WO2016120652A1 - Muscle differentiation - Google Patents

Abstract

PPARγ controls the expression of the TGFβ family member, GDF3, which in turn regulates the restoration of skeletal muscle integrity by promoting muscle progenitor cell differentiation. Thus, GDF3 as a secreted extrinsic effector protein acting on myoblasts and serving as an exclusively macrophage-derived differentiation promoting factor in tissue repair. Thus, the invention relates to a GDF3 compound for use in the treatment of a patient having a condition or disease associated with impaired muscle, said patient being in need of muscle differentiation, or in improving differentiation of muscle of a patient in need thereof by differentiation of newly formed muscle fibers. The invention also relates to pharmaceutical preparations and methods of treatment including cell therapies.

Description

MUSCLE DIFFERENTIATION

The present invention relates to the field of muscle regeneration.

BACKGROUND ART

Tissues suffer physical and biochemical damage during an organism's lifetime. In order to maintain the body's integrity and homeostasis, it is critically important to completely regenerate [ad integrum) bodily damage. Certain organs, such as skeletal muscle, possess excellent regenerative potential by which a complete regeneration is possible. In many cases a straightforward paradigm can be applied to these regenerative processes whereby organ injury induces expansion and differentiation of a quiescent population of tissue-specific stem cell-like progenitors. Strikingly, the immune system has an indispensible role in tissue regeneration. Impaired injury-related immune response has been shown to greatly influence regeneration in a wide variety of organs, including liver, central nervous system or skeletal muscle (Chazaud, 2014; Duffield et al, 2005; Laflamme and Murry, 2011; Rapalino et al, 1998). Importantly, immune cells and in particular M<t s have a dual role during damage and regeneration. First, these cells need to react to the injury, remove damaged tissues and in the second phase, initiate restoration of tissue integrity via promoting repair mechanisms. It can be presumed that during this latter phase the immune response to tissue injury regulates the reengagement of tissue progenitor cell populations to support cellular growth and differentiation. Our knowledge is fragmented on how macrophages (M<t s) change their phenotype, employ sensory and regulatory mechanisms and change their effector functions to serve such reparatory roles. This is particularly important because the proper signaling between the participating cell types can ensure precisely timed progression of repair, while avoiding asynchrony, which can lead to delay, fibrosis and/or chronic inflammation (Dadgar et al., 2014). We sought to identify such integrated sensory, regulatory and effector mechanisms equipping a macrophage with the capacity to contribute to timed progression of repair of muscle, in particular skeletal muscle.

Musculoskeletal Disorders (MSDs), including skeletal muscle disorders and injuries are a common and costly problem for people and companies worldwide. In particular, in case of skeletal muscle injuries, while the application of non-steroid anti-inflammatory drugs are applied and use of growth factors, particularly bFGF, NGF, and IGF-1, as a novel therapeutic approach is suggested, a medicinal treatment that enhances the repair of injured muscle could have significant clinical applications. It is advised that future research should focus on the use of growth factors that facilitate muscle regeneration in vivo and that the balance between growth and differentiation must be maintained in order to restore functional muscle structure [Baoge L et al., Treatment of Skeletal Muscle Injury: A Review. ISRN Orthopedics, Volume 2012 (2012), Article ID 689012]. Moreover, in a large number of muscle disorders, e.g. myopathies (diseases of skeletal muscle which are not caused by nerve disorders), wherein the skeletal or voluntary muscles became weak or wasted, regeneration of the impaired muscle would be highly desirable.

Skeletal muscle injury is a rapid but precisely timed and tightly regulated process in which a complete restoration of muscle structure can be achieved within weeks of an almost complete destruction of the tissue. The critical contribution of immune cells and principally M<t s to skeletal muscle regeneration is well documented (Arnold et al., 2007; Bryer et al., 2008; Burzyn et al., 2013). While many aspects of skeletal muscle development and regeneration are well understood and can be studied in vitro (Yin et al., 2013), the mechanism behind is only partly understood.

Several attempts have been made to treat muscle related disorders.

For example, growth and differentiation factor-8 (GDF-8), also known as myostatin, is a member of the transforming growth factor-beta (TGF-0 superfamily of structurally related growth factors, all of which possess important physiological growth-regulatory and morphogenetic properties) is a negative regulator of skeletal muscle mass, and there is considerable interest in identifying factors which regulate its biological activity.

WO2003072715 relates to the use of proteins comprising at least one follistatin domain to modulate the level or activity of growth and differentiation factor-8 (GDF-8). The invention is useful for treating muscular diseases and disorders, particularly those in which an increase in muscle tissue would be therapeutically beneficial.

WO2008030706 among other similar publications relates to anti-myostatin monoclonal antibodies that preferentially bind myostatin (GDF-8) over GDF-11 and are resistant to protein cleavage, and use of the antibodies for treatment, prophylaxis or diagnosis of various disorders or conditions in mammalian and avian species.

WO2010083034A1 discloses that ActRIIB (and its fusion proteins) can be used to increase circulating adiponectin levels in mouse models. Therefore, ActRIIB-derived agents can be used to treat or prevent hypoadiponectinemia. ActRIIB also has been identified as a type II serine/threonine kinase receptor (required for binding ligands and for expression of type I receptor) for activins and several other TGF- beta family proteins including GDF3 (mentioned in line with GDF8, i.e. myostatin), with which it can biochemically interact as a ligand. Thus, WO2010083034A1 disclosed that such ActRIIB-ligands, like GDF3 should be antagonized.

Similarly, W02014000042 Al relates to an endogenous activin A and/or activin B activity suppressor propeptide or nucleic acid is useful in composition, preferably pharmaceutical composition for treating or preventing activin-induced muscle wasting, cachexia-anorexia syndrome and conditions induced or exacerbated by over expression of active TGF-β family ligands chosen from activin B, activin A, activin C, activin E, bone morphogenetic protein 7 (BMP7), BMP5, BMP6, BMP8A, BMP8B, BMP2, BMP4, BMP10, growth differentiation factor 2 (GDF2), GDF5, GDF6, GDF7, BMP3, BMP3B, left-right determination factor (lefty) 1 , lefty2, GDF1 , GDF3, nodal growth differentiation factor (NODAL), BMP 15, GDF9, GDF 15, mullerian inhibiting substance (MIS) and inhibin, and in therapy.

Quite surprisingly, the present inventors have found that PPARy regulated the expression of a secreted factor, GDF3 (Growth/differentiation factor 3, also known as Vg-related gene 2 (Vgr-2), KFS3, MCOP7 or MCOPCB6), whose expression was induced during muscle regeneration in a PPARy-dependent manner in infiltrating M<t s and that GDF3 could enhance the differentiation of primary myogenic precursor cells fMPCs) in in vitro cultures. GDF3 also resulted in a robust increase on myotube fusion in primary myoblast cell lines. Thus GDF3. once released from ΜΦΒ within the injured /regenerating tissues, could regulate molecular pathways relevant to muscle differentiation in primary muscle cells and thereby could skew the balance between myoblast proliferation and differentiation towards the latter.

Several functions have been attributed to GDF3 so far.

In humans the GDF3 gene is located on the short (p) arm of chromosome 12 at position 13.1. More precisely, the GDF3 gene is located from base pair 7,689,784 to base pair 7,695,763 on chromosome 12.

GDF3 itself is part of the transforming growth factor beta (TGFP) superfamily, which is a group of proteins that help control the growth and development of tissues throughout the body. Within the TGF superfamily, the GDF3 protein belongs to the bone morphogenetic protein family, which is involved in regulating the growth and maturation (differentiation) of bone and cartilage. The proteins in this family are regulators of cell growth and differentiation in both embryonic and adult tissue. While the GDF3 gene is known to be involved in bone and cartilage development, its exact role has been unclear [see Levine AJ, Brivanlou AH. GDF3 at the crossroads of TGF-beta signaling. Cell Cycle. 2006 May;5(10):1069-73. Epub 2006 May 15. Review]

Expression of GDF3 occurs in ossifying bone during embryonic development and in the brain, thymus, spleen, bone marrow and adipose tissue of adults [Chen C, Ware SM, Sato A, Houston-Hawkins DE, Habas R, Matzuk MM, Shen MM, Brown CW (January 2006). "The Vgl-related protein Gdf3 acts in a Nodal signaling pathway in the pre-gastrulation mouse embryo". Development 133 (2): 319-29.]. It has been shown to negatively and positively control differentiation of embryonic stem cells in mice and humans [Levine A, Brivanlou A (2006). "GDF3, a BMP inhibitor, regulates cell fate in stem cells and early embryos". Development 133 (2): 209-16.]

