WO2018178030A1 - Methods and compositions for treating degenerative muscular and/or neurological conditions or diseases - Google Patents

Abstract

The present invention relates to a method for treating degenerative muscular and/or neurological conditions or diseases. The inventors found that TRF2 is decreased with age in human striated muscles which are post-mitotic cells. More particularly, in contrast to mitotic cells, downregulation of TRF2 in differentiated myotubes does not lead to telomere deprotection but triggers potent oxidative stress, along with increases in reactive oxygen species (ROS), mitochondrial dysfunction, FOXO3a activation, and autophagy. Thus, the invention relates to a method of treating diseases targeting post mitotic tissues such as muscle or neurodegenerative diseases in a subject in need thereof, comprising a step of administering said subject with a therapeutically effective amount of an activator, which activates the expression or activity of TRF2.

Description

METHODS AND COMPOSITIONS FOR TREATING DEGENERATIVE MUSCULAR AND/OR NEUROLOGICAL CONDITIONS OR DISEASES

FIELD OF THE INVENTION:

The invention is in the field of muscular and neurology fields. More particularly, the invention relates to a method for activating the expression or activity of TRF2 in a subject suffering from degenerative muscular and/or neurological conditions or diseases.

BACKGROUND OF THE INVENTION:

Recent findings indicate that the accumulation of senescent cells in tissues play a critical role in aging. However, this mechanism does not apply to the long-lived post-mitotic cells, such as myotubes and neurons, whose functioning progressively declines with age without any evidence of cellular senescence. The clock that leads to post-mitotic cell aging as well as the link to the « classical » senescence pathways are still largely unknown.

Among the ageing hallmarks¹, telomere instability and dynamics is well known to act as a mitotic clock for cellular senescence⁵'⁶. Interestingly, telomere changes also occurs during post-mitotic cell aging ¹⁰ (Carneiro et al, PLoS Genet. 2016 Jan 20;12(l):el005798). (Flores et al, 2008), (Carneiro et al., 2016; Daniali et al., 2013) (Mamdani et al., 2015; von Zglinicki, 2002). Accordingly, there is a need to understand telomere biology in post mitotic differentiated tissues and cells and thus find new therapy pathway to treat muscle and neurodegenerative diseases

Inventors choose skeletal muscle cells as a model to study telomeres in post-mitotic cell. Skeletal muscle represents 40% of body mass and fluctuates depending on age and health conditions. Skeletal muscle is composed mainly of post-mitotic differentiated multinucleated cells and contains quiescent satellite cells with renewal potential. Upon aging, exhaustion of this pool of proliferative cells limits regeneration or muscle repair, contributing to sarcopenia or age-dependent muscle wasting⁷'⁸, which represents one of the first causes of loss of independence in the elderly. Nevertheless, the cascade of events associated with functional and structural changes in aging muscle remains partially understood⁹.

SUMMARY OF THE INVENTION:

The present invention relates to a method of treating degenerative muscular and/or neurological conditions or diseases in a subject in need thereof, comprising a step of administering said subject with a therapeutically effective amount of an activator of the expression or activity of TRF2. In particular, the invention is defined by the claims. - -

DETAILED DESCRIPTION OF THE INVENTION:

For the first time, the inventors have reported that the level of the telomeric protein, TRF2, which protects cells from telomere instability and replicative senescence, decreases with age in human striated muscle. In contrast to mitotic cells, downregulation of TRF2 in differentiated myotubes (a cellular model used as proof of concept) does not lead to telomere deprotection but triggers potent oxidative stress, along with increases in reactive oxygen species (ROS), mitochondrial dysfunction, FOX03a activation, and autophagy. The inventors have thus identified a new target to use in treatments targeting tissues mainly composed of post mitotic cells (e.g., muscle and brain), hence muscle diseases and/or neurodegenerative diseases.

Accordingly, the present invention relates to a method of treating degenerative muscular and/or neurological conditions or diseases in a subject in need thereof, comprising a step of administering said subject with a therapeutically effective amount of an activator of the expression or activity of TRF2.

