucleotide sequence of the TSHR of SEQ ID NO: 2 or a functional variant having a nucleotide sequence that is at least 60% identical to the sequence SEQ ID NO: 2, more preferably an AAV8 or an AAV9.
• AAV adeno-associated virus
• Another object of the invention relates to an adenoviral vector comprising a protein sequence of the thyroid stimulating hormone receptor (TSHR) of SEQ ID NO: 1 or a functional variant having a protein sequence identical to at least 60% to the sequence SEQ ID NO: 1, or a nucleotide sequence of the TSHR of SEQ ID NO:2 or a functional variant having a nucleotide sequence that is at least 60% identical to the sequence SEQ ID NO:2 for its use as a medicament.
• TSHR thyroid stimulating hormone receptor
• the adenoviral vector preferentially used according to the invention is an adeno-associated virus (AAV), preferentially an AAV8 or an AAV9.
• AAV adeno-associated virus
• the adenoviral vector is preferentially used according to the invention to preserve and regenerate the structure and the muscular function by blocking senescence, more preferentially to prevent and/or treat myopathies, even more preferentially DMD.
• the adenoviral vector is preferably used according to the invention to prevent and/or treat sarcopenia.
• the R-DMDdel52 rat model is generated by deletion of 188 bp (base pairs) and generation of a premature stop codon at exon 52 of the Dystrophin (DMD) gene to produce a loss of function of the allele responsible for the production of dystrophin.
• DMD Dystrophin
• ENSRNQE00000518152 The sequence of rat exon 52 (ENSRNQE00000518152) and surrounding intronic sequences were selected to identify guide RNA sequences (sgRNAs) by the BWA algorithm for genome editing by the CRISPR method.
• F0 animals were screened by junction PCR genotyping. Genotyping is performed on DNA extracted from tail or ear biopsies.
• the PCR mix was prepared with 10X Dream Taq buffer (ThermoFisher Scientific, Cat#B65), 0.25mM dNTP (10mM), 0.3M forward primer ((5'- CTAACGCATTTAAAATATGCTGTCA-3'), 0.3 M of the antisense primer (5'-GTTGGCTTAGCTCAACAACCAAGAT-3', 100 pM), 0.03U/ ⁇ l DreamTaq and approximately 150ng of DNA.
• the PCR was carried out using the Thermal Cycler 2720 with the following thermal program: initial denaturation at 95°C for 5 minutes, 40 cycles of denaturation at 95°C for 10 seconds, annealing of the primers at 60°C for 30 seconds and elongation at 72°C for 45 seconds, and final elongation at 72°C for 5 minutes.
• exon 52 was detected on a 2% agarose gel.
• Rats were housed in a pathogen-free facility with 12-hour light and 12-hour dark cycles in accordance with European Directive 2010/63/EU.
• dystrophin was then evaluated by immunofluorescence (FIG. 1 A) and western blot [FIG. 1 B) in the muscle of WT and R-DMDdel52 rats.
• Western blot was performed by cryosections of frozen muscles which were homogenized using a dounce homogenizer in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5% NP40 and Halt protease inhibitor cocktail (Pierce).
• a survival curve was established in order to compare the probability of survival between WT and R-DMDdel52 rats as a function of the number of days. This made it possible to show that the R-DMDdel52 rats die after 14 months of life, unlike the WT rats.
• R-DMDdel52 rats show significantly lower body mass than WT controls from 3 months of age, with a large decrease in body mass index and body length at age 12 months (Table 1). Consistent with the progressive loss of muscle mass, a reduction in Tibialis anterior (TA) and soleus weight was observed in 12-month-old R-DMDdel52 rats compared to WT rats.
• TA Tibialis anterior
• TA muscles are isolated from 12-month-old WT and R-DMDdel52 rats. After isolation, the muscles are immediately frozen in isopentane cooled in liquid nitrogen and sectioned at 7 m.