A few mutations in the GDF3 gene have been found to cause Klippel-Feil syndrome, a condition characterized by the abnormal joining (fusion) of two or more spinal bones in the neck (cervical vertebrae).

Uniprot entry Q9NR23 - GDF3_HUMAN on GDF3 lists among the function of GDF3 the negative regulation of myoblast differentiation.

Apparently no hint can be found in the prior art for using GDF3 or related solutions in muscle differentiation.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to a GDF3 compound for use in the treatment of a subject, preferably a patient having a condition or disease associated with impaired muscle and said patient being in need of muscle differentiation. The invention also relates to a GDF3 compound for use in improving differentiation of muscle of a subject or a patient in need thereof.

Preferably the muscle differentiation comprises the differentiation of myoblasts to myotubes (myotube differentiation). Preferably the muscle differentiation is myotube differentiation.

Preferably the GDF3 compound stimulates or up-regulates differentiation of muscle cells. Preferably the GDF3 compound helps differentiation of newly formed muscle fibers. Preferably the GDF3 compound is a factor of macrophage origin.

In particular, impaired muscle implies or has impaired muscle structure and/or impaired muscle function. In particular, in the impaired muscle there is a need of myotube formation. In the impaired muscle of said patient to be treated in particular undifferentiated myoblasts are present which are optionally detected.

The invention also relates to a GDF3 compound for use in enhancing differentiation of myoblasts or myogenic precursor cells (MPCs) in a subject, preferably a patient.

In a particular embodiment the GDF3 compound decreases myoblast proliferation.

In a particular embodiment the GDF3 compound increases myotube fusion.

In a particular embodiment the GDF3 compound skews or shifts the balance between myoblast proliferation and differentiation towards differentiation.

The invention also relates to a GDF3 compound for use in improving regeneration or differentiation of muscle of a patient in need thereof.

The invention also relates to a use of GDF3 compound for improving muscle regeneration in a condition or disease that is characterized by a failure in muscle regeneration or muscle differentiation or for any condition as defined herein.

The invention also relates to a use of GDF3 compound in the preparation of a medicament or pharmaceutical preparation for use in improving muscle regeneration in a condition or disease that is characterized by a failure in muscle regeneration or for any condition as defined herein.

The invention relates to GDF3 for use in improving regeneration of muscle.

Muscle or tissue

Preferably the muscle is skeletal muscle.

Preferably the muscle comprises undifferentiated myoblasts.

Preferably the muscle is impaired muscle.

Preferably the impaired muscle is injured muscle.

Preferably the impaired muscle is diseased muscle.

Preferably the impairment of the muscle is due to a toxin.

Preferably the impairment of the muscle is due to a disease.

Preferably the impairment of the muscle is due to an injury.

The subject or patient

The subject, e.g. patient may be an animal having muscle, preferably skeletal muscle, e.g. a vertebrate animal.

Preferably, the patient is a mammal, optionally or in particular a human. In an embodiment the subject is a livestock animal preferably mammal. The compound of the invention may be used in food, feed or dietary supplement or other similar products as disclosed herein.

Conditions /diseases /disorders

Preferably in said impaired muscle said impairment is due to or associated with a medical disorder of the muscle, in particular a disease (diseased muscle) selected from the group consisting of myopathies, preferably selected from myopathies caused by an inflammatory condition, a viral or bacterial infection, biologically active compounds like medicaments, toxins, etc., myopathies associated with systemic disorders, myopathies caused by genetic disorders, preferably muscular distrophies, sarcopenia,

muscular atrophia,

muscular dystrophy, selected from 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,

or a condition with impaired muscle, e.g. an injury, selected from an injury due to a toxin, due to impaired supply of the muscle, such as hypoxia or hypoglycaemia or impaired blood supply, due to a functional muscle injury e.g. increased muscle tone, a structural muscle injury characterized by strained or pulled muscle, (i.e. when muscle is overstretched or torn) or when the muscle is cut or bruised, e.g. in case of a wound, etc.

The GDF3 compound

A GDF3 compound is a compound comprising or consisting essentially of GDF3.

In a preferred embodiment the GDF3 compound is a recombinant GDF3 compound.

In certain embodiments the recombinant GDF3 can be prepared e.g. in bacterial, insect or mammalian cells or in viral expression systems.

In a preferred embodiment the GDF3 compound is a wild type GDF3 compound.

Preferably the recombinant or wild type GDF3 is a mammalian GDF3, optionally a human GDF3.

Preferably the origin of GDF3 is the same species as that of the patient.

In a preferred embodiment the GDF3 compound is a mutant GDF3 compound (a mutant variant of a wild type GDF3).

In an embodiment GDF3 is a mutant variant of a wild type GDF3 the sequence of which is at least 70%, 80% or at least 90% identical with a wild type GDF3, preferably a mammalian, optionally a human GDF3. (As to exemplary sequences of wild type GDF3 see Table 1).

In an embodiment GDF3 is a fragment of a wild type GDF3 the length of which is at least 70%, 80% or at least 90% of that of a wild type GDF3, preferably a mammalian, optionally a human GDF3.

In an embodiment GDF3 is a mutant variant of a fragment of a wild type GDF3 the sequence of which is at least 70%, 80% or at least 90% identical with that of the corresponding fragment of the wild type GDF3, preferably a mammalian, optionally a human GDF3, based on a sequence alignment including any accepted sequence alignment method.

Preferably the recombinant or mutant or fragment maintains the fold of a wild type GDF3 compound.

In a preferred embodiment a mature GDF3 or a GDF3 free of a signal peptide and/or the propeptide, preferably both from the signal peptide and the propeptide is used herein.

In an embodiment a full length (precursor) GDF3 comprising a signal peptide (signal sequence) and a propeptide is expressed. The signal peptide and the propeptide are cleaved off during proteolytic processing at proteolytic processing sites. Alternatively, the full length GDF3 can be obtained and the signal peptide and the propeptide are cleaved off in vitro by proteolysis. Preferably the mature peptide is used or is acting as an active agent.

The GDF3 compounds of the invention may be fused to an other peptide or protein for example in order to facilitate targeting. Spacer sequences between fusion partners may also be applied. The GDF3 compounds of the invention may also be conjugated to a pharmaceutically acceptable nonproteinaceous polymer. In these embodiments the GDF3 compound is preferably recombinant.

The GDF3 compound may be glycosylated and unglycosylated.

Pharmaceutical composition or preparation

The invention relates to a composition for any use as defined above said composition comprising a GDF3 compound as an active ingredient.

In an embodiment the composition may be formulated as a pharmaceutical composition for systemic or topical administration, preferably a pharmaceutical composition formulated for administration selected from the group consisting of intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, parenteral and oral administration.

In an embodiment the composition may be formulated as a dietary supplement, nutraceutical, functional food, or as a food composition with a health claim.

Preferably the (pharmaceutical) composition is formulated for systemic administration and/or for local administration.

For example, in the composition, GDF3 can be prepared in the form of an injection, like intramuscular, subcutaneous injection or intravenous injection; e.g. in a lyophilized form which can be reconstituted.

Preferably the pharmaceutical composition comprises a GDF3 compound and is formulated for administration of GDF3 to the muscle, preferably to the impaired muscle as a target site.

Alternatively, GDF3 can be prepared in a spray formulation.

Alternatively, GDF3 can be prepared in the form of an ointments or gel.

In embodiments the composition of formulated wherein the GDF3 compound is present in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound.

Preferably the composition for any use as defined herein.

In an embodiment said composition comprises a GDF3 expression construct as an active ingredient, wherein preferably the GDF3 expression construct is useful for expressing GDF3 in mammalian immune cells. Preferably the composition is in the form of cells, said composition may comprise a manipulated macrophage overexpressing GDF3.

In an embodiment said composition being formulated for local administration to the impaired muscle. The invention relates to a pharmaceutical composition for any use as defined above said composition comprising a GDF3 expression construct as an active ingredient.

Preferably the GDF3 expression construct is useful for expressing GDF3 in mammalian immune cells, preferably in macrophages. Preferably the pharmaceutical composition comprises a manipulated macrophage overexpressing GDF3, preferably a recombinant macrophage as disclosed herein.

In a preferred embodiment the macrophage is a Ly6C- macrophage.

Method of treatment

The invention also relates to a method of treatment of a patient in a condition or disease as defined above, said method comprising the step of administration of GDF3 to said patient in an effective amount. In an embodiment the GDF3 compound is administered systemically.

