Methods for inducing muscle hypertrophy

Abstract

The Inventors hypothesized that the PIK3CA / AKT / mTOR pathway was abnormally affected in patients with HFMH. They identified a somatic gain-of-function mutation of PIK3CA in 5 pediatric patients with hemifacial myohyperplasia, a rare cause of facial asymmetry exclusively involving facial muscles. To understand the physiopathology of muscle hypertrophy, they created a mouse model carrying specifically a PIK3CA mutation in skeletal muscles. PIK3CA gain-of-function mutation led to striated muscle cell hypertrophy, mitochondria dysfunction and hypoglycemia with low circulating insulin levels. A PIK3CA inhibitor, namely Alpelisib, was able to prevent and to reduce muscle hypertrophy in the mouse model. They then concluded that PIK3CA is a relevant target to induce skeletal muscle hypertrophy. The present invention relates to a PIK3CA protein or fragment thereof and / or an agent for PIK3CA protein expression for use in promoting skeletal muscle hypertrophy in a subject in need thereof.

Description

[0001] METHODS FOR INDUCING MUSCLE HYPERTROPHY

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular rheumatology.

[0004] BACKGROUND OF THE INVENTION:

[0005] Muscular atrophy refers to a reduction in muscle mass, more particularly a reduction in striated skeletal muscle. The causes of this atrophy can be varied. Muscular atrophy is observed in pathological situations (e.g. neuromuscular diseases, metabolic disorders, hypercatabolism, eating disorders, cachexia, sarcopenia, medication) as well as physiological situations (e.g. ageing, lack of physical activity, malnutrition). Whatever the case, muscular atrophy can be deleterious leading to weakness, fatigue and reduced quality of life, as well as frailty and an increased risk of falls, fractures and loss of independence. Hemifacial myohyperplasia (HFMH) is a rare cause of facial asymmetry exclusively involving facial muscles, initially reported as ‘hypertrophy and asymmetry of the facial muscles’1. This disorder is reported in very few patients in the literature2'6. The clinical presentation of HFMH patients is strikingly consistent, with unilateral muscular hypertrophy mimicking spasm and orofacial dystonia, leading to diagnostic errors and inadequate management strategies, including aggressive attempts of surgical correction6. The genetic causes of HFMH are currently unknown. The recent discovery of the role played by somatic mutation of genes activating the PIK3CA / AKT / mTOR pathway has opened new treatment perspectives for patients7. Particularly, PIK3CA gain-of-function mutations explain the vast majority of overgrowth syndromes7. PIK3CA encodes the 110-Kd catalytic a-subunit of PI3K, a lipid kinase that controls signaling pathways involved in cell proliferation, motility, survival and metabolism8. Post-zygotic mosaic gain-of-function PIK3CA mutations result in protein activation, leading to abnormal cellular proliferation, tissue hyperplasia and organ overgrowth.

[0006] SUMMARY OF THE INVENTION:

[0007] The invention is defined by the claims. In particular, the present invention relates to a PIK3CA protein or fragment thereof and / or an agent for PIK3CA protein expression for use in promoting skeletal muscle hypertrophy in a subject in need thereof. DETAILED DESCRIPTION OF THE INVENTION:

[0008] The Inventors hypothesized that the PIK3CA / AKT / mTOR pathway was abnormally affected in patients with HFMH. They identified a somatic gain-of-function mutation of PIK3CA in 5 pediatric patients with hemifacial myohyperplasia, a rare cause of facial asymmetry exclusively involving facial muscles. To understand the physiopathology of muscle hypertrophy, they created a mouse model carrying specifically a PIK3CA mutation in skeletal muscles. PIK3CA gain-of-function mutation led to striated muscle cell hypertrophy, mitochondria dysfunction and hypoglycemia with low circulating insulin levels. A PIK3CA inhibitor, namely Alpelisib, was able to prevent and to reduce muscle hypertrophy in the mouse model. They then concluded that PIK3CA is a relevant target to induce skeletal muscle hypertrophy.

[0009] In a first aspect, the present invention relates to a PI3K protein or fragment thereof and / or an agent for PI3K protein expression for use in promoting skeletal muscle hypertrophy in a subject in need thereof. More particularly, the present invention relates to a PIK3CA protein or fragment thereof and / or an agent for PIK3CA protein expression for use in promoting skeletal muscle hypertrophy in a subject in need thereof.

[0010] As used herein, the term “subject” denotes a vertebrate such as mammal, bird, fish, amphibian or reptile. In some embodiments, the vertebrate is a warm-blooded vertebrate (i.e. mammal, bird, fish). In some embodiments, the subject is a mammal, such as a rodent, a feline, a canine (e.g. a dog), an equine (e.g. horse), a bovine (e.g. a beef), a sheep or a primate. More particularly, the subject according to the invention is a human. In some embodiments, the subject suffers from muscular atrophy, muscular dystrophy or muscular hypotrophy. In some embodiments, the subject suffers from muscle wasting, decreased muscle tone, muscle weakness or muscle fragility. In some embodiments, the subject suffers from sarcopenia (i.e. gradual loss of skeletal muscle mass and strength usually associated with advanced ageing). In some embodiments, the subject suffers from cachexia (i.e. multifactorial syndrome with involuntary progressive weight loss characterized by skeletal muscle loss). In some embodiments, the subject suffers from overweight or obesity (i.e. respectively body mass index over 25 or 30).

[0011] As used herein, the term “skeletal muscle hypertrophy” or “skeletal muscle development” refers to an increase in skeletal muscle fiber cross sectional area and skeletal muscle mass. In a more particular aspect, the invention also relates to the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression for use in promoting skeletal muscle hypertrophy in a subject suffering from muscular atrophy, muscular dystrophy or muscular hypotrophy.

[0012] In an even more particular aspect, the invention also relates to the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression for use in promoting skeletal muscle hypertrophy in a subject suffering from muscle wasting, decreased muscle tone, muscle weakness or muscle fragility.

[0013] In an even more particular aspect, the invention also relates to the PIK3CA protein or fragment thereof and / or the agent for PIKCA protein expression for use in the treatment of sarcopenia in a subject in need thereof.

[0014] In an even more particular aspect, the invention also relates to the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression for use in the treatment of cachexia in a subject in need thereof.

[0015] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects 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., disease manifestation, etc.]).

[0016] As used herein, the term “PI3K” refers to phosphoinositide 3-kinases also called phophatidylinositide 3-kinases. PI3K belongs to a family of enzymes which phosphorylate the 3 ’hydroxyl group of the inositol ring of the phosphatidylinositol (Ptdins). The PI3K signalling pathway can be activated, resulting in the synthesis of PIP3 from PIP2. PIK3CA is mainly recruited through tyrosine kinase receptors.

[0017] As used herein, the term “PIK3CA” refers to a protein encoded by the PIK3CA gene (Entrez Gene: 5290; Ensembl: ENSG00000121879). PIK3CA gene encodes the 110-kDa catalytic alpha subunit of PI3K (pl 10a), which converts, at the plasma membrane, phosphatidylinositol 4, 5 -bisphosphate (PtdIns(4,5)P2) to phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3; or PIP3) with subsequent recruitment of PDK1, which in turn phosphorylates AKT on the Thr308 residue to initiate downstream cellular effects. PIK3CA also regulates many other pathways, including the Rho / Racl signaling cascade.

[0018] An exemplary amino acid sequence for PIK3CA is shown as SEQ ID NO:1.