As used herein, the terms "treating" or "treatment" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the - - maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein, the term "degenerative muscular conditions or diseases" refers to a progressive and generalised loss of function of muscle which deteriorate the muscle over time or to disorders that affect the muscle system. The muscle system refers to an organ system consisting of skeletal, smooth and cardiac muscles. The muscle disease is selected from the group consisting of but not limited to muscular dystrophy (e.g., Becker's muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, oculopharyngeal muscular dystrophy, spinal muscular atrophy, Brown-Vialetto-Van Laere syndrome, Fazio -Londe syndrome); muscular atrophy (e.g., muscle atrophy associated with a cancer, muscle atrophy associated with AIDS, muscle atrophy associated with congestive heart failure, muscle atrophy associated with chronic obstructive pulmonary disease, muscle atrophy associated with renal failure, muscle atrophy associated with severe burns, and muscle atrophy associated with long bed rest); amyotrophic lateral sclerosis; Charcot-Marie-Tooth disease, Dejerine-Sottas disease, sarcopenia, myasthenia gravis or Kennedy's disease.

In a particular embodiment, the degenerative muscular condition is sarcopenia. The term "sarcopenia" refers to a syndrome which is characterised by a progressive and generalised loss of skeletal muscle mass and strength with a risk of adverse outcomes such as physical disability, poor quality of life and death.

As used herein, the term "degenerative neurological conditions or diseases" refers to a progressive loss of structure or function of neurons, including death of neurons. The degenerative neurological conditions or disease is selected from the group consisting of but not limited to: Alzheimer's disease (AD) and other dementias; Parkinson's disease (PD) and PD- related disorders; Prion disease; Motor neurone diseases (MND); Huntington's Disease (HD); Spinocerebellar ataxia (SCA) or Spinal muscular atrophy (SMA).

In a particular embodiment, the neurodegenerative disease is Alzheimer's disease. Alzheimer's disease is characterised by loss of neurons and synapses in the cerebral cortex and certain subcortical regions. - -

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human. More particularly, the subject according to the invention has or is susceptible to have degenerative muscular and/or neurological conditions or diseases as described above.

As used herein, the term "TRF2" refers to telomeric repeat binding factor 2. TRF2 is a protein that is present at telomeres throughout the cell cycle. It is also known as TERF2, TRF2, and TRBF2, and is encoded in humans by the TERF2 gene. The naturally occurring human TRF2 gene has a nucleotide sequence as shown in Genbank Accession number NM 005652.4 and the naturally occurring human TRF2 protein has an aminoacid sequence as shown in Genbank Accession number NP 005643.2. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers NM 001083118.2 and NP 001076587.1).

As used herein the term "activator of the expression or activity of TRF2" refers to any TRF2 activators that are currently known in the art or that will be identified in the future. It includes any chemical entity that, upon administration to a subject, results in activation of a biological activity of TRF2. In still another embodiment, the activator is able to modulate, induce or stabilize the expression of TRF2. More particularly, such activator activates the TRF2 gene expression. The term "activates the TRF2 gene expression" refers to a natural or synthetic compound that has a biological effect to activate or significantly increase the expression of the gene encoding for TRF2. Typically, the activator of TRF2 expression has a biological effect on one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.

In a particular embodiment, the activator of the expression or activity of TRF2 is peptide, petptidomimetic, small organic molecule, antibody or aptamers.

The term "peptidomimetic" refers to a small protein-like chain designed to mimic a peptide.

In a particular embodiment, the activator of the expression or activity of TRF2 is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

In a particular embodiment, the activator of the expression or activity of TRF2 is a small organic molecule. The term "small organic molecule" refers to a molecule of a size comparable - - to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

In some embodiments, the activator of the expression or activity of TRF2 is an antibody.

As used herein, the term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al, 2004; Reff & Heard, 2001 ; Reiter et al, 1996; and Young et al, 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a "chimeric" antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A "human antibody" such as described in US 6,075,181 and 6,150,584. In some embodiments, the - - antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388.

In some embodiments, the activator is an intrabody having specificity for TRF2. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term "single domain antibody" (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called "nanobody®". According to the invention, sdAb can particularly be llama sdAb.