In a preferred embodiment the GDF3 compound is administered locally to the impaired muscle.

In a preferred embodiment the macrophages are augologous macrophages.

In a preferred embodiment GDF3 is recombinantly expressed in the macrophages.

In a preferred embodiment the macrophages are M<t s differentiated into Ly6C- cells.

Preferably, the macrophages are CD45+ Ly6C- F4/80+ macrophages.

In an embodiment in case of muscle injuries the GDF3 compound or the manipulated macrophage overexpressing GDF3 is administered on day 1, 2, 3, 4, 5 or 6 after the injury, preferably on day 3, 4 or 5 after the injury, highly preferably on day 4 after the injury. Alternatively the macrophage are administered when the inflammatory phase of healing starts to decline or after it has started to decline.

Alternatively, in case of systemic diseases the GDF3 compound is administered continuously for the time required, e.g. typically for 1, 2, 3 or 3 weeks, from 2 to 12 months or for years.

Methods of introduction include (or selected from the group consisting of], but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous and oral routes.

In a preferred embodiment the GDF3 compound is co-administered with a further agent for use in muscle regeneration, preferably a further agent for use in muscle differentiation preferably differentiation of newly formed muscle fibers, in particular myotube differentiation.

Cell therapy and macrophage therapy

The invention also provides for (administration by) cell therapy wherein cells expressing GDF3 are administered to the site of muscle to be repaired. Typically cells expressing GDF3 at a high level are applied to target the muscle needing treatment or regeneration.

In case of a local injury the cells can be administered locally. GDF3 may be in principle any type of cells which are pharmaceutically acceptable or tolerable.

In a preferred embodiment the cells are preferably cells of the same species as the patient in order to avoid rejection.

In an embodiment the cells are separated from the injured muscle tissue e.g. by being embedded into the matrix.

Alternatively, the cells are applied intravenously and the blood of the patient carries the cells to the target cite, preferably in case of a systemic disease. In particular blood cells, preferably macrophages are used.

In an embodiment in case of muscle injuries the GDF3 compound is administered on day 1, 2, 3, 4, 5 or 6 after the injury, preferably on day 3, 4 or 5 after the injury, highly preferably on day 4 after the injury. Alternatively the macrophage are administered when the inflammatory phase of healing starts to decline or after it has started to decline. Alternatively, in case of systemic diseases the GDF3 compound is administered continuously for the time required, e.g. typically for 1, 2, 3 or 3 weeks, from 2 to 12 months or for years.

Preferably the therapeutic cells are macrophages.

Preferably genetically engineered or recombinant macrophages are used herein which express recombinant GDF3, preferably secreting GDF3, preferably mature GDF3.

Preferably viruses selected from the group consisting of adenoviruses, lentiviruses, adeno-associated viruses and poxviruses are used for GDF3 gene delivery into the macrophages.

In an embodiment a nonviral method is used for gene delivery into macrophages, for example a method selected from electroporation, nucleofection, lipofection, receptor-mediated gene transfer, microorganisms as vehicles for transfection.

For example intramuscular administration, e.g. an intramuscular injection can be applied. Alternatively the cells may be provided in a gel matrix, e.g. a hydrogel. In this case a topical administration is applicable.

In a preferred invention autologous gene therepy is used.

In an embodiment macrophages are isolated from muscle; CD45+ cells are isolated; CD45+ cells are sorted as necessary, preferably Ly6C-, more preferably Ly6C- F4/80+ macrophages are isolated. Preferably Ly6C- F4/80+ macrophages are used in the present invention e.g. for gene delivery.

Laboratory uses

The invention also relates to a use of a GDF3 compound for differentiation of myoblasts to myotubes ex vivo.

The invention also relates to a use of a GDF3 compound for differentiation of myoblasts to myotubes in a model animal.

An animal model of a condition/disease associated with impaired muscle wherein the gene of GDF3 is knocked out or knocked down.

Transgenic animal

The invention also relates to an animal model of a condition/disease associated with impaired muscle wherein the gene of GDF3 is knocked out or knocked down.

The invention also relates to a transgenic animal having increased muscle mass associated with an increased number of muscle fibers said transgenic animal having stable recombinant expression of GDF3 protein, wherein preferably recombinant expression of GDF3 protein is provided by macrophages and/or muscle cells of said animal.

DEFINITIONS

GDF3, as a natural molecule, is a member of the bone morphogenetic protein (BMP) family and the TGF-beta superfamily. Mammalian, including human variants of the gene product (full length GDF3) comprise a signal peptide (signal sequence) and a propeptide which are cleaved off during proteolytic processing at proteolytic processing sites, said signal peptide and propeptide being is cleaved off to produce a mature protein containing seven conserved cysteine residues forming three conserved disulphide bridges in the mature peptide fold. Since it lacks the cysteine, which is responsible for the formation of inter-molecular disulfide bond, GDF3 may exist as a non-covalent homodimer. The members of this family are regulators of cell growth and differentiation in both embryonic and adult tissues.

GDF3 undergoes processing to obtain an about 110 to 120 amino acids long mature peptide. This magure peptide is 114 amino acids long in humans. The full length GDF3 in humans is 364 amino acids long wherein the signal peptide consists of amino acids 1 to 24 and the propeptide amino acids 25 to 250. In humans, amino acids 251 to 364 form the mature peptide. In humans the full length GDF3 comprises two N-glycosylation sites at positions 112 and 306, i.e. the mature peptide comprises a single glycosylation site at position 306 (full length numbering). The mature peptide comprises three disulphide bonds, which are positioned in the human sequence between cysteine amino acids at positions 264 and 329, positions 293 and 361 and positions 297 and 363; or positions equivalent thereto in any vertebrate or preferably mammalian sequence. Unless indicated differently in case of natural variants the sequence numbering follows the numbering of the full length peptide sequence, preferably according to the human sequence.

The terms "GDF3" or "GDF3 protein" refer herein to a protein encoded by the gene of a vertebrate, preferably a mammal, or a processed form thereof. Said "GDF3" or "GDF3 protein" has preferably the sequence of any natural variant of a vertebrate, preferably mammalian species, said variant having a GDF3 activity. Preferably said "GDF3" or "GDF3 protein" comprises the sequence of a mature peptide fragment wherein the GDR3 signal peptide and propeptide have been cleaved. In a broader sense "GDF3" or "GDF3 protein" include protein fragments, variants and variants of fragments (fragment variants) having GDF-3 activity, preferably said variants or fragments or fragment variants having a sequence having at least 70%, at least 80% or at least 90% identity with a corresponding portion of a natural variant, preferably a natural mature peptide, said fragments or fragment variant having a length of at least 80 or 90 or 100 or 110 amino acids and at most 115 or 120 or 130 or 140 or 150 amino acids, preferably 80 to 150 or 90 to 140 or 100 to 130 or 110 to 120 amino acids. Highly preferably the length of the mature peptide is about 112, 113, 114, 115, 116, 117 or 118 amino acids.

Natural peptides of known sequences are listed hereinbelow.

In a broader sense the terms include the full length unprocessed precursor form of the protein, as well as the propeptide-linked and mature forms resulting from post-translational cleavage. The terms also refer to any fragments of GDF3 that maintain the known biological activities associated with the protein, including sequences that have been modified with conservative or non-conservative changes to the amino acid sequence.

These GDF3 molecules according to the invention may be derived from any source, e.g. natural, recombinant or synthetic. The protein may be human or derived from animal sources, in particular from vertebrates, including bovine, chicken, murine, rat, porcine, ovine, turkey, baboon, and fish. Mammalian sources are preferred. A "variant" GDF3 molecule or refers herein to a molecule which differs in amino acid sequence from a natural GDF3 molecule by virtue of addition, deletion and/or substitution of one or more amino acid residue(s) in the parent GDF3. In a preferred embodiment, the variant GDF3 comprises one or more amino acid substitution(s) in the mature peptide part of GDF3. A variant may be a natural variant or an artificial variant, wherein the amino acid sequence of the latter does not occur in nature.

"Mature GDF3" refers to the protein that is cleaved from the carboxy-terminal domain of the GDF3 precursor protein.

"GDF3 propeptide" refers to the polypeptide that is cleaved from the amino-terminal domain of the GDF3 precursor protein. The GDF3 propeptide is capable of binding to the propeptide binding domain on the mature GDF-8.

The phrase "GDF3 activity" refers to one or more of physiological function associated with active GDF3 protein.