[0019] SEQ ID NO : 1 >sp | P42336 | PK3CA HUMAN Phosphatidylinositol 4 , 5-bisphosphate 3- kinase catalytic subunit alpha isoform OS=Homo sapiens OX=9606 GN=PIK3CA PE=1 SV=2

[0020] MPPRPSSGEL WGIHLMPPRI LVECLLPNGM IVTLECLREA TLITIKHELF KEARKYPLHQ LLQDESSYI F VSVTQEAERE EFFDETRRLC DLRLFQPFLK VIEPVGNREE KILNREIGFA IGMPVCEFDM VKDPEVQDFR RNILNVCKEA VDLRDLNSPH SRAMYVYPPN VESSPELPKH IYNKLDKGQI IWIWVIVSP NNDKQKYTLK INHDCVPEQV IAEAIRKKTR SMLLSSEQLK LCVLEYQGKY ILKVCGCDEY FLEKYPLSQY KYIRSCIMLG RMPNLMLMAK ESLYSQLPMD CFTMPSYSRR I STATPYMNG ETSTKSLWVI NSALRIKILC ATYVNVNIRD IDKIYVRTGI YHGGEPLCDN VNTQRVPCSN PRWNEWLNYD IYI PDLPRAA RLCLSICSVK GRKGAKEEHC PLAWGNINLF DYTDTLVSGK MALNLWPVPH GLEDLLNPIG VTGSNPNKET PCLELEFDWF SSWKFPDMS VIEEHANWSV SREAGFSYSH AGLSNRLARD NELRENDKEQ LKAI STRDPL SEITEQEKDF LWSHRHYCVT I PEILPKLLL SVKWNSRDEV AQMYCLVKDW PPIKPEQAME LLDCNYPDPM VRGFAVRCLE KYLTDDKLSQ YLIQLVQVLK YEQYLDNLLV RFLLKKALTN QRIGHFFFWH LKSEMHNKTV SQRFGLLLES YCRACGMYLK HLNRQVEAME KLINLTDILK QEKKDETQKV QMKFLVEQMR RPDFMDALQG FLSPLNPAHQ LGNLRLEECR IMSSAKRPLW LNWENPDIMS ELLFQNNEI I FKNGDDLRQD MLTLQI IRIM ENIWQNQGLD LRMLPYGCLS IGDCVGLIEV VRNSHTIMQI QCKGGLKGAL QFNSHTLHQW LKDKNKGEIY DAAIDLFTRS CAGYCVATFI LGIGDRHNSN IMVKDDGQLF HIDFGHFLDH KKKKFGYKRE RVPFVLTQDF LIVI SKGAQE CTKTREFERF QEMCYKAYLA IRQHANLFIN LFSMMLGSGM PELQSFDDIA

[0021] YIRKTLALDK TEQEALEYFM KQMNDAHHGG WTTKMDWI FH TIKQHALN

[0022] As used herein, the term “an agent for PIK3CA protein expression” denotes a molecule that partially or totally enhances PIK3CA biological activity (e.g. kinase activity) or expression. The term also encompasses a molecule able to increase or restore PIK3CA gene expression. In order to test the functionality of a putative agent for PIK3CA protein expression a test is necessary. For that purpose, to identify agent for PIK3CA protein expression, a western blot analysis can be run on cell extracts to test the effect on the putative agent for PIK3CA protein expression on the level of PIK3CA. If the level of PIK3CA is increased, the putative agent for PIK3CA protein expression will have the desired effect.

[0023] In some embodiments, the agent for PIK3CA protein expression according to the invention is a low molecular weight compound, e. g. a small organic molecule (natural or not). In some embodiments, the small organic molecule is a PIK3CA agonist. In some embodiments, the small organic molecule is an activator of PIK3CA, in particular an allosteric activator of PIK3CA. The term "small organic molecule" refers to a molecule (natural or not) 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 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da. In some embodiments, the agent for PIK3CA protein expression is selected from a list consisting in 1,2,9, 10-tetram ethoxy-7H- dibenzo[de,g]quinolin-7-one (“57704”; Doyon G, et al. (2014) Discovery of a Small Molecule Agonist of Phosphatidylinositol 3-Kinase pl 10a That Reactivates Latent HIV-1. PLOS ONE 9(1): e84964) or aminopyridines, such as l-(7-((2-((4-(4-ethylpiperazin-l- yl)phenyl)amino)pyridin-4-yl)amino)indolin-l-yl)ethan-l-one (“UCL-TRO-1938”, “1938”; CAS N°: 2919575-27-0; Gong, Grace Q et al. “A small-molecule PI3Ka activator for cardioprotection and neuroregeneration.” Nature vol. 618,7963 (2023): 159-168). Other agents for PIK3CA protein expression are described in W02023 / 041905.

[0024] In some embodiments, the PIK3CA protein of the invention is an isolated, synthetic or recombinant PIK3CA protein. In some embodiments, said PIK3CA protein comprises a sequence as set forth by SEQ ID NO:1. In some embodiments, the PIK3CA protein of the present invention comprises or consists of an amino acid sequence having at least 70% of identity with SEQ ID NO:1. According to the invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99, or 100% of identity with the second amino acid sequence. Amino acid sequence identity is preferably determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990). In particular the PIK3CA protein of the invention is a functional conservative variant of the PIK3CA protein according to the invention. As used herein the term “function-conservative variant" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the PIK3CA protein, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Accordingly, a "functionconservative variant" also includes an PIK3CA protein which has at least 70 % amino acid identity and which has the same or substantially similar properties or functions as the native or parent PIK3CA protein to which it is compared. Functional properties of the PIK3CA protein of the invention could typically be assessed in any functional assay as described in the EXAMPLE.

[0025] A further aspect of the present invention relates to a fusion protein comprising the PIK3CA protein or peptide according to the invention that is fused to at least one heterologous polypeptide. The term “fusion protein” refers to the PIK3CA protein or peptide according to the invention that is fused directly or via a spacer to at least one heterologous polypeptide. According to the invention, the fusion protein comprises the protein or peptide according to the invention that is fused either directly or via a spacer at its C-terminal end to the N-terminal end of the heterologous polypeptide, or at its N-terminal end to the C-terminal end of the heterologous polypeptide. As used herein, the term “directly” means that the (first or last) amino acid at the terminal end (N or C-terminal end) of the protein or peptide is fused to the (first or last) amino acid at the terminal end (N or C-terminal end) of the heterologous polypeptide. In other words, in this embodiment, the last amino acid of the C-terminal end of said protein or peptide is directly linked by a covalent bond to the first amino acid of the N- terminal end of said heterologous polypeptide, or the first amino acid of the N-terminal end of said protein or peptide is directly linked by a covalent bond to the last amino acid of the C- terminal end of said heterologous polypeptide. As used herein, the term “spacer” refers to a sequence of at least one amino acid that links the protein or peptide of the invention to the heterologous polypeptide. Such a spacer may be useful to prevent steric hindrances. In some embodiments, the heterologous polypeptide is a cell-penetrating peptide, a Transactivator of Transcription (TAT) cell penetrating sequence, a cell permeable peptide or a membranous penetrating sequence. The term “cell-penetrating peptide” is well known in the art and refers to a cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).

[0026] The proteins, peptides or fusion proteins of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said proteins, peptides or fusion proteins, by standard techniques for production of amino acid sequences. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’s instructions. Alternatively, the proteins, peptides or fusion proteins of the invention can be synthesized by recombinant DNA techniques as is now well-known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly) peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired (poly) peptide, from which they can be later isolated using well- known techniques. The proteins, peptides or fusion proteins of the invention can be used in an isolated (e.g., purified) form or contained in a vector, such as a membrane or lipid vesicle (e.g. a liposome).