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a small molecule which activates the expression or activity of TRF2) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

By a "therapeutically effective amount" is meant a sufficient amount of an activator that activates the expression or activity of TRF2 for use in a method for the treatment of muscle or neurodegenerative disease at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at - - levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The TRF2 activators as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. - -

It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum- drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in - - a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

A further object of the present invention relates to a method of screening a drug suitable for the treatment of muscle diseases or neurodegenerative diseases comprising i) providing a test compound and ii) determining the ability of said test compound to activate the expression or activity of TRF2.

Any biological assay well known in the art could be suitable for determining the ability of the test compound to activate the activity or expression of TRF2. In some embodiments, the assay first comprises determining the ability of the test compound to bind to TRF2. In some embodiments, a population of cells is then contacted and activated so as to determine the ability of the test compound to activate the activity or expression of TRF2. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. It is to be understood that test compounds capable of activating the activity or expression of TRF2, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, aptamers or nucleic acids. For example the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules. - -

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: TRF2 level is decreased upon aging in human skeletal cells. A.

Immunoblots of whole protein extracts from human biopsies collected at different ages with antibodies against TRF2 (data not shown) and B-ACTIN used as loading control. TRF2 protein level was normalized to B-ACTN level. Means ± SD are shown. B. and C. Telomeric Induced Foci analysis (TIFs) in transduced myoblasts and myotubes, respectively. Fixed cells were stained with a telomeric probe (PNA, Green) and 53BP1 (Red) antibody indicating dsDNA damage. An average of 100 Z331 stacks images were taken and deconvo luted post acquisition (Deltavision Elite®, GE). Colocalization event corresponds to foci of DNA damage response at telomeres (TIFs) and are reported. Means ± SEM are shown, c. In myoblasts, transduction of ShTRF2 induced an increase in TIFs (ShTRF2 vs. ShScramble, p= 0.0005; ANOVA, Kruskal- Wallis' multiple comparisons test, =0.05) indicating that modulation of TRF2 expression in mitotic myoblasts increases telomeric damages d. In myotubes, only foci within multinucleated cells were counted (minimum of 40 nuclei per condition). No statistical difference is seen between the different conditions (TRF2 over expression or knock-down and respective controls; ANOVA, Kruskal- Wallis' multiple comparisons test, D=0.05) indicating that modulation of TRF2 340 expression in postmitotic myotubes does not increase telomeric damages. ^< 0.05 ; **< 0.01 ;342 ##*< 0.001.

Figure 2. TRF2 modifies mitochondrial content, autophagy and ROS in post mitotic tissues. A. Relative quantification of mitochondrial DNA content in transduced myotubes. Cells were collected 10 days after transduction and DNA extracted. Mitochondrial DNA (mtDNA) was quantified by qPCR and normalized to genomic DNA from three independent experiments (ΔΔΟ; method; tRNA-Leu and B2-microglobulin for mt and nuclear DNA; respectively). ShTRF2 transduced myotubes show an increase in mitochondrial DNA content (ShScramble vs. ShTRF2, p=0.0003; Kruskal- Wallis multiple comparisons test; □ =0.05). Mean ± SEM shown. B. We report the total number of ROS foci normalized to the number of nuclei. On average, the number of nuclei per cell and DAPI intensities were identical between conditions (data not shown). Means ± SEM are shown. Hydrogen peroxide treatment increases ROS foci number (Empty vs. Empty+ H202, p= 0.0274; Empty vs. TRF2+H202, p= - -

0.0291; Holm-Sidak's multiple comparisons test; D=0.05). Down regulation of TRF2 significantly increases ROS (ShScramble vs.ShTRF2, p< 0,0001; Holm-Sidak's multiple comparisons test; D=0.05). C. Detection of autophagy foci in transduced myotubes using the Cyto-iD® detection kit. An average of 100 z-stacks were taken for each condition (Deltavision Elite®, GE). Pictures were then treated post-acquisition and deconvo luted with IMARIS. Single nucleus-cells were excluded from the analysis. We report the total number of Autophagy- related foci normalized to the number of nuclei. On average, the number of nuclei per cell and DAPI intensities were identical between conditions (data not shown). Means ± SEM are shown. Down-regulation of TRF2 significantly increases the number of autophagy foci (ShScramble vs. ShTRF2, p= 0,0079; Man- Whitney comparisons test, D=0.05). D. Quantification of Foxo3a foci per nucleus. Single nucleus-cells were excluded from the analysis. Means ± SEM are shown. TRF2 knock-down is associated with an increase of nuclear Foxo3a foci (ShScramble vs. ShTRF2, p=0.028; Kruskal-Wallis' multiple comparisons test; D=0.05). EXAMPLE:

Material & Methods

Ethic statement

The collection of fetal muscle biopsies was approved by the "Agence Francaise de la Biomedecine" of the Ministery of Health for legal access to the biological material in full accordance with the law (research protocol number PFS 13-006). Samples were obtained after therapeutic abortion (Tumorotheque, Assistance publique des Hopitaux de Marseille, AC-2013- 1786). Parents have provided written informed consent for the use of biopsies for medical research in accordance with the Declaration of Helsinki. Muscle biopsies were processed by fetopathologists from foetuses not affected by a muscular pathology. Skeletal muscle biopsies from teens and adults were obtained from the Nice Hospital (CHU l'Archet) from healthy donors using standardized muscle biopsy protocol registered as protocol number DC-2015 2374.

Western Blot: Cells were scraped from culture dish in IX PBS complemented with protease inhibitors, mixture was spin down (800g, lOmin at 4°C) and pellet stored at -80°C for further use. Whole cell lysates were prepared from cells by adding cell lysis buffer (i.e., Mueller Buffer: 50mM Hepes pH7.4, 0.1% Triton X-100, 4mM EGTA pH8.0, lOmM EDTA pH8.0, 15mM Na₄P₂07, 25mM NaF, 5mM NaV0₄, lOOmM β-glycerophosphate) complemented with phosphatase inhibitors (Roche). Total protein concentration was determined using a Bradford Protein Assay reagent (BioRad), by measuring absorption at 750nm on a spectrophotometer. - -

For each sample, 30μ§ of protein was resolved in a 4-15% gradient mini-protean precast polyacrylamide gels (BioRad) and transferred to PVDF low fluorescence membranes (Millipore) for 5 hours at 4°C (Wet transfer). After blocking for 1 hour with 10% skim milk in PBST (0.2% Tween-20 in PBS), the membranes were incubated overnight at 4°C with primary antibodies diluted in 5% BSA in PBS. The following primary antibodies were used: TRF2, mouse monoclonal, (clone IMG-124A, 1 : 1000, Imgenex); Actin, goat polyclonal, (1 :3000, Santa Cruz); ATM, mouse monoclonal, (clone 2C1 , 1 : 1500, Abeam); Ser 1981 phospho-ATM, mouse monoclonal, (clone 10H1 1.E12, 1 : 1500, Cell Signaling Technology); Fox03a, rabbit monoclonal (clone 75D8, 1 : 1500, Cell Signaling Technology); ATG4A, rabbit monoclonal (clone D62C10, 1 : 1500, Cell Signaling Technology); LC3A/B, rabbit polyclonal (1 :2000, Cell Signaling Technology). The membranes were then rinsed three times in PBST for lOmin and incubated 1 hour at room temperature with appropriate secondary antibodies diluted (1 : 15000) in Li-COR blocking reagent (e.g., IRDye^® 800CW/680 Goat anti-mouse; Goat anti-rabbit; Li- COR). Following three rinses in PBST for 15min, membranes were imaged by IR fluorescence with the Odyssey^® imaging system (Li-COR). Quantifications between membranes were normalized using the intensity of the ladder (2.5μ1, PageRuler^Tm plus prestained protein ladder; Thermo S cientific) .

Cell culture: Cells used for this study were isolated¹ from patient 12 (Extended Data. 1) and produced as previously described². For day-to-day maintenance, human myoblasts were seeded in dishes coated with 0.1%> pigskin gelatin in 4: 1 Dulbecco modified Eagle medium/Medium 199 supplemented with 15% FBS, 0.02M HEPES, 1.4mg/l vitamin B 12, 0.03mg/l ZnS0₄, 0.055mg/l dexamethasone, 2.5μ^1 hepatocyte growth factor and 10μg/l basic fibroblast growth factor. Cultures were maintained in a 5% oxygen environment and passaged at -60% confluency. Population doublings (PDs) were calculated as PD = ln[(final number of cells)/(initial number of cells)]/In(2).

