WO2018178029A1 - 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 SIRT3 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 muscle diseases or neurodegenerative 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 SIRT3.

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 activate the expression and activity of SIRT3 in a subject suffering from degenerative muscular and/or neurological conditions or diseases.

BACKGROUND OF THE INVENTION:

Telomere attrition is recognized as a hallmark of aging¹. Although telomere instability and dynamics have been well described in mitotic cells⁵'⁶, but the incidence in post-mitotic differentiated tissues and cells remains ambiguous.

Other mechanisms than senescence that render a cell dysfunctional with age exist. For instance, this is the case for cells born during embryogenesis that are not or almost not replaced during life and that we name here long-life post-mitotic cells (LLPMC). Examples of LLPMC are myotubes, mature neurons, adipocytes and fibroblasts.

Aging neurons show progressively a decline in a onal transport, a focal accumulations of cytoplasmic and membrane proteins, synaptic transmission alteration, impaired synaptic plasticity, and altered calcium homeostasis that correlates with profound transcriptional changes. These cellular changes are likely to contribute to the progressive decline in neuronal plasticity and cognitive performances in the majority of healthy people but are also possibly contributing to neurodegenerative diseases when combined with specific disease-associated impairments.

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 wasting, which represents one of the first causes of loss of independence in the elderly. Characteristics of skeletal muscle aging a conspicuous reduction in myo fiber plasticity (due to the progressive loss of muscle mass and in particular of the most powerful fast fibers), alteration in muscle-specific transcriptional mechanisms, and muscle atrophy.

As in most tissues, telomere attrition has been reported in aging muscle¹⁰ (Carneiro et al, PLoS Genet. 2016 Jan 20;12(l):el005798). Likewise, the impact of telomere attrition in neurones remains is not yet known. Accordingly, there is a need to understand the telomere - - changes occurring during aging in post mitotic differentiated tissues and cells and thus find new therapy pathway to treat muscle and neurodegenerative diseases.

SUMMARY OF THE INVENTION:

The present invention relates to 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 SIRT3. In particular, the invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Inventors have shown for the first time in post-mitotic cells (e.g. myotubes) that the repression of sirtuin-3 (SIRT3) expression, induces mitochondrial dysfunction and ROS production. More particularly, they found that the telomeric repeat binding factor 2 (TRF2) was associated with the SIRT3 locus creating a long-distance regulatory loop⁴. TRF2 downregulation abolishes this loop and represses SIRT3 expression, inducing mitochondrial dysfunction and ROS production. Moreover, exogenous SIRT3 expression in this set up restored the mitochondrial network and decreased ROS production. The inventors have thus identified a new target to use in the treatments of muscle diseases and/or neurodegenerative diseases. Accordingly, the present invention relates to 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 SIRT3. 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 disease is selected from the group consisting of but no 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 a progressive loss of structure or function of neurons, including death of neurons. The neurodegenerative 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 susceptible to have a muscle or neurodegenerative diseases as described above.

As used herein, the term "SIRT3" refers to NAD-dependent deacetylase sirtuin-3, mitochondrial also known as SIRT3 is a protein that in humans is encoded by the SIRT3 gene. SIRTS (SIRT1-SIRT7) belong to class III histone deacetylases, which are dependent on NAD+ for their activity. Of the seven SIRTs, SIRT3 is the only SIRT analog whose increased expression has been shown to be associated with longevity in humans. SIRT3 is mainly located in mitochondria and involved in the regulation of metabolic processes. The naturally occurring human SIRT3 isoform b gene has a nucleotide sequence as shown in Genbank Accession number NM 001017524.2 and the naturally occurring human SIRT3 isoform b protein has an aminoacid sequence as shown in Genbank Accession number NP 001017524.1. The naturally occurring human SIRT3 isoform a gene has a nucleotide sequence as shown in Genbank Accession number NM 012239.5 and the naturally occurring human SIRT3 isoform a protein has an aminoacid sequence as shown in Genbank Accession number NP 036371.1. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers: NM 001127351.1 and NP 001120823.1 (SIRT3 isoform 1); NM 001177804.1 and NP_001171275.1 (SIRT3 isoform 3); NM_022433.2 and NP_071878.2 (SIRT3 isoform 1 with alternate 5'exon)).