[0027] In specific embodiments, it is contemplated that proteins, peptides or fusion proteins according to the invention may be modified in order to improve their therapeutic efficacy and their stability using well-known techniques. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. A strategy for improving drug stability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. For example, Pegylation is a well-established and validated approach for the modification of a range of polypeptides (Chapman, 2002). The benefits include among others: (a) markedly improved circulating half-lives in vivo due to either evasion of renal clearance as a result of the polymer increasing the apparent size of the molecule to above the glomerular filtration limit, and / or through evasion of cellular clearance mechanisms; (b) reduced antigenicity and immunogenicity of the molecule to which PEG is attached; (c) improved pharmacokinetics; (d) enhanced proteolytic resistance of the conjugated protein (Cunningham-Rundles et.al., 1992); and (e) improved thermal and mechanical stability of the PEGylated polypeptide. Therefore, advantageously, the proteins, peptides or fusion proteins of the invention may be covalently linked with one or more polyethylene glycol (PEG) group(s). One skilled in the art can select a suitable molecular mass for PEG, based on how the pegylated polypeptide will be used therapeutically by considering different factors including desired dosage, circulation time, resistance to proteolysis, immunogenicity, etc. In one embodiment, the PEG of the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CH3 ("methoxy PEG"). In addition, such a PEG can consist of one or more PEG sidechains which are linked together. PEGs with more than one PEG chain are called branched PEGs. Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol. For example, a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide. One form of PEGs includes two PEG side-chains (PEG2) linked via the primary amino groups of a lysine (Monfardini et al., 1995). To effect covalent attachment of PEG groups to the polypeptide, the hydroxyl end groups of the polymer molecule must be provided in activated form, i. e. with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl proprionate (SPA), succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehyde, nitrophenyl carb onate (NPC), and tresylate (TRES)). Suitable activated polymer molecules are commercially available, e. g. from Shearwater Polymers, Inc., Huntsville, AL, USA, or from PolyMASC Pharmaceuticals pic, UK. Alternatively, the polymer molecules can be activated by conventional methods known in the art, e. g. as disclosed in WO 90 / 13540. Specific examples of activated linear or branched polymer molecules for use in the present invention are described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Polymers for Research and pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference). Specific examples of activated PEG polymers include the following linear PEGs : NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM- PEG), and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs such as PEG2-NHS.

[0028] The conjugation of the proteins, peptides or fusion proteins and the activated polymer molecules is conducted by use of any conventional method. Conventional methods are known to the skilled artisan. The skilled person will be aware that the activation method and / or conjugation chemistry to be used depends on the attachment group(s) of the polypeptides as well as the functional groups of the PEG molecule (e.g., being amine, hydroxyl, carboxyl, aldehyde, ketone, sulfhydryl, succinimidyl, maleimide, vinylsulfone or haloacetate). In one embodiment, the proteins, peptides or fusion proteins of the invention are conjugated with PEGs at amino acid D and E (for COOH), T, Y and S (for OH), K (for NH2), C (for SH if at least one cysteine is conserved) or / and Q and N (for the amide function). In one embodiment, additional sites for PEGylation can be introduced by site-directed mutagenesis by introducing one or more lysine residues. For instance, one or more arginine residues may be mutated to a lysine residue. In another embodiment, additional PEGylation sites are chemically introduced by modifying amino acids on proteins, peptides or fusion proteins of the invention. In one embodiment, PEGs are conjugated to the polypeptides or fusion proteins through a linker. Suitable linkers are well known to the skilled person. A preferred example is cyanuric chloride ((Abuchowski et al., 1977); US 4,179, 337). Conventional separation and purification techniques known in the art can be used to purify pegylated polypeptides of the invention, such as size exclusion (e.g. gel filtration) and ion exchange chromatography. Products may also be separated using SDS- PAGE. In one embodiment, the pegylated polypeptides provided by the invention have a serum half-life in vivo at least 50%, 75%, 100%, 150% or 200% greater than that of an unmodified polypeptide.

[0029] In some embodiments, the agent for PIK3CA protein expression of the invention is selected from the group consisting of an isolated, synthetic or recombinant nucleic acid encoding for PIK3CA protein, a nucleic acid sequence encoding for the fusion protein, a nucleic acid encoding a fragment of a PIK3CA protein, a nucleic acid encoding a fragment of a peptide according to the invention, a cell expressing PIK3CA protein, and / or an agent inducing PIK3CA gene expression and their combinations. In some embodiments, the agent for PIK3CA protein expression of the invention is a polynucleotide encoding a polypeptide that comprises the amino acid sequence as set forth in SEQ ID NO:1. In some embodiments, the agent for PIK3CA protein expression of the invention is a polynucleotide encoding a polypeptide having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identity with the amino acid sequence set forth in SEQ ID NO:1.

[0030] As used herein, a sequence "encoding" an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon. These nucleic acid sequences can be obtained by conventional methods well known to those skilled in the art. Typically, said nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector. So, a further object of the present invention relates to a vector and an expression cassette in which a nucleic acid molecule encoding for a proteins, peptides or fusion proteins of the invention is associated with suitable elements for controlling transcription (in particular promoter, enhancer and, optionally, terminator) and, optionally translation, and also the recombinant vectors into which a nucleic acid molecule in accordance with the invention is inserted. These recombinant vectors may, for example, be cloning vectors, or expression vectors. As used herein, the terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Any expression vector for animal cell can be used. Examples of suitable vectors include pAGE107 (Miyaji et al., 1990), pAGE103 (Mizukami and Itoh, 1987), pHSG274 (Brady et al., 1984), pKCR (O'Hare et al., 1981), pSGl beta d2-4 (Miyaji et al., 1990) and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vectors include adenoviral, lentiviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami and Itoh, 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana et al., 1987), promoter (Mason et al., 1985) and enhancer (Gillies et al., 1983) of immunoglobulin H chain and the like.

[0031] A further aspect of the invention relates to a host cell comprising a nucleic acid molecule encoding for a protein, peptide or a fusion protein according to the invention or a vector according to the invention. In particular, a subject of the present invention is a prokaryotic or eukaryotic host cell genetically transformed with at least one nucleic acid molecule or vector according to the invention. The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed". In a particular embodiment, for expressing and producing proteins, peptides or fusion proteins of the invention, prokaryotic cells, in particular E. coli cells, will be chosen. Actually, according to the invention, it is not mandatory to produce the proteins, peptides or fusion proteins of the invention in a eukaryotic context that will favour post-translational modifications (e.g. glycosylation). Furthermore, prokaryotic cells have the advantages to produce protein in large amounts. If a eukaryotic context is needed, yeasts (e.g. saccharomyces strains) may be particularly suitable since they allow production of large amounts of proteins. Otherwise, typical eukaryotic cell lines such as CHO, BHK-21, COS- 7, C127, PER.C6, YB2 / 0, HEK293, mononuclear macrophage / monocyte-lineage hematopoietic precursors, Haematopoietic stem cells, Mononuclear precursor cells, osteoblast or inactive osteoclast could be used, for their ability to process to the right post-translational modifications of the fusion protein of the invention. The construction of expression vectors in accordance with the invention, and the transformation of the host cells can be carried out using conventional molecular biology techniques. The protein, peptide or the fusion protein of the invention, can, for example, be obtained by culturing genetically transformed cells in accordance with the invention and recovering the proteins, peptides or fusion proteins expressed by said cell, from the culture. They may then, if necessary, be purified by conventional procedures, known in themselves to those skilled in the art, for example by fractional precipitation, in particular ammonium sulfate precipitation, electrophoresis, gel filtration, affinity chromatography, etc. In particular, conventional methods for preparing and purifying recombinant proteins may be used for producing the proteins in accordance with the invention. A further aspect of the invention relates to a method for producing a protein, peptide or a fusion protein of the invention comprising the step consisting of: (i) culturing a transformed host cell according to the invention under conditions suitable to allow expression of said protein, peptide or fusion protein; and (ii) recovering the expressed protein, peptide or fusion protein. In some embodiments, the agent for PIK3CA protein expression of the invention is an agent inducing PIK3CA gene and peptide expression selected from the group consisting of, but not limited to, Human Cytomegalovirus (HCMV), VHL / E HCMV strain, and TB40 / E HCMV strain.