Myogenicity of the cells was verified by myotube formation following a change to differentiation medium (2% horse serum in 4: 1 Dulbecco modified Eagle medium: Medium 199) when 70-90% confluent.

RT-qPCR: Cells were lysed (RNeasy plus kit (Qiagen)) after washing with PBS, scraped (BD Biosciences) and sheared by centrifugation through Qiashredder columns (Qiagen). Total RNA purified according to the manufacturer's instructions was quantified on a Nanodrop 1000 spectrophotometer (Thermo Scientific). For Reverse Transcription (RT) 2x 500ng RNA was reverse transcribed in technical duplicates using two separate kits (SuperScriptlll, invotrogen; High Capacity cDNA RT Kit, Applied Biosystem). The cDNA was - - diluted 1 :4 in water for quantitative RT-PCR (qRT-PCR) in triplicates using FastStart universal SYBR Green master Mix (Roche) and a 7900HT Fast Real-time PCR system with 384 well block module (Applied Biosystem). Melting curves were analyzed (SYBR green) to exclude non-specific amplification products. We confirmed amplicon size at least once on agarose gels. Crossing-threshold (Ct) values were normalized by subtracting the geometric mean of three housekeeping genes (GAPDH, PPIA and HPRT1). All Ct values were corrected by their PCR efficiency, determined by 1 :2 or 1 :4 cDNA dilution series.

Immunofluorescence Assays: Traditional immunofluorescence assays were performed as followed: cells were grown on cover slides and fixed for lOmin on ice with 4% paraformaldehyde in PBS. After PBS washes, cells were incubated for lh at room temperature in blocking solution (l%Triton X-100, 1%BSA, 5% donkey serum in PBS). Cells were then stained overnight at 4°C in blocking solution containing the respective primary antibodies (TRF2, 1 : 100; Foxo3A, 1 :250; LaminB, 1 :200). After three washes with PBS/ 0.1% Triton X- 100, slides were incubated for lh30min at room temperature with secondary antibodies (AlexaFluor 1 :500) in PBS containing 0.5% Triton X- 100, 5% BSA. Slides were mounted with Vectashield with DAPI (Vector Laboratories, Burlingame, USA). Images were taken using a Delta vision elite system (GE) with a 60X oil-immersed lens (60X/TRIF - Plan Apochromat; Olympus).

Telomeric Induced Foci (TIFs): Cells were grown on cover slides and fixed for lOmin on ice with 4% paraformaldehyde in PBS. After PBS washes, cells were incubated for lh at room temperature in blocking solution (l%Triton X-100, 1%BSA, 5% donkey serum in PBS). To perform the PNA-Fish staining, cells were washed twice with SSC2X for 5min at RT and subsequently treated with RNaseA for 45min at 37°C. After additional SSC2X washes (5min, 4°C), coverslides were dried and incubated upside-down with a hybridization solution containing the PNA-probe (20μ1 H₂0; 70μ1 formamide; 7μ1 10% blocking B (Roche); Ιμΐ 1M Tris pH7.2; Ιμΐ probe) and sealed on coverslips using rubber cement. Slides were then heated at 85°C for 4min, and incubated in the dark at 37°C in a humidification chamber for 2 hours. After removal of the rubber cement, cells were washed in a serie of three different solutions. Twice for 15min at RT with a washing solution I (lOmM Tris pH 7.2; 70% formamide); twice for 15min at RT with a washing solution II (150mM NaCl; 50mM Tris pH7.2; 0.05% Tween 20) and twice with PBS for 5min at RT. Cells were then blocked for 1 hour with blocking solution and immuno-stained overnight at 4°C in blocking solution containing the primary rabbit polyclonal anti-53BPl antibody (Novus Biologicals, 1 :500). After three washes with PBS/ 0.1% Triton X-100, slides were incubated for lh30min at room temperature with Alexa - -

555 Donkey anti-rabbit secondary antibody in PBS containing 0.5% Triton X-100, 1% BSA, 2.5% donkey serum. Slides were mounted with Vectashield with DAPI (Vector Laboratories, Burlingame, USA). Images were taken using a Delta Vision elite system (GE). Co-localization events, representing telomeric DNA damages (TIFs), were counted in at least 30 nuclei per condition from three independent experiments using IMARIS software.