As used herein the term "activator of the expression or activity of SIRT3" refers to any

SIRT3 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 SIRT3. In still another embodiment, the activator is able to modulate, induce or stabilize the expression of SIRT3. More particularly, such activator activates the - -

SIRT3 gene expression. The term "activates the SIRT3 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 SIRT3. Typically, the activator of SIRT3 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 SIRT3 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 SIRT3 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 SIRT3 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 macromolecules (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 a particular embodiment, the small organic molecule is polyphenol or polyphenol precursor. Polyphenol also known as polyhydroxyphenols are well known in the art and refers to a class of natural or synthetic molecules characterized by the presence of large multiples of phenol structural unit. In a particular embodiment, the small organic molecule is resveratrol or an analog thereof. Resveratrol, also known as 3,5,4-trihydroxy-trans-stilbene is a stilbenoid, a type of natural phenol, and a phytoalexin, particularly, it is a plant polyphenol found in high concentrations in red grapes. Resveratrol has the formula C14H1203, CAS Number 501-36-0 and the following structure in the art: - -

The polyphenol or polyphenol precursor can be selected from the group consisting of chlorogenic acid, epigallocatechin gallate, resveratrol, caffeic acid, cinnamic acid, ferulic acid, piceatannol, ellagic acid, epigallocatechin gallate, grape seed extract, and any analog thereof. The SIRT3 activator can be selected from the group consisting of cinnamic acid, quinic acid, fucoxanthin, a biguanide, rosiglitazone, or any analog thereof. The biguanide can be metformin.

In some embodiments, the activator of SIRT3 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 SIRT3. 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 SIRT3) 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 SIRT3 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 SIRT3 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 SIRT3.

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 SIRT3. In some embodiments, the assay first comprises determining the ability of the test compound to bind to SIRT3. 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 SIRT3. 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 SIRT3, 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 depletion modifies higher-order conformation of the subtelomeric SIRT3 gene. A. Quantification of TRF2 enrichment by ChlP-ddPCR^® at the subtelomeric SIRT3 region. Cells were collected 10 days after transduction. ChlP was performed using a TRF2 antibody and results were normalized to Alu repeats. At distal sites, TRF2 enrichment is decreased upon TERF2 knock-down (ShScramble vs. ShTRF2: l lpITSl, p=0.041; SIRT3 (prom), p= 0.0386; Mann- Whitney comparison test, a=0.05), whereas the most subtelomeric site (CICp23) is enriched (ShScramble vs. ShTRF2, p= 0.032). Moreover, TRF2 enrichment is increased upon TERF2 overexpression (Empty vs. TRF2, p= 0.024). Primers encompassing the HS3ST4 TRF2 binding site were used as positive controls (ShScramble vs. ShTRF2, p= 0.0095; Extended Data). B, C: Myotubes were collected 10 days after transduction. Each measure represents the amplification of interactions involving a fixed primer and a second primer along the 2Mb of the locus, both located in proximity of a Hindlll restriction site. Quantification was performed by ddPCR^®. All assays were done in biological duplicate and run in technical triplicate (6 measures per point). TRF2 modulation impacts the chromatin structure in the vicinity of the SIRT3 gene. B. TERF2 overexpression enhances subtelomeric looping and interaction between the distal part of the 1 lp subtelomere and the SIRT3 locus (Empty vs. TRF2 position: 164360, p=0.0195; 166712, p=0.0039; 171528, p=0.0008; Unpaired t Test; α=0.05) whereas C. TERF2 down- regulation decreases these interactions (ShScramble vs. ShTRF2 position: 157640, p= 0.0017; 164360, pO.0001 ; 166712, p=0.074; 171528, p=0.4785; Unpaired t Test; α=0.05). - -