[0032] In a further aspect, the present invention relates to the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression according to the invention in combination with one or more compound promoting skeletal muscle hypertrophy (e.g. steroids).

[0033] In a further aspect, the present invention relates to the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression according to the invention in combination with one or more compound promoting skeletal muscle hypertrophy for use in the treatment of sarcopenia in a subject in need thereof.

[0034] In a further aspect, the present invention relates to the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression according to the invention in combination with one or more compound promoting skeletal muscle hypertrophy for use in the treatment of cachexia in a subject in need thereof.

[0035] In some embodiments, the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression of the present invention is administered sequentially or concomitantly with one or more therapeutic active agent. In one embodiment, said additional active compounds may be contained in the same composition or administrated separately.

[0036] Typically the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression according to the invention as described above are administered to the subject in a therapeutically effective amount. By a "therapeutically effective amount" of the PIK3CA protein or fragment thereof and / or of the agent for PIK3CA protein expression of the present invention as above described is meant a sufficient amount of the PIK3CA protein or fragment thereof and / or of the agent for PIK3CA protein expression for treating the disease at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the PIK3CA protein or fragment thereof and / or an agent for PIK3CA protein expression 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 disorder being treated and the severity of the disorder; activity of the specific the PIK3CA protein or fragment thereof and / or of the agent for PIK3CA protein expression employed; the specific composition employed, 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 PIK3CA protein or fragment thereof and / or of the agent for PIK3CA protein expression employed; the duration of the treatment; drugs used in combination or coincidental with the PIK3CA protein or fragment thereof and / or with the agent for PIK3CA protein expression employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the PIK3CA protein or fragment thereof and / or of the agent for PIK3CA protein expression 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 PIK3CA protein or fragment thereof and / or of the agent for PIK3CA protein expression of the present invention 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 PIK3CA protein or fragment thereof and / or of the agent for PIK3CA protein expression of the present invention, preferably from 1 mg to about 100 mg of the PIK3CA protein or fragment thereof and / or of the agent for PIK3CA protein expression of the present invention. 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. In a particular embodiment, the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression according to the invention may be used in a concentration between 0.01 pM and 20 pM, particularly, the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 pM. According to the invention, the PIK3C A protein or fragment thereof and / or the agent for PIK3CA protein expression of the present invention is administered to the subject in the form of a pharmaceutical composition. Thus, the invention also relates to a therapeutic composition comprising the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression for use in promoting skeletal muscle hypertrophy in a subject in need thereof.

[0037] Typically, the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic 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. In 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.

[0038] 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 the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression 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 PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression of the present invention 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 compounds 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 agent of the present inventions 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 typical methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression of the present invention plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. 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. 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.

[0039] 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.

[0040] FIGURES:

[0041] Figure 1: A mouse model of PZ 3C4-related skeletal muscle overgrowth. A. Male and female body weights of PIK3CAWTand PIK3CAHSA~CreER(n= 15 per group) mice following Cre recombination. B. Adipose tissue and skeletal muscle volume quantification. C. Strength measured using grip test (n= 3 per group). D. Representative pictures of PIK3CAWTand PIK3CAHSA~CreERmice. E. Western blot of pl 10a and GFP and P-AKTSer473in skeletal muscles of PIK3CAWTand PIK3CAHSA~CreERmice. F. GFP immunofluorescence in skeletal muscles of PIK3CAWTand PIK3CAHSA~CreERmice. G. Representative Hematoxylin and eosin (H&E) staining of striated muscles of PIK3CAWTand PIK3CAHSA~CreERmice. Scale bar: 20pm. H. Western blot of P-AKTSer473, total AKT, P-S6RP and S6RP in skeletal muscles of PIK3CAWTand PIK3CAHSA~CreERmice and quantification at 8 and 24 weeks of age. I. Representative immunofluorescence of P-AKTl lir308and P-S6RP in skeletal muscle of PIK3CAWTand PIK3CAHSA~CreERmice. Scale bar: 10 pm. J. Representative immunofluorescence of KI67 in skeletal muscle of PIK3CAWTand PIK3CAHSA~CreERmice. Scale bar: 10 pm. K. Quantification of skeletal muscle cell area of PIK3CAWTand PIK3CAHSA~CreERmice (n = 5 mice per group). L. Twelve-hour fasted glycemia in PIK3CAWTand PIK3CAHSA~CreERmice. M. Insulin circulating levels in PIK3CAWTand PIK3CAHSA~CreERmice. N. Circulating IGF-1 levels in PIK3CAWTand PIK3CAHSA~CreERmice. O. Oral tolerance test (GTT) in PIK3CAWTand PIK3CAHSA~CreERmice (n= 6 mice per group). P. 18F-fluorodeoxy glucose uptake in skeletal muscle of PIK3CAWTand PIK3CAHSA~CreERmice. Q. TMRE S. Mitotraker and R. 10 NAO staining in skeletal muscle of PIK3CAWTand PIK3CAHSA~CreERmice.

[0042] Figure 2: Mouse model characterization and quantitative histological analysis of tibialis anterior (TA) muscle changes in PIK3CAWTand PIK3CAHSA'CreERmice treated with either vehicle or alpelisib. A. Representative immunofluorescence of PAX7 satellite cells and quantification. B. Quantification of number of fibers per muscle and TA muscle cross section area (CSA) in PIK3CAWTand PIK3CAHSA~CreERmice treated with either vehicle or alpelisib. C. Complete blood count in PIK3CAWTand PIK3CAHSA~CreERmice. D. p Islet area in in PIK3CAWTand PIK3CAHSA~CreERmice.

[0043] Figure 3: Alpelisib prevents and reverses skeletal muscle overgrowth in PIK3CAHSA'CreERmice. A. Male body weights of PIK3CAWT(n= 6) and PIK3CAHSA~CreERtreated with either vehicle (n= 12) or preventive (n= 12) or curative alpelisib (n= 12). B. Whole-body T2-weighted magnetic resonance images (MRI) of PIK3CAWTtreated either with vehicle (n= 4) or alpelisib (n= 4), PIK3CAHSA~CreERvehicle-treated (n= 5), PIK3CAHSA~CreER-treated with preventive alpelisib (n= 4) and PIK3CAHSA~CreER-treated with therapeutic alpelisib (n= 4) mice. C. Representative H&E staining of skeletal muscle of PIK3CAWTand PIK3CAHSA~CreERmice treated with either vehicle or preventive or curative alpelisib. Scale bar: 10pm. D. Representative immunofluorescence of P-AKTl lir308and P-S6RP in skeletal muscle of PIK3CAWTand PIK3CAHSA~CreERmice treated with either vehicle or alpelisib. Scale bar: 10 pm. E. Western blot and quantification of Pl 10, P-AKTSer473, AKT, P-S6RP and S6RP in skeletal muscle of P / K3CA‘nand PIK3CAHSA~CreERmice treated with either vehicle or alpelisib (n=4-5 mice per group). F. Twelve-hour fasted glycemia in PIK3CAWTand PIK3CAHSA~CreERmice treated with vehicle, preventive or curative alpelisib (n= 4 mice per group). G. Insulin and H. IGF-1 circulating levels in PIK3CAWTand PIK3CAHSA~CreERmice treated with vehicle, preventive or curative alpelisib (n= 4 mice per group).