• the slides are immersed in Mayer's hematoxylin (Sigma-Aldrich) for 3 minutes and after rinsing in deionized water, they are immersed in a solution of lithium carbonate for 3 seconds, then incubated in eosin for 30 seconds. After washing with distilled water, the slides are immersed in 50% and 70% ethanol, then equilibrated in 95% and 100% ethanol.
• Mayer's hematoxylin Sigma-Aldrich
• a severe fibrotic deposit is confirmed by Sirius red staining.
• Figl D lower panels.
• the extent of fibrosis is quantified by measuring the percentage of the area occupied by connective tissue, showing statistically significant differences between WT and R-DMDdel52 rats at the anterior tibia (Fig. 1E). In fact, R-DMDdel52 rats show approximately 2.5 times more fibrosis than WT rats.
• central nuclei is also evaluated, as a pathological marker of a muscle undergoing a degeneration/regeneration event.
• the R-DMDdel52 muscle showed a considerable number of central core fibers compared to the controls. (Fig.l F)
• EOM extraocular muscles
• R-DMDdel52 MEOs exhibited a high number of central nucleus myofibers compared to WT muscles (Fig. 1J), but to a lesser extent compared to TAs (Fig. 1F). Furthermore, fiber size analysis did not show any significant variation between the MEOs of 12-month-old WT and R-DMDdel52 rats (Fig. 1K).
• R-DMDdel52 exhibit progressive and severe muscular dystrophy characterized by well-described features of DMD pathophysiology, including the fact that the extraocular muscles are specifically unaffected by the disease.
• Example 2 The extraocular muscles do not show progressive muscle regeneration defects in the rat DMD model
• the temporal course of muscle regeneration in the anterior tibia of R-DMDdel52 rats was assessed, using immunofluorescence for embryonic myosin heavy chain (eMHC), a specific marker of developing and regenerating myofibers on histological sections. of skeletal muscles.
• eMHC embryonic myosin heavy chain
• the following primary antibodies were used: Pax7 (Santa Cruz Biotechnology, sc-81648), ki67 (Abeam, sp6 abl 6667), Cdkn2a (Abeam Anti-CDKN2A/pl 6INK4a, Abl 08349), Cdknl a (Thermofisher, bs-10129R ) and yH2AX (Abeam, abl 1174). After repeated washings, the slides were incubated with Alexa fluorine secondary antibodies for 45 minutes at room temperature. Laminin staining was performed after incubation of the secondary antibodies, for 1 h at 37°C using a conjugated antibody (NB300-144AF647).
• FIG. 2A-B A sharp decrease in the number of regenerating fibers was observed over time (Fig. 2A-B), indicating a progressive reduction in muscle regeneration during the progression of muscular dystrophy.
• a co-immunolabeling is carried out for Pax7 and Ki67, a proliferation and activation marker, on histological sections of skeletal muscles isolated from the TA and MEO of WT and R-DMDdel52 rats.
• MSCs The cell cycle status of MSCs is then assessed in WT and R-DMDdel52 rats. It can be said that in the TA muscle of WT and R-DMDdel52 rats, MSCs only divide at early stages (3 weeks), while remaining arrested in their cell cycle at later stages of pathology, including in the pathological context of DMD.
• MEO MSCs are highly active in a WT context and retain their regenerative capacity in R-DMDdel52 animals.
• Example 3 The R-DMDdel52 TA MSCs exhibit an altered myogenic potential in vitro:
• the cells were fixed with 4% PFA for 10 minutes at 4° C. and permeabilized with 0.5% Triton in 1 X PBS for 6 minutes. After blocking with 10% BSA for 30 minutes at room temperature, the cells were incubated O/N at 4°C with the following primary antibodies: Pax7 (Santa Cruz Biotechnology, sc-81648), MyoD (Dako, clone 5.8A, M3512), ki67 (Abeam, sp6 abl 6667), sarcomeric myosin heavy chain (MF20, DSHB, AB_2147781).