Biological functions of GDF3 identified so far can be selected from the group of functions listed herein: BMP signaling pathway, cell development, endoderm development, formation of anatomical boundary, in utero embryonic development, mesoderm development, regulation of apoptotic process, regulation of

MAPK cascade etc.

A "functional" GDF3 compound, e.g. a fragment or variant of GDF3 exerts GDF3 function, in particular increases myotube formation and/or myoblast differentiation.

The terms "isolated" refer to a molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it is derived. The phrase "substantially purified" or "purified" refers to preparations where the isolated protein is at least 70% to 80% (w/w) pure, at least 80%-89% (w/w) pure, at least 90-95% pure, or at least 96%, 97%, 98%, 99% or 100% (w/w) pure.

The term "treating" or "treatment" refers to both therapeutic treatment and prophylactic treatment such as prevention. Those in need of treatment may include subjects already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e., those needing preventative measures). The term treatment in a broader sense includes both measures that address the underlying cause of a disorder and measures that reduce symptoms of a medical disorder without necessarily affecting its cause.

Musculoskeletal Disorders or MSDs disorders that affect the human body's movement or musculoskeletal system (i.e. muscles, tendons, ligaments, nerves, discs, blood vessels, etc.).

Myopathies are diseases of skeletal muscle, which cause the skeletal muscles to become weak or wasted, and wherein the primary defect or cause of the disease is within the muscle (as opposed e.g. to the nerves), preferably which are not caused by nerve disorders.

There are many different types of myopathies, some of which are inherited, some inflammatory, and some caused by endocrine problems. Myopathies are rare and not usually fatal. Typically, effects are mild, largely causing muscle weakness and movement problems, and many are transitory. Only rarely will patients become dependent on a wheelchair. However, muscular dystrophy (which is technically a form of myopathy) is far more severe. Some types of this disease are fatal in early adulthood.

Myopathies are usually degenerative, but they are sometimes caused by drug side effects, chemical poisoning, or a chronic disorder of the immune system.

The term "medical disorder" refers herein in particular to disorders of muscle ("muscle disorder"), in particular disorders associated with muscle differentiation. Such disorders may be, among others, muscle and neuromuscular disorders such as muscular dystrophy (including but not limited to severe or benign X-linked muscular dystrophy, limb-girdle dystrophy, facioscapulohumeral dystrophy, myotinic dystrophy, distal muscular dystrophy, progressive dystrophic ophthalmoplegia, oculopharyngeal dystrophy, Duchenne's muscular dystrophy, and Fakuyama-type congenital muscular dystophy; amyotrophic lateral sclerosis (ALS); muscle atrophy; frailty; congenital myopathy; myotonia congenital; familial periodic paralysis; myasthenia gravis; Eaton-Lambert syndrome; secondary myasthenia; paroxymal muscle atrophy; and sarcopenia, cachexia and other muscle wasting syndromes.

According to the invention preferably upon treatment of the medical disorder differentiation of the muscle (myoblasts) and/or myotube formation is required.

A myopathy may be

- systemic disease or due to a systemic disease and may results from several different disease processes including endocrine, inflammatory, paraneoplastic, infectious, drug- and toxin-induced processes or myopathies with other systemic disorders, and/or

- an inherited or familial myopathy resulting in the degeneration of muscle tissue, i.e. a dystrophy, which is generally present in a chronic fashion, or

- metabolic myopathies where symptoms on occasion can be precipitated acutely.

"Impaired" muscle is understood herein as muscle, preferably skeletal muscle being in a condition wherein myotube formation and/or muscle fiber formation, including or preferably myoblast differentiation is required. The condition may be e.g. a disorder or an injury. Many disorders are associated with injuries at specific or several sites of the body. Thus, the impaired muscle may be injured or diseased muscle.

An "injury" of the muscle is a condition wherein the muscle structure is detectably damaged in particular at a site of the body, preferably locally. An injury of the muscle is preferably selected from an injury due to a toxin, due to impaired supply of the muscle, such as hypoxia or hypoglycaemia or impaired blood supply, due to a functional muscle injury e.g. increased muscle tone, a structural muscle injury by strained or pulled muscle, (i.e. when muscle is overstretched or torn) or when the muscle is cut or bruised, e.g. in case of a wound, etc. Skeletal muscle injury is particularly a condition eliciting a rapid but precisely timed and tightly regulated process in which a restoration of muscle structure can be achieved within weeks form the time of the injury, typically of destruction of the tissue.

"Toxin" as used herein is a poisonous substance produced synthetically or within living cells or organisms resulting in impairment of cells, in particular muscle cells.

The term "therapeutically effective" refers to a treatment which results in improvement of symptoms of a disorder, a slowing of the progression of a disorder, or a cessation in the progression of a disorder. The therapeutic benefit is determined by comparing an aspect of a disorder, such as observation of muscle regeneration, differentiation of muscle cells, observation of inflammation, the amount of muscle mass, before and after GDF3 of the invention is administered.

As used herein "pharmaceutically acceptable" or "pharmaceutically tolerable" carrier or excipient or medium etc. includes any and all solvents, media, coatings, physiological media, matrix and the like that are physiologically compatible. In embodiments the carrier is suitable for systemic, e.g. intravenous or intramuscular administration. The use of such media and agents, such as for the administration, like injection or application of proteins, implantation, injection or application of cells, is well known in the art.

The term "subject" refers to an animal, preferably a vertebrate species, more preferably mammalian (including a nonprimate and a primate) or avian species, including, but not limited to, murines, simians, humans, mammalian farm animals (e.g., bovine, porcine, ovine), mammalian sport animals (e.g., equine), and mammalian pets (e.g., canine and feline); preferably the term refers to humans. The term also refers to avian species, including, but not limited to, chickens and turkeys.

A "patient" is a subject who is a target of therapy. In an embodiment the subject, preferably a mammal, more preferably a human, is further characterized with a disease or disorder or condition that would benefit from the administration of GDF3.

The„percent identity" is the percent of amino acids or nucleobases in a first sequence (e.g. an original or a modified sequence) present in a second sequence (e.g. an original or a modified sequence) wherein the first sequence is aligned with the second sequence, and wherein, if after alignment any of the first or second sequence has an additional unaligned (overhanging) portion, then percent identity is defined for the aligned portions only wherein the reference sequence is the sequence having identical number of or more amino acids or nucleobases than the other sequence. Therefore, a 30 amino acids or nucleobases long sequence comprising the full sequence of a sequence of interest (a modified sequence or in this case a fragment) of 20 amino acids or nucleobases long would have a portion of 100% identity with the sequence of interest, while further comprising an additional 10 amino acids or nucleobases portion. Or if the sequence of interest of 20 amino acids or nucleobases has e.g. 2 or 4 amino acids or nucleobases mutated or deleted in comparison with the aligned portion of the reference sequence then the sequence of interest has a 90% or 80% identity, respectively, with the reference sequence.

Two nucleic acid or amino acid sequences are considered substantially identical if, when optimally aligned according to a know alignment algorithm (see e.g. the computerised implementations of these algorithms, such as GAP, BESTFIT, FASTA and TFASTA or the BLAST algorithm) with parameters suitable for that kind of alignment, they share at least about 70% identity. In alternative embodiments, sequence identity in optimally aligned substantially identical sequences may be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

The term "recombinant" when made in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques. "Recombinant nucleic acid" constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Referring to a nucleic acid construct as 'recombinant' therefore indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation of the host cell. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination and/or repair events.

"Comprising" something means including something wherein something else which is not listed or given, may be included, too. Comprising may be limited to "consisting essentially of", wherein what is not included is functionally not essential for carrying out the invention, or "consisting of wherein nothing else is included but those which are listed or given.

As used in this specification, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

BRIEF DESCRIPTION OF THE FIGURES

Fig 1. Impaired regeneration of skeletal muscle in PPARy deficient animals. (A) Representative images of HE stained skeletal muscle from WT and PPARg MacKO animals prior (day 0) or post cardiotoxin (CTX) induced injury (day 8) are shown. Asterisk labels phagocytic/necrotic fibers and arrow points to foci of inflammatory infiltrations. Immunfluorescent detection of Desmin (red), F4/80 (green) and nuclei (blue) at day 8 post CTX injury is also shown. Scale bars in the upper left represent 50 μηι. (B) Ratio of necrotic/phagocytic fibers relative to all regenerative fibers (+/- SD) at day 8 of regeneration in WT and PPARg MacKO muscle sections (C) Fiber size repartition of regenerating muscle in WT or PPARg MacKO animals (+/- SEM) at day 8 and day 21 post CTX injury. (D) Average fiber cross section area (CSA) of regenerating muscle (+/- SEM) at indicated timepoints post CTX injury. (E) Representative images of HE stained skeletal muscle 22 days after CTX injury from bone marrow transplanted animals that received either WT or Ppargf - Sox2Cre+ KO bone marrow. (F) Muscle fiber CSA of BMT animals 22 days post CTX injury (+/-SEM). For cumulative CSA figures, Pparg expression levels in M<t s and for full body Ppargf - Sox2Cre+ KO images, see Fig 8 and 9.