[0044] EXAMPLE:

[0045] Material & Methods

[0046] Animals

[0047] Following previously described procedures10, we interbred homozygous R26StopFLP 110* (Stock# 012343) mice with HSA Cre-ER mice (Stock# 025750), both obtained from The Jackson Laboratory. We obtained R26StopFLP110*+ / ~ x HSA Cre-ER+ (henceforth PIK3CAHSA~CreER) and R26StopFLPl 10*+ / - x HAS Cre-ER- (henceforth PIK3CAWTmice. The pl 10* protein expressed by R26StopFLP 110* mice is a constitutively active chimera that contains the iSH2 domain of p85 fused to the NH2- terminus of pl 10 via a flexible glycine linker11. To generate tissue-specific pl l0*-transgenic mice, a cloned loxP- flanked neoR-stop cassette was inserted into a modified version of pROSA26-l, followed by cDNA encoding pl 10* and then a frt-flanked IRES-EGFP cassette and a bovine polyadenylation sequence (R26StopFLP 170*)12. To follow Cre recombination, PIK3CAWTand PIK3CAHSA~CreERmice were then interbred with Gt(ROSA)26SoPm4(ACTBAdTomato’-EGFP)Luo / Jmice10,13. These mice express a cell membrane-localized tdTomato fluorescent protein in all tissues that is replaced by GFP after Cre recombination. Animals were fed ad libitum and housed at a constant ambient temperature in a 12-h light cycle. Animals were fed with regular chow food (2018 Teklad global 18% protein rodent diets, 3.1 Kcal / g, Envigo). Animal procedures were approved by the Ministere de lEnseignement Superieur, de la Recherche et de Plnnovation (APAFIS N°20439-2018121913526398 and 2021110914486827). All appropriate procedures were followed to ensure animal welfare. At the age of 6 weeks, PIK3CAWTand PIK3CAHSA~CreERmice received a daily dose of 40 mg. kg'1tamoxifen for 5 consecutive days to induce Cre recombination. Tamoxifen was administered through oral gavage. I)IK3( 'i'! [and PIK3CAHSA~CreERmice were treated with the PIK3CA inhibitor alpelisib (MedChem Express, Germany; 50 mg.kg-1 in 0.5% carboxymethylcellulose (Sigma Aldrich), daily p.o.) or vehicle (0.5% carboxymethylcellulose (Sigma Aldrich), daily p.o.). Treatment was started either immediately (preventive study) or 6 weeks (therapeutic study) following Cre induction. The last dose of alpelisib or vehicle was administered approximately 3 hours before sacrifice. All mice were fasted for 12 hours before blood glucose measurement (Accuchek Performa, Roche Diagnostic), Magnetic Resonance Imaging and sacrifice.

[0048] Grip test

[0049] Grip strength performance of the mice was evaluated using a grip strength dynamometer obtained from Bioseb (Chaville, France). To assess hindlimb muscle strength, the mice were positioned on a grid surface. Gentle traction was applied to the mice's tails in the opposite direction, and the maximum strength exerted by each mouse before releasing their grip was measured five times. A recovery period of 30 seconds was provided between each measurement. The average value from the five measurements was calculated as the indicator of hindlimb grip strength. Magnetic Resonance Imaging (MRI) evaluation

[0050] All images were acquired with a 4.7-T small-animal MRI system (BioSpec USR47 / 40; Bruker BioSpin, Ettlingen, Germany) on the “Plateforme Imageries du Vivant, Universite de Paris, PARCC, INSERM, Paris, France.” Mice underwent whole-body magnetic resonance imaging using 3D T2-weighted sequences with and without fat saturation. Volumetric evaluation by MRI was performed with 3D Slicer software14and Intelli Space Portal software (Philips Healthcare). Whole body adipose tissue segmentation was obtained by thresholding on T2 weighted sequence and then removing hypersignal related to water on T2 weighted sequence with fat saturation. Muscles of each limb were manually segmented according to their low signal intensity and anatomy on T2 weighted sequence. For both adipose and muscle tissues, volume was calculated by summing images based on 2D contours and slice thickness.

[0051] FDG PET-CT imaging

[0052] Mice were fasted overnight with free access to water. Mice were then anesthetized (2±0.5% isoflurane in dioxygen) and weighed, and glycemia was measured in blood drawn from the caudal ventral artery using an Accu-Chek® Aviva Nano A (Accu-Chek, France). A 29G needle catheter (Fischer Scientific, France) connected to 5 cm polyethylene tubing (Tygon Microbore Tubing, 0.010" x 0.030"OD; Fisher Scientific, France) was inserted into the caudal vein for radiotracer injection. 9.2±1.5 MBq of 2'-deoxy-2'-[18F]fluoro-D-glucose (FDG; Advanced Applied Applications, France) in 0.2 mL saline was injected via the catheter. Mice were left awake in their cage for 45 min and then installed into the PET-CT dedicated bed. Respiration and body temperature were registered. Body temperature was maintained at 34±2 °C, and anesthesia was controlled according to the breathing rate throughout the entire PET-CT examination. CT was acquired in a PET-CT scanner (nanoScan PET-CT; Mediso Medical Imaging Systems, Hungary) using the following acquisition parameters: semicircular mode, 50 kV tension, 720 projections full scan, 300 ms per projection, and binning 1 :4. CT projections were reconstructed by filtered retroproj ection (filter: cosine; cutoff: 100%) using Nucline 3.00.010.0000 software (Mediso Medical Imaging Systems, Hungary). Fifty-five minutes post tracer injection, PET data were collected for 10 min in list mode and binned using a 5 ns time window, with a 400-600 keV energy window and a 1 :5 coincidence mode. Data were reconstructed using the Tera-Tomo reconstruction engine (3D-OSEM-based manufactured customized algorithm) with expectation maximization iterations, scatter, and attenuation correction. Volumes of interest (VOIs) were delineated on the organs or anatomical structure of interest on PET / CT fusion slices using the PMOD software package (PMOD Technologies Ltd, Zurich, Switzerland). FDG accumulation was quantified as the standard uptake value (SUV), which measures the ratio of the radioactivity concentration in VOI to the whole-body concentration of the injected radioactivity.

[0053] Blood and Plasma analysis

[0054] At the end of each experiment, blood samples were collected from the mice in EDTA- coated tubes. To measure blood count, fresh blood samples were analyzed on a hematology analyzer (ProCyte Dx; IDEXX Laboratories) and centrifuged at 500*g for 15 min. The collected plasma concentration was used to determine insulin (U-PLEX mouse insulin Assay (Meso Scale Discovery) ref# K1526HK) and IGF-1 (Novus Biologicals, ref# MG100) circulating levels using enzymatic methods from commercially available kits.

[0055] Oral glucose test

[0056] Mice were fasted overnight (12 h) with access to drinking water. All body weights were measured, and tails were carefully cut for blood glucose determination (time point 0). During glucose tolerance test (GTT), freshly prepared glucose solution was administered orally (1 g glucose / kg body weight of 20% glucose solution in water, (Sigma, ref# G8270)). After 15, 30, 45, 60, 90 and 120 minutes, blood glucose was measured again at all time points.

[0057] Morphological analysis

[0058] Mouse tissues were fixed in 4% paraformaldehyde and paraffin embedded. Tissue sections (4 pm thick) were stained with hematoxylin and eosin (H&E).

[0059] Measurement of muscle fibers size

[0060] Hematoxylin and eosin (H&E) slides were scanned with a NanoZoomer 2.0HT (Hamamatsu) and analyzed with Qupath-0.2.313. Muscles were segmented using deep learning Cellpose algorithm14. For both, areas were then measured with Fiji15.