Autophagy: Cells were grown on cover slides and treated as indicated in the Cyto-iD autophagy kit (Enzo). Briefly, transduced differentiated cells were incubated for 30min at 37°C with a solution composed per well of: 1ml of IX Assay Buffer, 200μ1 Horse Serum, 2μ1 Cyto- iD and Ιμΐ nuclear stain. Cells were washed twice with IX Assay Buffer prior and after the incubation. Cells chemically treated (e.g., EGCG: lOmM; H2O2: ΙΟΟμΜ), were incubated first for 6hours in differentiation media at 37°C. Cells were then fixed in 4% PFA for lOmin at RT and 15μ1 of vectashield (vector laboratories) was used to mount the cover slides on coverslips. Pictures were taken using a Delta Vision Elite system (60X/TRIF - Plan Apochromat; Olympus) (GE). An average of 100 stacks and 50 nuclei were taken per conditions. Images were then treated using IMARIS. All intensities of Autophagy related foci and nuclei were used for the analyses. Excluding single-nuclei cells for myotubes analysis.

Reactive Oxygen Species (ROS): Cells were grown on cover slides and treated as indicated in the ROS kit (Enzo). Briefly, transduced differentiated cells were washed twice and lml of fresh differentiation media was added, with or without drugs and incubated for 30min at 37°C (N-Acetyl-L-cystein: 5mM; EGCG: lOmM; H₂0₂: ΙΟΟμΜ). After additional wash and media renewal (lml), cells were incubated for 1 hour at 37°C with a solution composed 2X ROS detection and 4μ1 of Oxidative stress reagent per 10ml (5mM; dilution 1 :2500). The solution was added to the lml of fresh media to adjust to appropriate concentration. Cells were then washed three times with IX PBS and directly mounted using 15μ1 of vectashield+DAPI (no fixation). Pictures were taken using a DeltaVision Elite system (GE). An average of 100 stacks and 50 nuclei were taken per conditions. Images were then treated using IMARIS. All intensities of Autophagy-related foci and nuclei were used for the analyses, excluding single- nuclei cells for myotubes analysis.

Mitochondrial DNA quantification: DNA from cells was extracted using a high salt precipitation and resuspended in 50μ1 of TE (lOmM Tris HC1 pH8.0; ImM EDTA). After DNA quantification, all samples were diluted to a concentration of 3ng^l in TE. A qPCR assay was then performed using Ιμΐ of the prepared diluted samples. All samples were run in triplicates using FastStart universal SYBR Green master Mix (Roche) and a 7900HT Fast Real-time PCR system with an 384 well block module (Applied Biosystem). Melting curves were analyzed to - - exclude nonspecific amplification products. The program used is as followed: denaturation 95°C - 5min, followed by 40 cycles of 95°C - 30 sec, 60°C - 30s annealing, 72°C - 30s extension. PCR was stopped with a final 98°C - 10 min step. We used two sets of primers as described elsewhere³, to measure the relative mitochondrial content (mtDNA and nucDNA, respectively; See Extended primer list). To determine the mitochondrial DNA content, we used the following equations: D CT = (nucDNA CT - mtDNA CT); Relative mitochondrial DNA content = 2x2 ^DCT.