• TA-derived MSCs of 6-month-old R-DMDdel52 rats show a decrease in the percentage of MyoD-positive myoblasts co-expressing Ki67 compared to WT control cells (Fig. 31-J).
• MSCs are purified from limb muscles by FACS sorting with CD45, CD31 , Terl 19 as exclusion markers and a7-integrin as a positive selection surface marker (Fig.S2A), allowing the culture of pure CSM-derived myoblasts (Figure 3O-P).
• the FACS sorting is done by the following method: After dissociation of the cells, the cells isolated from the hind limb were incubated with CD45-PE-Cy7 (BD Pharmingen, 552848), Terl 19-PE-Cy7 (BD Pharmingen, 557853), CD31-PE-Cy7 (BD Pharmingen, 561410), ⁇ 7-integrin-FITC (CliniSciences, Cl 79570-100) for 45 minutes at 4°C. After washes with HBSS supplemented with DNase (Sigma-Aldrich, DN25), the cells are incubated with the vital dye 7-AAD (BD Pharmingen, 559925) and sorted with the BD FacsAria device. The MSC population was identified as CD45:Ter119:CD31-negative and ⁇ 7-integrin-positive.
• a decrease in proliferative potential is observed in sorted R-DMDdel52 cells, as shown by the lower number of myoblasts (Pax7 ⁇ :MyoD ⁇ ) labeled with EdU (Fig. 3Q) in cultured cells from dystrophic limb muscles .
• R-DMDdel52 sorted muscle satellite cells also show strong deficiencies in myogenic differentiation with the formation of small myotubes with fewer nuclei compared to WT cells (Fig. 3R-S).
• Example 4 The satellite cells of the Tibialis anterior R-DMDdel52 muscle express markers of senescence
• a scRNA-seq is performed from TA muscles of 12-month-old R-DMDdel52 and WT rats.
• AT and extra-ocular muscle cells are extracted from 12-month-old WT and R-DMDdel52 rats and immediately digested with 3U/ml of Dispose II (Roche, 4942078001) and 0.5u/ml of Collagenase A (Roche, 10103586001) for 20 minutes in HBSS at 37° C. with slight agitation.
• the cell suspension is passed through cell filters and centrifuged at 600g/minute for 5 minutes, at 4°C. The supernatant was discarded and the cell pellet was resuspended in 20% fetal bovine serum (Cat#S1810-500 Kevin Dutscher) in DMEM (Cat#41966-029 Gibco) supplemented with 25ng/ml bFGF ( RP4037, Sigma-Aldrich) with penicillin/streptomycin 100 mg/mL. (354234, Life Sciences) coated with matrigel (Sarstedt, #94.6140.802). Cells were grown for 48 hours to assess proliferation. After 48 h of proliferation, the cells were cultured for 5 days in 5% fetal bovine serum to induce differentiation.
• cells were filtered through 100 and 70 ⁇ m cell strainers and sorted by FACS after incubation in DAPI (D9542, Sigma-Aldrich) to obtain a clean cell suspension removing dead cells and debris.
• DAPI D9542, Sigma-Aldrich
• the snRNA-seq is carried out using the Chromium Next GEM Single Cell 3'Gene Expression V3 kit (10X Genomics) following the manufacturing protocol. Sequencing was performed on the Illumina NexSeq 550 System with the High Output Kit v2.5 (75 cycles, Illumina 20024906).
• WT and R-DMDdel52 cells are labeled on the t-SNE map, highlighting disease-related changes at the transcriptomic level (Fig. 4B). These changes were evident for MSCs that exhibited distinct gene expression signatures between WT and R-DMDdel52 samples (Fig. 4C).
• Cdkn1a and Cdkn2a are observed in the MSCs at the protein level by co-immunolabeling with Pax7 on muscle sections isolated from 6-month-old R-DMDdel52 and WT rats.