[0061] Immunohistochemistry and immunofluorescence

[0062] Paraffin-embedded tissue sections (4 pm) were submitted to antigen retrieval protocols using high temperature (120 °C) and high pressure in citrate buffer and a pressure cooker. Sections were then incubated with primary antibodies. For the immunofluorescence procedure, appropriate Al exafluor-conjugated secondary antibodies (Thermo Fischer Scientific) were incubated on the samples and analyzed using an LSM 700 confocal microscope (Zeiss) or Eclipse Ni-E (Nikon). Immunohistochemistry revelation was performed with appropriate horseradish peroxidase (HRP) linked secondary antibodies and analyzed with E800 (Nikon).

[0063] Western blot

[0064] Tissues were crushed and then lysed in RIPA lysis buffer supplemented with phosphatase and protease inhibitors. Protein concentrations were determined through the bicinchoninic acid method (Pierce). Then, protein extracts were resolved by SDS-PAGE before being transferred onto the appropriate membrane and incubated with the primary antibody followed by the appropriate peroxidase-conjugated secondary antibody (dilution 1 : 10,000). Chemiluminescence was acquired using Chemidoc MP, and bands were quantitated using Image Lab Software (Bio-Rad Laboratories).

[0065] Tissue digestion

[0066] Muscles of PIK3CAWTand PIK3CAHSA~CreERmice were harvested and rinsed with PBS IX (Gibco). After cutting them into small pieces, digestion buffer was added with DNAse (0,lmg / mL), Dispase I (0,8mg / mL), Collagenase P (0,2mg / mL) in lOmL of RPMI (Gibco) and incubated for 40minutes at 37°C on GentleMACS (Miltenyi) with the appropriate program. Following dissociation, tissues were filtered (70pm, Clearline), centrifuged 5 min at 250g, and resuspended in PBS.

[0067] Mitotracker cytometry

[0068] Following treatment with either vehicle or alpelisib, muscle of PIK3CAWTand PIK3CAHSA~CreERmice were collected and digested. Cells were resuspended in lOOpL of PBS in a 96-wells round-bottomed plate (ThermoFisher). Cells were incubated with fluorescent buffer in the dark for 10 minutes at 37°C and rinsed with PBS-SVF 2%. Cells were analyzed using Sony Spectral ID7000 and all flow data was processed with Sony ID7000 software and Kaluza software.

[0069] Imaging Flow Cytometry (ImageStream)

[0070] Skeletal muscles from PIK3CAWTand PIK3CAHSA~CreERmice were isolated, rinsed in phosphate buffered saline IX, cute in small pieces and digested as detailed below. After dissociation, cell suspensions were filtered (70pm, Clearline), centrifuged 10 min at 350g, and resuspended in phosphate buffered saline solution IX supplemented with 2% fetal bovine serum and EDTA at 0.5 m / mol. Cells were then transferred in microtube. Samples were run on an ImageStream ISX mkll (Amnis part of Luminex) that combines flow cytometry with detailed cell imaging. Magnification (40X) was used for all acquisitions. Data were acquired with INSPIRE software (Amnis part of Luminex) and analyzed with IDEAS software (v.6.2, Amnis part of Luminex).

[0071] Targeted LC-MS metabolites analyses

[0072] Plasma and serum were obtained after centrifugation of the blood at 500 g for 10 min. Blood samples were obtained in EDTA tubes for plasma analysis and EDTA-free tubes for serum analysis. Cells, plasma and serum samples were immediately snap-frozen in liquid nitrogen. For the LC-MS analyses, metabolites were extracted as previously described9. Briefly, the extraction solution was composed of 50% methanol, 30% ACN, and 20% water. The volume of the added extraction solution was adjusted to the cell number (1 ml per 1 million cells) or plasma and serum volume (200 pl per 10 pl of plasma or serum). After the addition of extraction solution, samples were vortexed for 5 min at 4 C and then centrifuged at 16,000 g for 15 min at 4 C. The supernatants were collected and stored at -80 C until the analyses were performed. LC-MS analyses were conducted using a QExactive Plus Orbitrap mass spectrometer (Thermo) equipped with an Ion Max source and a HESI II probe coupled to a Dionex UltiMate 3000 UPLC system (Thermo). External mass calibration was performed using the standard calibration mixture every 7 d as recommended by the manufacturer. Five microliters of each sample was injected onto Zic-pHilic (150 mm * 2.1 mm i.d. 5 pm) with the guard column (20 mm * 2.1 mm i.d. 5 pm) (Millipore) for liquid chromatography separation. Buffer A was 20 mM ammonium carbonate and 0.1% ammonium hydroxide (pH 9.2); buffer B was acetonitrile. The chromatographic gradient was run at a flow rate of 0.200 pl / min as follows: for 0-20 min, linear gradient from 80 to 20% B; for 20-20.5 min, linear gradient from 20 to 80% B; for 20.5-28 min, hold at 80% B. The mass spectrometer was operated in full-scan polarity switching mode with the spray voltage set to 2.5 kV and the heated capillary held at 320 C. The sheath gas flow was set to 20 units, the auxiliary gas flow was set to 5 units, and the sweep gas flow was set to 0 units. The metabolites were detected across a mass range of 75- 1000 m / z at a resolution of 35,000 (at 200 m / z) with the AGC target set to 106 and a maximum injection time of 250 ms. Lock masses were used to ensure mass accuracy below 5 ppm. Data were acquired with Thermo Xcalibur 4.0.27.131 software (Thermo). The peak areas of metabolites were determined using Thermo TraceFinder 3.3 SP1 software (Thermo) and identified by the exact mass of each singly charged ion and by known retention time in the HPLC column. Patients

[0073] HFMH was defined as congenital facial asymmetry with thickening of soft tissues leading to a set of specific clinical features: narrow palpebral fissure, eyebrow ptosis, nose deviation with small alar rim, external ear asymmetry and displacement with prominent concha, chin deviation with skin dimpling, and lip commissure canting. Following procedures previously described10, the study was conducted on 5 pediatric patients, including 3 females who were followed at Hopital Necker Enfants Malades. This protocol was approved by the Agence Nationale de Securite du Medicament et des Produits de Sante and the local ethical committee (Comite d’Ethique de Necker - Enfants Malades). Written informed consent was obtained from adult patients and from the parents of pediatric patients. Alpelisib was compassionately offered by Novartis. Biopsies of the buccinator and / or masseter muscles of the affected and control sides were performed in 5 / 5 HFMH patients for diagnosis. Control buccinator and masseter muscles were harvested on pediatric patients admitted for facial lacerations and / or mandibular trauma, that required surgical approaches to this anatomical region.

[0074] Data analysis and statistics

[0075] Data were expressed as the means ± s.e.m. Differences between the experimental groups were evaluated using ANOVA, followed by the Tukey-Kramer post hoc test when the results were significant (P < 0.05). When only two groups were compared, Mann-Whitney tests were used. Statistical analyses were performed using GraphPad Prism software (version 10.0.0).