Figure 2 : SIRT3 over-expression restore ROS production and partially restore mitochondrial content. SIRT3 rescue experiments. Gene expression quantified by RT-qPCR in transduced Myotubes A, B, C. Cells were collected 10 days after transduction. Each measure represents the average expression fold-change of six independent repetitions (Biological triplicate in technical RT duplicate) normalized to housekeeping genes (HPRT, PPIA and GAPDH; Δ A Ct method). Means + SEM with associated statistical significance are reported (Kruskal-Wallis multiple comparison test; a=0.05). SIRT3 overexpression reduces transcription of FOX03A stimulated by modulation of TRF2 level and H2O2 treatments. D. We report the total number of ROS foci normalized to the number of nuclei (Extended Data). Means + SEM are shown. SIRT3 overexpression decreases ROS foci number under TERF2 downregulation (ShScramble vs. ShTRF2-SIRT3, p= 0.496; ShScramble vs. ShTRF2, < 0.0001 ; Holm-Sidak's multiple comparisons test; a=0.05) and protects myotubes under H2O2 treatment (Empty+H₂02 vs. SIRT3+H₂0₂, p= 0.0034; ShScramble+H₂0₂ vs. SIRT3-ShTRF2, p=0.039; Holm- Sidak's multiple comparisons test; a=0.05). *< 0.05; ** < 0.01; *** < 0.001 ; **** < 0.0001 ; #< 0.05; ## < 0.01 (H2O2 conditions). E 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 (AACt method; tRNA-Leu and B2-microglobulin for mitochondrial and nuclear DNA; respectively). ShTRF2 and SIRT3 transduced myotubes show an increase in mitochondrial DNA content (ShScramble vs. ShTRF2, p<0.0001 ; ShScramble vs. SIRT3, p=0.03; ShScramble vs. ShTRF2-SIRT3, p=0.025; Kruskal-Wallis multiple comparisons test; D=0.05). Importantly, the results suggest a partial rescue of TRF2 downregulation by SIRT3 overexpression (ROS foci, average mitochondrial DNA content). Mean ± SEM shown.

EXAMPLE:

Material & MethodsCell 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 B12, 0.03mg/l ZnS0₄, 0.055mg/l dexamethasone,

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[(fmal 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 HPRTl). 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).

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 1ml 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 (1ml), 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 1ml 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; 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

We previously showed that TRF2 decrease with age in skeletal muscle and leads to increase in ROS production and mitochondrial DNA. Taking into account recent findings regarding extratelomeric TRF2 binding sites^17"20, we analyzed TRF2 binding in post-mitotic myotubes by chromatin immunoprecipitation (ChIP) followed by genome-wide sequencing (ChlP-Seq) in transduced myotubes (e.g., up- and down-regulation ofTERF2) and controls (e.g., Scramble and Empty construes) using peak calling detection tools¹⁷ (data not shown). TRF2 ChlP-Seq peaks revealed a conserved distribution of TRF2 binding across the genome, especially at interstitial telomeric sequences (ITS) between cancer cells¹⁷'²¹ and our post-mitotic model (i.e., HS3ST4). In the set of genes associated with peaks modified by TRF2 levels, we found the SIRT3 locus encoding the mitochondrial Sirtuin-3 NAD-dependent protein deacetylase involved in mitochondrial homeostasis, ROS production, and senescence³, located 50 kb from a TRF2-bound ITS and downregulated upon TERF2 knock-down (Fig. 2). Strikingly, this sub-telomeric localization is well-conserved throughout evolution (data not shown) and linked to longevity in polymorphism studies.

We then used ChIP followed by droplet digital PCR (ChlP-ddPCR) and chromatin conformation capture (3C) to further investigate the association between TRF2 and the SIRT3 - - region and its consequences on long-range chromatin structure, as the SIRT3 gene is located approximately 200 kb from the l ip telomere and may be regulated by long-range loop formation²² and telomeric position effects²³'²⁴ . By ChlP-ddPCR, we confirmed changes in TRF2-binding at the SIRT3 locus and control (e.g., HS3ST4) upon TRF2 modulation (Fig. 2). Noteworthy, upon TERF2 downregulation, the CICp23 subtelomeric pseudogene promoter was enriched. We speculated that TRF2 depletion triggers subtelomeric higher-order chromatin changes that exposes the CICp23 gene, localized between the 1 lp telomere and the SIRT3 locus, to the spreading and relocalization of the shelterin complex , an explanation consistent with the dynamic chromatin boundaries described at other telomeres²³'²⁴. Indeed, by 3C, we found a TRF2-dependent chromatin loop encompassing the 1 lp telomere and the SIRT3 locus (position 164360, Fig. 2), less frequent in TRF2-compromised myotubes (/?<0.0001) and reciprocally more abundant upon TRF2 overexpression (/?=0.0195).

We then assessed if SIRT3 overexpression could rescue the oxidative stress in TRF2- compromised myotubes (Fig. 3). Remarkably, SIRT3 expression significantly reduces FOX03A transcription and ROS production upon TERF2 downregulation, showing that SIRT3 downregulation is responsible, at least in part, of the oxidative stress generated by TRF2 downregulation.

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.

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 activity or expression of SIRT3.

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.

5. The method according to claim 1, wherein the activator is resveratrol.