Fig 2. Number and fate of infiltrating cells in injured muscle. For gating strategy, see Fig 10. (A) Total number of infiltrating CD45+ hematopoietic cells (+/- SD) isolated from CTX injured muscles of WT and PPARg MacKO animals at day 1, day 2 and day 4. (B) Percentage of neutrophils and Ly6C+ M<t s and the (C) calculated neutrophil and ΜΦ numbers (derived from the % of the total numbers) at day 1 in injured muscles. (D and E) Percentage of Ly6C+ and Ly6C- M<t s in injured muscles at day 2 and day 4.

Fig 3. Impact of PPARy on ΜΦ functions (A) Experimental strategy to measure in vitro phagocytosis in BMDMs. (B) Percentage of phagocytic BMDMs and the Median Fluorescence Intensity (MFI) in the phagocytic BMDM compartment in BMDMs derived from WT vs. PPARg MacKO or WT BMT vs PPARg KO BMT animals. (C) Effect of BMDM derived conditioned media on the proliferation of primary myoblasts (+/- SD). n= 4 and 3 for WT or PPARg MacKO derived medium, respectively. (D) Effect of BMDM derived conditioned media on the differentiation of primary myoblasts (+/- SD). n= 4 and 3. For the myoblast cell line independence, see Fig. IOC

Fig 4. Transcriptional analysis of Ly6C+ and Ly6C- ΜΦ populations derived from WT and PPARg MacKO animals. For schematics of comparisons, see Fig 11. (A) Heatmap representation of genes that show differential (p=0.05, min. 1.5X FC) expression in the four sorted WT vs PPARg MacKO M<t s in day 1 Ly6C+ (labeled as D lLy6C+ etc.), D2 Ly6C+ and D2 Ly6C-, and D4 Ly6C- cells. In each heatmap, the differently expressed genes are highlighted within a red square and the expression pattern of these genes in the other macrophage subtypes is also shown for reference. Blue and red arrows label genes that are downregulated or upregulated in the KO vs WT cells, respectively. The blue/red arrows point to the direction of increasing fold change difference. For RT-qPCR validation of the expression of selected PPARy dependent genes in muscle derived M<t s, see Fig 12. (B) Top 5 up and downregulated genes in the four sorted ΜΦ populations in PPARg MacKO vs. WT cells. Table lists gene symbols and fold change differences (FC). Labels Down 1 to 5 and Up 1 to 5 show the top 5 FC ranked genes that are down-, or upregulated in PPARy MacKO M<t s, respectively. The Downl/UPl labels represent the highest FC difference. GDF3 and Apoldl, the genes that are down, - or upregulated in PPARg MacKOs in all four subtypes, are highlighted. (C) Venn diagramms showing the overlap of genes that are down-, or upregulated in PPARg MacKO M<t s in the four analyzed ΜΦ subtypes. (D) Heatmap representation of the expression pattern of the genes that are RSG regulated in WT Ly6C- cells at day 2 in all isolated ΜΦ subtypes. Different Hist2h3 isoforms are labeled as histone genes.

Fig 5: Gd/3 is a PPARy target gene in BMDMs (A) Upper panel: mRNA expression of Angptl4, a canonical PPARy target gene, Pparg, Gd/3 and Apobecl, a nearby, not regulated gene, are shown in WT and PPARg MacKO BMDMs. Significant differences based on biologically relevant paired or unpaired t tests are shown. n=4 for WT and 5 for PPARg MacKO cells. (B) Identification of possible enhancers around the Gd/3 locus. The enhancer identification strategy around Angptl4 is shown in Fig 13A. The selection criteria for enhancers possibly involved in Gd/3 regulation are described in the text and in Fig 13B. Putative enhancers are labeled by vertical lines. Blue verticals highlight enhancers without PPARy ChIP enrichment, red verticals label enhancers where enrichment in PPARy binding in WT BMDMs was detected by PPARy ChIP (see Fig 5C). (C) ChIP on the putative enhancer regions reveal PPARy binding at +7.3 Kb, -21 Kb, -25 Kb, -44 Kb and -47 Kb enhancers around the Gd/3 locus. Representative graphs showing PPARy, RXR or IgG ChlPs carried out on 2 samples are shown. Angptl4 enhancer and Gd/3 +16 kB enhancer are shown as positive and negative controls, respectively.

Fig 6. GDF3 is a regulator of muscle regeneration. (A) Representative HE stained muscle sections of WT BMT and GDF3 KO BMT animals, 16 days post CTX injury. (B) Average myofiber CSA measurement in WT BMT and GDF3 KO BMT animals, 16 days post CTX injury. (C) Myofiber CSA repartition in WT BMT and GDF3 KO BMT animals at 16 days post CTX injury. (D) GDF3 protein expression in whole muscle lysates of regenerating WT muscles at different timepoints. For densitometric analyses, see Fig 14A and 14B. Specificity of the anti-GDF3 antibody is shown in Fig 14C. (E) GDF3 protein expression in whole muscle lysates at day 4 post CTX injury in WT and PPARg MacKO animals. (F) mRNA expression of Gd/3 in CD45+ and CD45- cells isolated from injured muscles at days 1, 2 and 4 post CTX in WT and PPARg MacKO animals. (G) GDF3 Protein expression in CD45+ and CD45- cells cells isolated from injured muscles at day 4 post CTX in WT and PPARg MacKO animals. Day 4 whole muscle lysate from WT mouse is loaded as a positive control (D4).

Fig 7. (A) in vitro proliferation (left panel) and differentiation (right panel) assays on primary myoblasts carried out with recombinant GDF3 reveal a pro-differentiation effect of GDF3 on muscle progenitor cells. (B) IF against desmin (red) and DAPI (blue) shows a drastic enhancement of myotube fusion in the presence of recGDF3 in in vitro primary myoblast differentiation assay (C) Heatmap representation of the expression pattern of selected genes validating the utilized in vitro primary differentiation myoblast assay (D) Heatmap representation of genes that are differently expressed (min. fold change difference of 1.2X between differentiated myoblasts +/- recGDF3) in the presence of GDF3 during myoblast differentiation (E) Heatmap representation of members of the TGF family signaling system that are expressed and regulated or expressed but not regulated in muscle derived macrophages. For non-expressed members, see Fig 14D.

Fig 8. PPARy in muscle infiltrative macrophages during skeletal muscle regeneration (A) GO analysis of the genes that are upregulated as inflammatory Ly6C+ M<t s differentiate into repair Ly6C- M<t s during muscle regeneration at day 2 past CTX injury. (B) Expression of Pparg in various macrophages and dendritic cells. Microarray data derived from muscle derived macrophages isolated for this study and various myeloid cell populations isolated within the Immunological Genome Project were pooled and normalized together (per gene normalization to the median expression level of Pparg). A selected set of samples and their normalized expression value are shown. The commonly used ΜΦ model, bone marrow derived macrophages, are highlighted in light blue, while the high Pparg expressor lung M<t s and splenic red pulp M<t s are highlighted in medium and dark blue. The most likely precursor for muscle derived macrophages, Ly6C+ monocytes, is shown in orange and labeled with an orange asterisk. The detailed description of all cell types is available upon request. (C) Expression of Pparg mRNA in day 1 WT and day 2 WT or PPARg MacKO CD45+ cells and in (D) day 2 Ly6C+ and Ly6C- M<t s isolated from CTX injured muscle.

Fig 9. Additional analysis of the impact of PPARy on muscle regeneration (A) Cumulative CSA analysis of muscle section derived from WT or PPARg MacKO animals at day 8 or day 21 post CTX injury. (B) IF of desmin (red), F4/80 (green) and DAPI (blue) on muscle sections from full body Pparg¹¹/*, Sox2Cre- (controls) and Pparg¹¹/-, Sox2Cre+ (KO) animals isolated at day 8 post CTX.