[0076] Results

[0077] PIK3CA gain-of-function mutations explain hemifacial myohyperplasia

[0078] We identified 5 pediatric patients including 3 girls, ranging from 4 to 15 years old with clinical presentation of HFMH (data not shown). In more details, 5 / 5 patients had chin skin dimpling and small nasal vestibule, 4 / 5 had chin deviation, dysmorphic ear, narrow palpebral fissure and eyebrow ptosis, 3 / 5 had lip commissure canting and finally 3 / 5 nasal deviation. All patients had MRI imaging demonstrating muscular hypertrophy affecting in various combinations facial, masticatory (pterygoids and masseter) and neck (scalene and sternocleidomastoid) muscles (data not shown). All patients had benefited from CT-scans imaging before HFMH diagnosis that showed bone anomalies in 4 / 5 cases including mild chin deviation in 3 / 4 and major skeletal deformation with homolateral maxillary and mandibular growth impairment in 1 / 5. All patients had available electroneuromyography (ENMG) assessment that showed normal results, including normal blink reflex responses and needle electromyogram (EMG) of facial muscles. 2 / 5 patients had been treated with botulinum toxin with injections performed every 6 months. Patient 5 benefited from 3 injections in mentalis, zygomaticus minor and major and orbicularis oculi muscles (130 then twice 200 I.U.) and patient 3 benefited from 9 injections in the same three muscles (I.U. injected from 50 to 400). For both patients, no relevant esthetic or functional results were observed. We performed muscular biopsies from the affected zones (oral mucosa, buccinator muscle and masseter muscle) in 5 / 5 patients, under general or local anesthesia depending on age, through an intraoral approach. Standard histology was normal. Genotyping of the biopsies revealed the presence of PIK3CA variants in all tested samples. Pathogenic E545K variant was found in patient 1 and 5 with a variant allelic frequency (VAF) of 15% and 14% respectively. The E542K variant was detected in patient 3 (VAF 12%) and 4 (VAF 21%). Lastly, the H1047R variant was identified in patient 2 (VAF 25%) (Table 1). All variants were already characterized as pathogenic7. Thus, we concluded that PIK3CA gain-of-function mutations explained all 5 HFMH cases.

[0079] A mouse model ofPIK3CA gain-of-function mutations in skeletal muscles

[0080] In order to gain insight into the mechanisms of PIK3CA -related muscle overgrowth we designed a mouse model carrying specifically a PIK3CA gain-of-function mutation in striated muscle. To this aim, we interbred the R26StopFLPl 10* mouse strain with HSA Cre mice to generate PIK3CAHSA~CreERanimals that express a constitutively overactivated form of PIK3CA upon tamoxifen administration. To follow Cre recombination, PIK3CAHSA~CreERmice were then interbred with Gt(ROSA)26Sortm4(ACTB-tdTomato-EGFP)Luo / J mice. In all tissues, these mice express a cell membrane-localized tdTomato fluorescent protein that is replaced by GFP after Cre recombination. To overcome developmental issues, we used six-week-old mice that were treated with a daily dose of 40 mg.kg-1 tamoxifen for five days to induce Cre recombination. We observed that starting 3 weeks after Cre recombination, PIK3CAHSA~CreERmice progressively gained weight compared to their wild-type littermates (PIK3CAWT) (Figure 1A). This was the case for both males and females. Around 11 weeks post induction in males and 24 weeks in females, body weight of controls mice became higher than in PIK3CAHSA~CreER(Figure 1A). Whole-body MRI at 15 weeks post tamoxifen, revealed skeletal muscle hypertrophy in PIK3CAHSA~CreERmice that was approximately twice as in controls (Figure IB and data not shown). Fat content in PIK3CAHSA~CreERwas significantly reduced compared to controls (Figure IB and data not shown). Skeletal muscle hypertrophy was associated with gain in muscle strength compared to controls (Figure 1C). We sacrificed 20 controls and 20 PIK3CAHSA~CreERmice 15 weeks after tamoxifen administration. Necropsy examination confirmed diffuse skeletal muscle hypertrophy with adipose tissue shrinkage compared to control littermates (Figure ID). We confirmed that mutant pl 10a (pl 10*) and GFP were expressed in striated muscles of PIK3CAHSA~CreERmice (Figure IE and data not shown). Histological examination revealed hypertrophic striated cells (Figure IF and Figure 2A, 2B and data not shown). We did not observe any other histological anomalies. Western blot and immunofluorescence studies showed AKT / mTOR pathway activation in striated muscles of PIK3CAHSA~CreERmice (Figure 1G and 1H). Mechanistically, PIK3CA is involved in cell growth and proliferation but KI67 immunostaining did not show hyperplasia (Figure II). However, using the Amnis ImageStream® system, we confirmed that cells isolated from striated muscle of PIK3CAHSA~CreERmice were hypertrophic compared to controls (Figure 1J). Blood cell count did not reveal any anomalies (Figure 2C). However, we observed that PIK3CAHSA~CreERmice were hypoglycemic compared to controls (Figure IK). We recently identified an endocrine loop leading to chronic hypoglycemia in another mouse model carrying specifically PIK3CA gain-of-function mutation in adipose tissue9. Indeed, PIK3CA gain-of- function mutation led to permanent GLUT transporters addressing at the cell membrane allowing glucose entry and subsequent hypoglycemia. The latter reduced insulin secretion, which in turn dramatically increased IGFBP production followed by liver IGF-1 sequestration and a reduction in IGF-1 circulating levels similar to clinical observations. We obtained similar results in PIK3CAHSA~CreERmouse model with low insulin and IGF1 levels (Figure IL, IM and Figure 2D) but conserved insulin secretion capacities as demonstrated using oral glucose tolerance test (Figure IN). Consistently, PET TDM showed higher FDG muscular uptake in PIK3CAHSA~CreERmice compared to controls (Figure IO). Mitochondria play a major role in energy for striated muscle metabolism. PI3K have been involved in mitochondrial function16, we therefore explored the impact of PIK3CA gain-of-function on mitochondria number and activity in the PIK3CAHSA~CreERmouse model. Flow cytometry of isolated striated muscle cells revealed alteration in the mitochondrial transmembrane potential as assessed by tetramethylrhodamine ethyl ester (TMRE) staining with reduced mitotracker expression (Figure IP and IQ). 10-N-nonyl acridine orange (NAO) staining showed reduced mitochondrial mass in straited muscle cells of PIK3CAHSA~CreERcompared to controls (Figure 1R) Plasma metabolites metabolomics analysis revealed energy production with increased ATP production with mitochondria and anabolic activities (data not shown). Hence, we created a mouse model of PIK3CA-related skeletal muscle overgrowth affecting both the gross morphology, the cellular structure and the function of striated muscles.

[0081] A PIK3CA inhibitor prevents and reduces striated muscle hypertrophy in a PIK3CAHSA~CreERmouse model

[0082] We decided to test whether a PIK3CA inhibitor, namely alpelisib, was efficient at reducing skeletal muscle overgrowth in our mouse model. To this end, we used two different approaches. We first administered alpelisib daily starting 48 hours after Cre induction as a preventive study. We observed that alpelisib-treated PIK3CAHSA~CreERmice had an overtly normal appearance during the 22 weeks of treatment and a body weight increase similar to that of controls (Figure 3A). MRI performed 8 weeks after alpelisib initiation showed no skeletal muscle overgrowth compared to vehicle PIK3CAHSA~CreERtreated mice (Figure 3B and data not shown). Mice were then sacrificed, and necropsy examination confirmed that alpelisib- treated PIK3CAHSA~CreERmice had no muscle hypertrophy and were indistinguishable from controls. Histology showed no difference between PIK3CAHSA~CreERmice treated with preventive alpelisib and PIK3CA'Tm cc (Figure 3C). Western blot and immunofluorescent studies showed blunted AKT and S6RP phosphorylation (Figure 3D and 3E). PIK3CAHSA~ CreERmjcetreated with preventive alpelisib demonstrated normal twelve-hour fasted glycemia compared to PIK3CAHSA~CreERvehicle treated mice (Figure 3F). Consistently, insulin and IGF- 1 circulating levels were corrected (Figure 3G and 3H). Next, we administered alpelisib to PIK3CAHSA~CreERmice 2 weeks after Cre induction when global muscle hypertrophy was already prominent for 20 additional weeks, as a therapeutic study. Following alpelisib introduction, we noticed rapid body weight decrease in alpelisib-treated PIK3CAHSA~CreERmice comparted to control (Figure 3A). MRI performed at 8 weeks showed significant reduction in muscular volume compared to PIK3CAHSA~CreERvehicle mice (Figure 2B, 3B and data not shown). Similarly to previous experiments with early alpelisib introduction, we observed that the histology of the striated muscle of PIK3CAHSA~CreERmice treated in the therapeutic study was conserved (Figure 3C), with reduced phosphorylation of the AKT and S6RP proteins (Figure 3D and 3E). Blood glucose, insulin and IGF-1 circulating levels were increased in the therapeutic alpelisib group (Figure 3F, 3G and 3H). We then concluded that PIK3CA is a relevant target to induce striated muscle hypertrophy. CONCLUSION

[0083] Here, we report for the first time that HFMH is due to a somatic gain-of-function mutation of PIK3CA in striated muscles. We further created a mouse model harboring a PIK3CA gain-of-function mutation in striated muscles that demonstrates muscles overgrowth. Muscle overgrowth was reversed by PIK3CA inhibition using alpelisib.