ChIP and ChlP-Seq: Samples for chromatin immunoprecitpitation (IP) were prepared as followed. IP using TRF2 antibody (TRF2- Imgenexl24A) were crosslinked for 10 min at RT and 20 min at 4°C with 0.8% formaldehyde (methanol free, ultrapure EM grade, Polysciences, Inc; Warrington PA). Reaction was stop at RT for 10 min with the addition of Glycine to a final concentration of 0.125 M. Cells were rinsed twice with ice-cold IX PBS, scraped from the dish and pelleted after centrifugation (800g, 5min at 4°C). Next, cells were treated according to the manufacturers guidance (Pierce Classic Protein G IP Kit, Thermo Scientific). For sonication, we used a total processing time of 15min per sample in a Bioruptor (Diagenode) using the following settings: 14 cycles; 30 Sec ON/30 Sec OFF on High power. Sonicated DNA was controlled on 2% agarose gel, valid sonication translated into a smear ranging from 200-700bp. IPs were processed using a 4°C O/N incubation (concentration of TRF2 antibody at l ^g); Ιμΐ of each preparation: IP, IgG, Rabbit non-immune Serum, No crosslink control, no Antibody control and 1% input were used as controls for ddPCR analysis. Primers were designed for the promoter region of each gene, results are normalized to Alu repeats. Each PCR primer pairs were tested on genomic DNA to verify specificity and efficiency (see primer list file).

ChlP-Seq analysis: DNA was sequenced on an Illumina HiSeq in single-end mode with a read length of 49nt, producing an average amount of 2.5M reads per replicate and 3.8M for the 1% input replicates. Raw data were filtered and trimmed using Trimmomatric⁴ reducing the reads set to -3.8% per file. Reads from each file were aligned to the human reference genome hg38 using Bowtie2⁵ with default parameters. The aligned files from same sample (replicates) were then merged together and all subsequent analysis were performed using MACS⁶ and a suits of tools including BEDTools⁷ and BEDOPS⁸. Significant peaks (p<0.05) were identified and annotated using the UCSC database (hg38). Data, including raw files and annotated peaks have been deposited on NCBI Gene Expression Omnibus (GEO; http ://www.ncbi.nlm.nih. gov/ geo/), accession GSE88983 - -

Statistical Analysis: All experiments were repeated at least three times, with three biological replicates (with the exception of human biopsies). Quantitative data are displayed as means□ standard error of the mean. Sample sizes as well as the statistical test used of each experiment are described in each corresponding figure legends or methods. Results from each group were treated with GraphPad prism (6) software for all statistical tests. All tests were two- sided and alpha set at 0.05. Only p-values less than 0.05 were considered statistically significant.

Results

Telomere attrition is recognized as a hallmark of aging¹. Although telomere instability and dynamics have been well described in mitotic cells ⁵'⁶, the incidence in post-mitotic differentiated tissues and cells remains ambiguous. Skeletal muscle represents 40% of body mass and fluctuates depending on age and health conditions. Skeletal muscle is composed mainly of post-mitotic differentiated multinucleated cells and contains quiescent satellite cells with renewal potential. Upon aging, exhaustion of this pool of proliferative cells limits regeneration or muscle repair, contributing to sarcopenia or age-dependent muscle wasting7,8, which represents one of the first causes of loss of independence in the elderly. Nevertheless, the cascade of events associated with functional and structural changes in aging muscle remains partially understood9. As in most tissues, telomere attrition has been reported in aging musclelO (Carneiro et al, PLoS Genet. 2016 Jan 20;12(l):el005798). However, the consequences of this attrition and whether telomeric proteins are modulated in human tissues are not yet known. Therefore, we evaluated TRF2 protein levels in skeletal biopsy specimens from donors of different ages (Fig. la). We found that the TRF2 protein level was negatively correlated with age (correlation reported by goodness of fit, R2 = 06967), decreasing abruptly during the third decade of life, in parallel with the rate of telomere shortening¹⁰. To determine unequivocally the implications of TRF2 in skeletal post-mitotic muscle cells, we established a procedure to mimic our in vivo observations by moderate modulation of TERF2 expression in non-dividing cells. Myoblasts (from deltoid muscle biopsies) that had been switched to differentiation medium (day 0; 2%> horse serum) upon reaching confluence formed multinucleated myotubes on day 5 and were transduced with lentiviral particles on day 7. On day 17, myotubes were collected (data not shown). Concomitantly, transduction was performed in subconfluent proliferating myoblasts, and cells were collected on day 5 (data not shown). As observed with other mitotic cells¹¹, decreases in TERF2 expression in myoblasts triggered telomere deprotection, i.e., the recruitment of DNA damage response (DDR) factors to telomeres, as - - monitored by telomeric dysfunction-induced foci (TIFs, Fig. lb; Extended Data 2). Surprisingly, similar TERF2 downregulation in post-mitotic myotubes triggered neither TIF nor ATM activation (Fig. lc, Extended Data 3), suggesting a myotube-specific mechanism of telomere protection. Importantly, no major differences in cell myogenesis, morphology, or apoptosis were observed upon TRF2 downregulation (data not shown).