Fig 10. Additional analysis of the impact of PPARy on macrophage numbers, fate and function. FACS strategy to enumerate (A) day 1 and (B) day 2 neutrophils and macrophages isolated from CTX injured muscle. (C) The pro-differentiation effect of conditioned medium derived from IL4 treated macrophages is diminished in PPARg MacKO macrophage supernatants in a myoblast cell line independent manner.

Fig 11. Schematics of transcriptomic analyses of muscle derived ΜΦ populations

Fig 12. mRNA expression of PPARy dependent genes in muscle derived sterile inflammatory ΜΦΒ detected by RT-qPCR.

Fig 13. Schematics of active enhancer identification (A) Identification of the active, PPARy regulated enhancer around the Angptl4 locus. Red vertical line labels the relevant enhancer. (B) Enhancer selection scheme for identifying active enhancers around the Gd/3 locus.

Fig 14. Additional analysis of the expression of TGFft family members in muscle and in muscle derived macrophages (A and B) Densitometric evaluation of GDF3 protein expression from western blots in Fig 6D and E, respectively. (C) Western blot detection of GDF3 in day 4 whole muscle lysates derived from WT and GDF3-/- animals show high specificity of the anti-GDF3 antibody. (D) List of members of the TGF family signaling system that are not expressed in muscle derived macrophages.

DETAILED DESCRIPTION OF THE INVENTION

Tissue regeneration is an indispensable part of life that ensures survival. The involvement of M<t s in tissue regeneration appears to be an evolutionary conserved process. M<t s are infiltrating myeloid cells that are equipped with by an extensive range of cell surface and intracellular molecules that enable them not only to specifically sense and interpret the nature of the damage and also to monitor the progress of repair. Moreover, M<t s are transcriptionally plastic with the capacity to assume dramatically different cellular phenotypes depending on environmental cues (Kaikkonen et al., 2013; Lavin et al., 2014; Okabe and Medzhitov, 2014; Ostuni et al., 2013). These properties, along with the ability to secrete proteins, could enable M<t s to orchestrate regenerative processes in a rapidly changing milieu of injured/regenerating tissues; however, the details of their supporting functions in regeneration have remained elusive. Particularly little is known about how M<t s instruct myoblasts to form muscle fibers.

The present inventors have applied cardiotoxin (CTX) induced skeletal muscle injury model and carried out an unbiased transcriptomic analysis of the involved ΜΦ populations. It has been found that the fatty acid regulated transcription factor, Peroxisome Proliferator-Activated Receptor gamma (PPARy) (Tontonoz et al., 1998), was induced and highly expressed in the infiltrative ΜΦ populations within injured muscles. Importantly, mice with a deletion of PPARy in their myeloid lineages showed a pronounced delay in skeletal muscle regeneration. Thus, the present inventors have identified PPARy as a regulator of myoblasts formation from muscle fibers. Most surprisingly, the present inventors have also found that PPARy regulated the expression of a secreted factor, GDF3, which is a member of the TGFft family, in repair M<t s. Strikingly, GDF3 has proved to be a ΜΦ derived protein whose expression is induced during muscle regeneration in a PPARy-dependent manner and could enhance the differentiation of primary myogenic precursor cells (MPCs) in in vitro cultures. More specifically, GDF3 slightly but significantly decreased myoblast proliferation in primary myoblast lines whereas resulted in a robust increase on myotube fusion. Thus GDF3. once released from ΜΦΒ within the injured /regenerating tissues, could regulate molecular pathways relevant to muscle differentiation in primary muscle cells and thereby could skew the balance between myoblast proliferation and differentiation.

Apparently, no such function of GDF3 has been suggested so far.

In summary, the data provided herein reveal a novel integrated pathway with sensory, gene regulatory and effector components in which PPARy in repair M<t s responds to signals and, via GDF3 as a repair ΜΦ-derived novel pro-differentiation factor, supports the timely promotion of tissue repair during muscle regeneration.

The present inventors have made serious effort to identify integrated sensory, regulatory and effector mechanisms equipping a macrophage with the capacity to contribute to timed progression of muscle tissue repair.

In more detail, as skeletal muscle possesses excellent regenerative capacity, in the early phase of this study it was especially interesting for the inventors that the delay in muscle regeneration in ΜΦ PPARy-deficient animals was detectable as long as three weeks after the initial injury, well beyond the timeframe that is required to regenerate muscle structure. One of the best characterized genetic models for the involvement of ΜΦΒ in muscle regeneration is the ΜΦ selective AMPK-deficient mouse, which exhibits a profound delay in muscle regeneration in the same experimental system (Mounier et al., 2013). The phenotype seen in the ΜΦ PPARy deficient mice was comparable in its extent to the delay reported in ΜΦ AMPK deficient animals, thus appearing to be among the most dramatic reported deficiencies caused by impairments in ΜΦ functions. This suggests that PPARy is likely to play an important and integrated sensory and regulatory function to synchronize cellular interactions during regeneration.

Our analysis did not reveal an obvious difference in ΜΦ numbers or fate in PPARg MacKO animals, as opposed to two other reported experimental systems where AMPK or IGF1 deficiency in muscle infiltrative ΜΦ5 led to altered ΜΦ polarization (Mounier et al., 2013; Tonkin et al., 2015). This is somewhat surprising, as PPARy has been reported to be a regulator of ΜΦ polarization (Odegaard et al., 2007). Either the Ly6C+ to Ly6C- ΜΦ transition in this system does not correspond to canonical polarized ΜΦ populations, or PPARy is not a critical component of alternatively polarized ΜΦΒ in this setting. Phagocytosis was normal in PPARg MacKO ΜΦΒ, and is therefore unlikely to explain the observed delay in regeneration. Systematic transcriptomic analyses, however, provided clues about both the sensory and the regulatory roles of PPARy in muscle infiltrating ΜΦΒ. Three relevant questions were answered by gene expression analysis: the identity of the PPARy responsive cell type, the genes regulated by PPARy and the functional consequences of PPARy deficiency in ΜΦΒ. By a combination of in vivo ligand activation of PPARy in WT animals and gene expression comparison of WT vs. PPARg MacKO ΜΦΒ the following picture emerged. First, PPARy deficiency gave consistent changes in gene expression status of WT vs. PPARg MacKO ΜΦΒ, as several apparent PPARy dependent genes were regulated in more than one ΜΦ subtypes. It is possible that a subset of the apparent PPARy dependent genes are not direct transcriptional targets of the receptor, as control of gene expression by transrepression is a well known feature of PPARy activity, especially with regard of its anti-inflammatory effects (Pascual et al., 2005). Second, an extensive set of genes was regulated by PPARy in a more restricted manner, showing differential expression in only one or a few ΜΦ subtypes. Either the presence of successive waves of downstream regulatory events initiated by PPARy or unrelated genomic events could have confounded the identification of direct PPARy targets. Third, in vivo treatment with RSG identified the Ly6C- ΜΦΒ as an in situ ΜΦ subtype that could be activated by a synthetic ligand for PPARy. The surprising fact that RSG treatment elicited characteristically different gene expression changes in Ly6C+ and Ly6C- M<t s isolated from the same tissue/timepoint could be explained by the inability of RSG to activate PPARy in the Ly6C+ ΜΦΒ or by a divergent regulatory repertoire for the two relevant ΜΦ subtypes [e.g. different cofactors or lineage specific factors). The most intriguing interpretation of the available data would be the involvement of a yet unidentified endogenous ligand/activator for PPARy whose activity is restricted to the Ly6C- compartment, which could explain the tendency of otherwise RSG inducible genes to be upregulated in the Ly6C- M<t s even in the absence of the synthetic ligand. In vivo treatment with RSG affects the systemic homeostasis, generating a plethora of off-targets and indirect genomic events. Therefore, our results are consistent but not conclusive regarding the precise mechanisms that lead to the observed gene expression changes. Irrespective, since PPARy demonstrates a characteristically divergent activity in the Ly6C- ΜΦ subtype, which dominates the regenerative phase of muscle injury/regeneration, it is plausible that PPARy activity is a licensing factor for repair M<t s and an unidentified lipid might serve as a switch on a functional regulatory circuit. The regulatory function of the receptor is likely to include many downstream elements.