• RNA-Seq histone H2A gamma X
• Example 5 MSCs from human patients with DMP express markers of senescence
• DMD muscle biopsies divided by age groups (1 -2, 3-4, 5-6, 7-8 years), and control biopsies are stained for embryonic myosin heavy chain (eMHC) to mark the regenerating myofibers (Fig. 5A).
• eMHC embryonic myosin heavy chain
• Pax7 co-immunostaining with Cdkn2a and yH2AX are performed on histological sections from DMD biopsies and control biopsies to assess the senescence status of human muscle satellite cells.
• Muscle sections from DMD patients showed substantial numbers of Pax7-positive MSCs that co-expressed Cdkn2a (Fig.5C-E) and yH2AX (Fig.5F-H) compared to control biopsies that barely expressed Cdkn2a and exhibited low levels of yH2AX (Fig.5C-H).
• human DMD MSCs express an early and progressive senescent signature, associated with a gradual decline in muscle regeneration potential.
• the same protocol as for the TA samples is used to characterize the MEO samples, identifying an average of 2505 cells.
• the transcriptomic profile of MSCs from TA and MEO from WT and R-DMDdel52 animals is compared and the specific expression of the Pitx2 gene is detected (Evano, Brendan et al. "Dynamics of Asymmetric and Symmetric Divisions of Muscle Stem Cells In Vivo and on Artificial Niches.” Cell reports vol. 30,10 (2020): 3195-3206. e7. doi:10.1016/j.celrep.2020.01.097; Noden, Drew M, and Philippa Francis-West. "The differentiation and morphogenesis of craniofacial muscles.” Developmental dynamics: an official publication of the American Association of Anatomists vol. 235.5 (2006): 1 194-218. doi:10.1002/dvdy.20697) in MEO WT and R- DMDdel52, confirming the specific identity of these cells (Fig. 6A-C).
• MSCs isolated from MEOs did not show a signature of senescence, with Cdkn1a and Cdkn2a being selectively expressed only in MSCs from R-DMDdel52 TA (Fig. 6B).
• RNA is extracted using the kit RNAqueous-Micro total RNA isolation (Invitrogen) and reverse transcribed using SuperscriptTM III reverse transcriptase (Invitrogen, 18080093) according to the manufacturer's instructions.
• qPCR is performed using the StepOnePlus real-time PCR system (Applied Biosystems) and SYBR Green detection tools (Applied Biosystems).
• Results are reported as relative gene expression (2-DDCT) using vehicle-treated cells or extraocular-derived myoblasts as reference. Gene expression is normalized to
• Co-immunostaining for Pax7 and senescence markers are also performed: Cdkn2a, Cdknl a and yH2AX (Hernandez-Segura et al. 2018; Wang et al, 2009; Bernadotte et al, 2016; Dungan et al, 2020) (Fig .6E-G), showing the absence of their expression in Pax7-positive satellite cells of MEOs.
• Example 7 TSHR signaling regulates senescence in myoblasts isolated from MEOs of R-DMDdel52 rats.
• TSHR the thyroid-stimulating hormone receptor
• MSCs of MEOs Fig. 6A-C
• Specific protein expression of TSHR in MEO MSCs is validated by immunofluorescence of TSHR on freshly isolated myogenic cells from extraocular muscles and limbs.
• TSHR staining cells were permeabilized for 5 minutes with 0.1% Triton in 1 X PBS, then incubated O/N with TSHR antibody (CliniSciences, BS-0003R). The following day, the cells were washed in IX PBS and incubated with Alexa fluorine secondary antibodies for 45 minutes at room temperature. Finally, the nuclei were counterstained with Hoechst (Sigma-Aldrich, B2261). Fluorescence was analyzed with an LSM800 confocal device.
• TSHR is highly expressed in MEO-derived myoblasts at the cell membrane, whereas myoblasts isolated from limb muscles express low or undetectable levels of TSHR.