[0084] TABLE 1 : Patient characteristics a VAF: Variant allele frequency, corresponds to the percentage of alternate or mutant reads to total reads detected by next generation sequencing. b COSMIC: Catalogue Of Somatic Mutations In Cancer

[0085] REFERENCES:

[0086] 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.

[0087] 1 Lee, S., Sze, R., Murakami, C., Gruss, J. & Cunningham, M. Hemifacial myohyperplasia: description of a new syndrome. American journal of medical genetics 103, 326-333 (2001).

[0088] 2 Castillo Taucher, S., Pardo Vargas, R. A. & Caivi Leon, D. Hemifacial myohyperplasia: an additional case. Am J Med Genet A 116A, 103-104 (2003). https: / / doi.org: 10.1002 / ajmg. a.10839 3 Pereira-Perdomo, D. F., Velez-Forero, J. & Prada-Madrid, R. Hemifacial myohyperplasia sequence. Am J Med Genet A 152A, 1770-1773 (2010). https: / / doi.org: 10.1002 / ajmg.a.33428

[0089] 4 Miranda, R. T., Barros, L. M., Santos, L. A., Bonan, P. R. & Martelli, H., Jr. Clinical and imaging features in a patient with hemifacial hyperplasia. J Oral Sci 52, 509-512 (2010). https: / / doi.org: 10.2334 / josnusd.52.509

[0090] 5 Siponen, M., Sandor, G. K., Ylikontiola, L., Salo, T. & Tuominen, H. Multiple orofacial intraneural perineuriomas in a patient with hemifacial hyperplasia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 104, e38-44 (2007). https: / / doi.org: 10.1016 / j.tripleo.2006.12.030

[0091] 6 Zissman, S., Cooperman, Y., Leshem, D. & Gur, E. Progressive Surgical

[0092] Management of Hemifacial Myohyperplasia for Improved Functional and Aesthetic Results. Plast Reconstr Surg Glob Open 8, e2724 (2020). https: / / doi.org: 10.1097 / GOX.0000000000002724

[0093] 7 Canaud, G., Hammill, A. M., Adams, D., Vikkula, M. & Keppler-Noreuil, K. M. A review of mechanisms of disease across PIK3CA-related disorders with vascular manifestations. Orphanet J Rare Dis 16, 306 (2021). https: / / doi.org: 10.1186 / sl3023-021- 01929-8

[0094] 8 Madsen, R. R. & Vanhaesebroeck, B. Cracking the context-specific PI3K signaling code. Science signaling 13 (2020). https: / / doi.org: 10.1126 / scisignal.aay2940

[0095] 9 Ladraa, S. et al. PIK3CA gain-of-function mutation in adipose tissue induces metabolic reprogramming with Warburg-like effect and severe endocrine disruption. Sci Adv 8, eade7823 (2022). https: / / doi.org: 10.1126 / sciadv.ade7823

[0096] 10 Delestre, F. et al. Alpelisib administration reduced lymphatic malformations in a mouse model and in patients. Science translational medicine 13, eabg0809 (2021). https: / / doi.org: 10.1126 / scitranslmed.abg0809

[0097] 11 Klippel, A. et al. Membrane localization of phosphatidylinositol 3-kinase is sufficient to activate multiple signal-transducing kinase pathways. Molecular and cellular biology 16, 4117-4127 (1996).

[0098] 12 Srinivasan, L. et al. PI3 kinase signals BCR-dependent mature B cell survival. Cell 139, 573-586 (2009). https: / / doi.org: 10.1016 / j.cell.2009.08.041

[0099] 13 Bankhead, P. et al. QuPath: Open source software for digital pathology image analysis. Sci Rep 7, 16878 (2017). https: / / doi.org: 10.1038 / s41598-017-17204-5 14 Stringer, C., Wang, T., Michaelos, M. & Pachitariu, M. Cellpose: a generalist algorithm for cellular segmentation. Nature methods 18, 100-106 (2021). https: / / doi.org: 10.1038 / s41592-020-01018-x

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[0101] 16 Li, M. E. et al. Role of pl 10a subunit of PI3-kinase in skeletal muscle mitochondrial homeostasis and metabolism. Nat Commun 10, 3412 (2019). https: / / doi.org: 10.1038 / s41467-019-11265-y

Claims

CLAIMS:

1. A PIK3CA protein or fragment thereof and / or an agent for PIK3CA protein expression for use in promoting skeletal muscle hypertrophy in a subject in need thereof.

2. A therapeutic composition comprising the PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression for use according to claim 1.

3. The PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression for use according to any of claims 1 or 2, wherein the subject suffers from muscular atrophy, muscular dystrophy or muscular hypotrophy.

4. The PIK3CA protein or fragment thereof and / or the agent for PIK3CA protein expression for use according to any of claims 1 to 3, wherein the subject suffers from muscle wasting, decreased muscle tone, muscle weakness or muscle fragility.

5. The PIK3CA protein or fragment thereof and / or the agent for PIKCA protein expression for use according to claim 1 or 2, wherein the subject suffers from sarcopenia.

6. The PIK3CA protein or fragment thereof and / or the agent for PIKCA protein expression for use according to claim 1 or 2, wherein the subject suffers from cachexia.

7. The PIK3CA protein or fragment thereof and / or the agent for PIKCA protein expression for use according to claim 1 or 2, wherein the subject suffers from obesity.

8. The PIK3CA protein or fragment thereof and / or the agent for PIKCA protein expression for use according to any of claims 1 to 7, wherein the PIK3CA protein comprises a sequence as set forth by SEQ ID NO:1.

9. The PIK3CA protein or fragment thereof and / or the agent for PIKCA protein expression for use according to any of claims 1 to 7, wherein the agent for PIK3CA protein expression is a polynucleotide encoding a polypeptide that comprises the amino acid sequence as set forth in SEQ ID NO:1.

10. The PIK3CA protein or fragment thereof and / or the agent for PIKCA protein expression for use according to any of claims 1 to 7, wherein the agent for PIK3CA protein expression is a small organic molecule.

11. The PIK3CA protein or fragment thereof and / or the agent for PIKCA protein expression for use according to any of claims 1 to 7, wherein the agent for PIK3CA protein expression is selected from a list consisting in 1, 2,9,10-tetram ethoxy-7H- dibenzo[de,g]quinolin-7-one and l-(7-((2-((4-(4-ethylpiperazin-l- yl)phenyl)amino)pyridin-4-yl)amino)indolin-l-yl)ethan-l-one.

12. A method for promoting skeletal muscle hypertrophy in a subject in need thereof comprising a step of administering said patient with a therapeutically effective amount of a PIK3CA protein or fragment thereof and / or an agent for PIK3CA protein expression.

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