From the perspective of muscle regeneration, the most important finding was the identification of GDF3. which showed consistent regulation by PPARy in all relevant ΜΦ subtypes. Importantly. GDF3 is a ΜΦ derived paracrine factor with muscle regenerative functions, whose diminished ΜΦ expression is consistent with the delayed regeneration seen in PPARy deficient animals. To ascertain that GDF3 is not only a PPARy dependent factor, but also a direct PPARy target, we analyzed an extensive range of genomic/epigenomic data. Although it is clear that GDF3 is expressed in a PPARy-dependent fashion and can be induced by ligand in muscle derived Ly6C- ΜΦ5, direct regulation by PPARy has been challenging to prove, since ligand dependent regulation appears to be ΜΦ subtype specific and not detectable in BMDMs. However, we have provided data that are consistent with direct regulation, even in BMDMs. The fact that several putative enhancers bind the lineage specific transcription factor PU.l along with the signal specific PPARy:RXR heterodimer and fall within the CTCF/cohesin bordered genomic region even in BMDMs suggests that GDF3's PPARy-dependence is the consequence of direct binding of the receptor heterodimer to the GDF3 locus. Since ligand dependence is not evident in BMDMs, a subtype specific co- factor that confers selective regulation in Ly6C- repair ΜΦΒ might be missing from BMDMs. This observation along with the precise regulatory functions and relative importance of the five PPARy bound enhancers is unclear. The final proof would come from the identification of subtype selective co- activators and/or genetic elimination of putative enhancers in vivo. Additional in vitro and transfection- based experiments would be informative, but would still remain inconclusive.

To link ΜΦ biology to tissue regeneration, we analyzed the role of ΜΦ derived GDF3 in muscle regeneration in a combination of in vivo and in vitro approaches. Foremost, animals with GDF3 deficient bone marrow exhibited a delay in muscle regeneration. This suggests that endogenous GDF3-expressing cells of hematopoietic origin are likely contributing to muscle regeneration. By using BMT animals we circumvented the potential compensatory physiologic mechanisms that rescue the embryonic lethal phenotype in approximately 65% of mice with global GDF3 deficiency, as no compensatory mechanism has been implicated in immune cells. In our experiments. GDF3 protein expression peaked on day 4. at the time when the presumed switch from acute inflammation to active regeneration is completed in injured muscle and at the time of the beginning of myogenic precursor differentiation in vivo (Bentzinger et al., 2013; Parisi et al., 2015). It is noteworthy, that GDF3 expression at both the gene expression and protein levels was much lower in the CD45- fraction isolated from injured muscle than in the hematopoietic compartment. Considering that the separation of CD45+/- cells is inherently incomplete, our results indicate that ΜΦΒ are the predominant, if not the only source of GDF3 within the injured tissue. This notion is in line with the observation that in vitro established primary myoblasts cell lines do not express Gd/3 neither in their undifferentiated or differentiated stages. This exclusivity sets GDF3 apart from other ΜΦ derived regenerative factors, such as IGF1 (Tonkin et al.. 2015). which is also produced by muscle and in the liver upon injury. Our data further reinforce the contention that PPARy controls GDF3 protein expression as part of the repair synchronizing circuit linking reparatory M<t s to myoblast differentiation.

While our observations firmly establish that the PPARy dependent, ΜΦ derived GDF3 is required for tissue regeneration, the mechanism of action remains unclear. As our in vitro results with BMDM derived supernatants and myoblasts indicated the presence of a regulatory circuit between M<t s and muscle cells, we hypothesized that ΜΦ derived GDF3 is a tissue regeneration factor that regulates MPC expansion/differentiation within the injured tissue. Indeed, recombinant GDF3 enhanced myotube fusion and in vitro differentiation of established primary myoblasts. Importantly, the same effect was seen when GDF3 was added to independently isolated primary myoblast cell lines, excluding a cell line dependent effect.

Taken together. GDF3. which is expressed in and secreted by muscle infiltrating ΜΦΒ within injured and regenerating muscles has the capacity to elicit biologically relevant responses in primary myoblasts and differentiating myotubes and is a regulator of both in vitro muscle proliferation /differentiation and muscle regeneration in vivo.

Thus. GDF3 is a protein suitable for treatment if impaired muscle by promoting muscle cell, e.g. myoblast differentiation and myotube fusion.

In this study we describe a new regulatory circuit by which muscle infiltrating ΜΦΒ regulate tissue regeneration. This regulatory axis involves PPARy, a ligand activated transcription factor that appears to have distinct, but overlapping functions in the inflammatory and repair ΜΦΒ within the injured tissue. PPARy then regulates muscle regeneration through the action of GDF3, a secreted factor that regulates MPC differentiation. Thus, the PPARy-GDF3 regulatory axis orchestrates muscle regeneration by sensing and interpreting inflammation upon tissue injury and enhancing the execution phase of regeneration.

It is to noted that regarding the molecular effects of GDF3 on primary myoblasts, we found that GDF3 regulated several genes whose functions are closely linked to muscle biology. Whether these genes are directly regulated by GDF3 or they were only markers of the enhanced differentiation, is presently unclear.

Our study has left open several questions relevant to PPARy and GDF3 biology. First, gene expression data suggest that a switch from the inflammatory to the regenerative actions of PPARy occurs in M<t s during regeneration. Does that switch merely allow PPARy to fulfill its regenerative effector functions or is PPARy itself driving the functional switch? Related to this question, what are the molecular mechanisms that allow PPARy to regulate characteristically different genes in the two canonical ΜΦ subtypes? Second, GDF3 expression is induced during the regenerative phases. Whether PPARy acts alone or in unison with other regulators to ensure an adequate level of GDF3 expression in the injured tissue is unknown. Finally, finding the whole spectrum of regulatory actions of GDF3 in MPCs and the potential involvement of other TGF family members in this process requires further investigations (Sartori et al., 2013). Our transcriptomic analyses of relevant M<t s alone indicated that other ΜΦ derived TGF superfamily members could also play a role in muscle regeneration. As other cell types are also involved in the regeneration process (Heredia et al., 2013; Joe et al., 2010; Uezumi et al., 2010), it cannot be excluded that, besides GDF3, other TGF family members are active during regeneration

It is remarkable, though, that macrophages and myoblasts and their interactions are critical to supporting regeneration in themselves and the key elements of the complex delay phenotype can be modeled in vitro using these two cell types only. Therefore, irrespective of the answers to any open questions on mechanism, our findings demonstrate and duly support applications in pathological circumstances in which there is a need to improve muscle differentiation and myotube formation. In particular, the invention can be used in conditions in which recurrent muscle damage and asynchrony in repair due to genetic conditions leads to debilitating degenerative muscle diseases, such as Duchenne Muscular Dystrophy (DMD) and other conditions e.g. those listed herein. Therefore it is contemplated that GDF3 is also a useful regulator of muscle regeneration in myopathies, which are most of the time associated with the permanent presence of inflammatory cells, especially ΜΦΒ.

GDF3 can be used according to various aspects of the invention.

Sequences and sources of human and mouse orthologs are given in table 1 below.

Table 1 (Source: Wikipedia, January 30, 2015)

[1] Caricasole AA, et al. Oncogene 16 (1): 95-103.

[2] Chen C, et al. Development 133 (2): 319-29.

Several further GDF3 variants are known which are summarized according to their Gene ID in the

NCBI Gene database (http://www.ncbi.nlm.nih.gov/gene) and listed in Table 2. Table 2 - Natural GDF3 variants

102182110 9925 Capra hircus 100013947 13616 Monodelphis domestica

102146403 9541 Macaca fascicularis 101347960 127582 Trichechus manatus latirostris

102018279 34839 Chinchilla lanigera GDF3

102002321 79684 Microtus ochrogaster 100474590 9646 Ailuropoda melanoleuca

101964649 43179 Ictidomys tridecemlineatus PANDA 019372

101840876 10036 Mesocricetus auratus

101690832 9669 Mustela putorius furo

101650420 9371 Echinops telfairi

101622609 143302 Condylura cristata

101569058 10160 Octodon degus

101553430 42254 Sorex araneus

101534905 9978 Ochotona princeps

101472614 106582 Maylandia zebra

101413306 9361 Dasypus novemcinctus

101389918 73337 Ceratotherium simum simum

101375684 9708 Odobenus rosmaras divergens

101337368 9739 Tursiops truncatus

101273040 9733 Orcinus orca

101155973 8090 Oryzias latipes

101151622 9593 Gorilla gorilla

101103967 9940 Ovis aries

101097251 9685 Felis catus

101039799 27679 Saimiri boliviensis

101015395 9555 Papio anubis

100988098 9597 Panpaniscus

100942326 30611 Otolemur garnettii

100932896 9305 Sarcophilus harrisii

100763238 10029 Cricetulus griseus

100733509 10141 Cavia porcellus

100696395 8128 Oreochromis niloticus

100656112 9785 Loxodonta africana

100606028 61853 Nomascus leucogenys

100510963 9823 Sus scrofa

100461404 9601 Pongo abelii

100394876 9483 Callithrixjacchus

100346534 9986 Oryctolagus cuniculus

100061071 9796 Equus caballus

Alternative names are given in italics.