• myoblasts were isolated from WT MEOs and treated with a selective TSH receptor antagonist, ML224 (Neumann et al., 2014).
• the myoblasts derived from CSM were treated with 10 ⁇ M of ML224 (HY12381-S, CliniSciences) for 2 days (proliferation) or 7 days (differentiation) or 20 M of Forskolin (F3917, Sigma-Amdrich) (experiment following) only for 48 hours.
• Control cells were treated with DMSO (D8418, Sigma Aldrich) as vehicle for ML224 and Forskolin.
• the proliferation was measured after 24 h of culture by adding to the culture medium a pulse of 10 mM of 5-ethynyl-20-deoxyuridine (EdU) for 24 h (EdU Click-IT PLUS Kit C10640, Life Technologies).
• EdU 5-ethynyl-20-deoxyuridine
• myoblasts from ML224-treated MEOs exhibited differentiation defects and compromised myotube formation associated with a significant reduction in fusion index compared to vehicle-only-treated control cells (Fig. 7D- E).
• WT and R-DMDdel52 MSCs isolated from limb muscles were treated with Forskolin, a well-known activator of adenylate cyclase [Alasbahi & Melzing, 2012) and TSHR signaling.
• R-DMDdel52 myoblasts exposed to forskolin abolished aberrant expression of Cdkn1a (p21) and Cdkn2a (p16), while R-DMDdel52 myoblasts treated with vehicle maintained expression. of these markers of senescence (Fig. 51-J).
• myoblasts from forskolin-treated R-DMDdel52 limbs exhibited enhanced differentiation potential compared to control R-DMDdel52 cells, forming significantly larger myotubes with a higher fusion index (Fig. 7K-L).
• Muscles from the hind limbs of adult rats were dissected and digested with 3U/ml of Dispose II (Roche, 4942078001) and 0.5u/ml of Collagenase A (Roche, 10103586001) for 1 hour in HBSS at 37°C with a slight agitation.
• the cell suspension was passed through cell filters and centrifuged at 600g/min for 5 minutes, at 4°C.
• Cells were fixed with 4% PFA for 10 minutes at 4°C and permeabilized with 0.5% Triton in 1X PBS for 6 minutes. After blocking with 10% BSA for 30min at RT, the cells were incubated O/N at 4°C with the following primary antibodies: MyoD (Dako, clone 5.8A, M3512), and pl 6 (Abeam Anti-CDKN2A/ pl 6INK4a, Ab 108349). The following day, the cells were washed in 1X PBS and incubated with Alexa fluorine secondary antibodies for 45 min at room temperature. Finally, the nuclei were counterstained with Hoechst (Sigma-Aldrich, B2261).
• qPCR was performed using the StepOnePlus real-time PCR system (Applied Biosystems) and SYBR Green detection tools (Applied Biosystems). Results are reported as relative gene expression (2-DDCT) using vehicle-treated cells or extraocular-derived myoblasts as reference.
• the gene expression of the rat transcript was normalized to the expression levels of (Sactin.
• a lentiviral vector carrying the coding sequence of TSHR and the Tomato reporter gene under the control of the CMV promoter (TSHR Tomato, Fig.8A) have been generated.
• Wild-type (WT) myoblasts and DMD myoblasts were isolated from 6-month-old rats and cultured.
• Transduction with the TSHR Tomato coding sequence or with a control lentivirus carrying only the Tomato gene was carried out 24 hours after culturing.
• Cells were analyzed 72 hours after transduction and the efficiency of transduction of WT and DMD myoblasts with TSHR Tomato or a control lentivirus (Fig. 8B) was verified.
• TSHR overexpression was validated by qPCR, showing greater transgene expression in cells transduced with TSHR Tomato compared to control cells (Fig.SC).
• DMD cells overexpressing TSH R have lower expression of the senescence marker p! 6 (Fig.SD) thus demonstrating that TSHR prevents the entry into senescence of DMD myoblasts.

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