We investigated whether TRF2 inhibition impacted other hallmarks of aging¹'¹². We first analyzed the mitochondrial network and contents (Fig. 2A) and quantified reactive oxygen species (ROS) upon TRF2 modulation (Fig. 2B). In TRF2-compromised myotubes, the mitochondrial network was punctuated and not as connected as it is under control conditions (Fig 2A). Moreover, quantitative analysis of mitochondrial DNA (Fig. 2A) demonstrated a higher level of mitochondrial DNA upon TRF2 depletion (8-fold, p = 0.0003), which could constitute a compensatory mechanism for mitochondrial dysfunction¹³. Accordingly, decreased TERF2 expression was correlated with high levels of ROS production, indicating mitochondrial dysfunction, up to levels identical to those seen with H202 treatment (Fig. 2B). Moreover, antioxidant treatment reversed both ROS production and mitochondrial network disconnection. As mitochondria can regulate autophagy via ROS productionl4, autophagic activity was evaluated, and a significant increase in the number of autophagic foci was observed upon TERF2 downregulation (p = 00079; Fig. 2C). Next, we evaluated the transcriptional impact of TRF2 modulation in muscle cells by focusing on genes involved in muscle homeostasis (anabolism and catabolism) and stress sensing. Briefly, we observed increased expression of FOX03a and PGCI D genes upon TRF2 downregulation, which was probably a direct consequence of increased ROS production triggered by TRF2 inhibition. The elevated level and nuclear localization of FOX03a in TRF2-compromised cells was further confirmed by western blotting and immunofluorescence (data not shown).

Finally, in agreement with increased autophagic flux (Fig. 2C), the expression levels of several genes involved in autophagy were altered (SQSTM1, ATG2, ATG9b, ATG14, and ATG16). Importantly, most of these genes were also modulated in mouse models of muscle atrophy¹⁵. These observations are reminiscent of previous studies indicating a role of mitochondria in the regulation of autophagy through ROS generation¹⁶'¹⁴. Briefly, unlike observations in mitotic cells, TRF2 depletion in myotubes does not trigger telomere deprotection but rather induces mitochondrial dysfunction, ROS production, and autophagy (Fig. 1, Fig. 2). These findings suggest that extratelomeric functions of TRF2 are required for redox homeostasis of myotubes. - -

Taking into account recent findings regarding extratelomeric TRF2 binding sites, where TRF2 has been shown to function as a regulator of gene expression^17"20, we analyzed TRF2 binding in post-mitotic myotubes by chromatin immunoprecipitation (ChIP) followed by genome-wide sequencing (ChlP-Seq) in transduced myotubes and controls (Fig. 1). We used peak calling detection tools, using previously published parameters¹⁷, to compare data sets between conditions and respective controls (e.g., ShTRF2 vs. ShScramble; TRF2 vs. Empty). Analyses of TRF2 ChlP-Seq peaks revealed a conserved distribution of TRF2 binding across the genome, especially at internal telomeric sequences (ITS) between mitotic cells¹⁷'²¹ and our post-mitotic model. Among them, ITS were associated with HS3ST4, a gene previously shown to be regulated by TRF2 in cancer cells¹⁸.

Overall, these findings indicate that a high TRF2 level protect the post mitotic cells from triggering the cascades of pathways leading to mitochondria dysfunction and ROS production.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method for treating degenerative muscular and/or neurological conditions or diseases in a subject in need thereof, comprising a step of administering said subject with a therapeutically effective amount of an activator of the expression or activity of TRF2.

2. The method according to claim 1, wherein the degenerative muscular condition is sarcopenia.

3. The method according to claim 1, wherein the degenerative neurological condition is

Alzheimer.

4. The method according to claim 1, wherein the activator is a small molecule.