The sequence of full length human GDF3 is as follows (SEQ ID NO: 1):

10 2 0 30 4 0 50

MLRFLPDLAF S FLLI LALGQ AVQFQEYVFL QFLGLDKAPS PQKFQPVPYI

60 7 0 8 0 90 100

LKKI FQDREA AATTGVSRDL CYVKELGVRG NVLRFLPDQG FFLYPKKI SQ

110 12 0 130 14 0 150

AS SCLQKLLY FNLSAI KERE QLTLAQLGLD LGPNSYYNLG PELELALFLV

160 17 0 18 0 190 2 00

QEPHVWGQTT PKPGKMFVLR SVPWPQGAVH FNLLDVAKDW NDNPRKNFGL

2 10 22 0 230 24 0 250

FLEI LVKEDR DSG FQPED TCARLRCSLH ASLLWTLNP DQCHPSRKRR

2 60 27 0 2 8 0 2 90 300

AAI PVPKLSC KNLCHRHQLF INFRDLGWHK WI IAPKGFMA NYCHGECPFS

310 32 0 330 34 0 350

LTI SLNS SNY AFMQALMHAV DPEI PQAVCI PTKLS PI SML YQDNNDNVI L

360

RHYEDMWDE CGCG

The GDF3 compound of the invention comprises or consists essentially of any of the above sequences or an at least 70%, 75%, 80%, 85%, 90% or 9% percent long functional fragment of those sequences or an essentially identical variant of any of those sequences. Preferably, in a subject a GDF3 compound is applied which is of the same species as the subject or an essentially identical variant thereof. Preferably the subject is an animal with skeletal muscles, preferably a vertebrate animal, preferably a fish, an amphibian, a reptile, a bird or a more preferably mammal, in particular a human.

Protein variants e.g. modified proteins

Protein variants according to the present invention can be prepared by protein engineering techniques of the art [see for example Stefan Lutz, Uwe T. Bornscheuer Eds., Protein Engineering Handbook, Volume 1 & Volume 2, 2009 Wiley-VCH Verlag GmbH & Co. KgaA Print ISBN: 9783527318506; Online ISBN: 9783527634026.].

Nucleic acid sequences encoding GDF3 can be expressed in vitro by transformation of a suitable host cell. "Host cells" are cells in which a vector can be propagated and its DNA expressed.

Upon protein engineering certain amino acids can be changed or mutated which may include deletions, insertions, truncations to fragments, fusions and the like. Such mutation may render the GDF3 compound e.g. more stable, e.g. against denaturation or aggregation or decomposition in the gastrointestinal system or in the blood dependent on the route of administration or oxidation in particular in case of topical administration (i.e. increase their half life). I also can be rendered more resistant against proteolytic cleavage while preserving its function as disclosed herein. Such techniques are generally known in the art.

For example, aspects of modifying proteins for therapeutic purposes are described e.g. in Khudyakov, Yury E. Medicinal Protein Engineering December 1, 2008 by CRC Press, ISBN 9780849373688 - CAT# 7368

Recombinant GDF3

Recombinant GDF3 can be obtained from a number of provider, for example R&D Systems (Minneapolis, MN 55413, USA) provides

- Recombinant Mature Human GDF-3 Protein (CHO-derived, sequence: Ala251-Gly364),

- Recombinant Mature Mouse GDF-3 Protein (CHO-derived, sequence: Ala253-Gly366),

- Recombinant Mature Mouse GDF-3 Protein (prepared in E. coli-derived, sequence: Ala253- Gly366).

Human proteins are also provided in carrier free version.

Appropriate antibodies recognizing the mature peptide sequence also in the full protein are also provided.

For example, MyBioSource, Inc. (San Diego, California, USA) also provides Recombinant Human Mature GDF3 with a Met-Lys-His6 tag at the N-terminal.

Recombinant human GDF3 is also available at BioVision, Inc. (San Francisco, CA, USA).

Manufacturers often prepare the protein in a lyophilized e.g. from a concentrated solution and recommend to reconstitute the lyophilized GDF3 in sterile buffer, e.g. lOOmM Acetate buffer. Storage is proposed desiccated and frozen (e.g. below 18 degree C) and after reconstitution at 4 degrees C e.g. for between 2 to 7 days, unless stabilizer is added.

Recombinant GDF3 can be prepared e.g. in bacterial, insect or mammalian cells e.g. as taught in US20070259807A1 (Examples 1 to 3). Brifly, the DNA sequence of GDF3 may be amplified using appropriate PCR oligonucleotide primers. If needed coding sequences for restriction enzymes may be added. The amplified sequence is ligated into an expression vector appropriate for transformation of the desired cell intended for expressing the protein.

As an other example, mature recombinant GDF3 can be prepared by the method as described by 0. Andersson et al. [Developmental Biology 311 (2007) 500-511]. Briefly, a DNA fragment encoding the mature region of GDF3 was fused downstream to a Xenopus Activin B pro-domain and a hemagglutinin (HA)-tag, such that, after processing, the HA-tag remained at the N-terminus of mature GDF3. This construct was cloned into a pCDNA3.1 vector backbone for expression in mammalian cells. GDF3 was processed and secreted as a mature protein of the expected size when expressed from this construct.

If a mature peptide is to be prepared for example the coding sequence of the mature sequence should be used.

Alternatively, e.g. a full length (precursor) GDF3 comprising a signal peptide (signal sequence) and a propeptide can be expressed. If the GDF3 is expressed in cells of an appropriate species, e.g. mammalian cells, for example in cells of the same species as the origin of GDF3, the signal peptide and the propeptide are cleaved off during proteolytic processing at proteolytic processing sites and preferably the mature peptide is secreted from the cells.

Alternatively, the full length GDF3 can be obtained and the signal peptide and the propeptide are cleaved off in vitro by proteolysis. Preparation of GDF3 propeptide is taught e.g. in EP1771557B1 (Example 1). By the same method, mutatis mutandis, the full length protein can be prepared.

In an embodiment the cells expressing full length GDF3 and duly processing it are used for treatment. In a preferred embodiment these cells are macrophages.

Viral expression of GDF3

Viral expression of GDF3 can be carried out in any known viral expression system.

For example, adenoviral expression of GDF3 is disclosed in US20070259807A1 (Example 6).

GDF3 producing viruses are also commercially available. For example e.g. GDF3 precursor producing adenoviruses (without tag) can be purchased from Applied Biological Materials Inc (Richmond, BC CAN, Cat.No.: 274037A).

Preparation of GDF3 propeptide is taught in EP1771557B1 (Example 1) in a human CMV - HEK293 expression system. Recombinant GDF3pro-Fc fusion was expressed in HEK293 cells and purified by standard techniques.

Monoclonal and polyclonal antibodies recognizing GDF3 are available from several manufacturers and can be used e.g. for Western blotting, Elisa etc. e.g. for monitoring expression or presence of GDF3 in vitro.

Protein therapies and therapeutic proteins

The GDF3 compounds of the invention may be fused to an other peptide or protein for example in order to facilitate targeting. This can be e.g. a peptide signal (signal peptide) or a propeptide enabling transport through a cell membrane or a physiological barrier.

When a signal peptide is part of the polypeptide, in principle any signal peptide known to the art may be used, including synthetic or natural sequences, for example, from a secreted or membrane bound protein. Generally, a signal sequence is placed at the beginning or amino-terminus of the fusion polypeptide of the invention and may be used to target the GDF3 compound to a specific site.

Spacer sequences between fusion partners may also be applied.

The GDF3 compounds of the invention may also be conjugated to a pharmaceutically acceptable nonproteinaceous polymer e.g. polyethylene glycol, polypropylene glycol or polyoxyalkylene to e.g. increase their half life in the subject's body, e.g. in circulation or gastrointestinal tract or muscle.

The GDF3 compound may be glycosylated and unglycosylated.

Methods for elaborating protein therapies and therapeutic proteins are well known in the art.

Targeting can be facilitated by fusion proteins which also can be prepared for therapeutic purposes e.g. in Schmidt, Stefan R. (Editor) "Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges" ISBN: 978-0-470-64627-4, April 2013.

In certain embodiments, the fusion component is a targeting ligand, or derivative or fragment thereof, capable of binding specifically to a pre-selected cell surface protein, and thereby delivering the GDF3 compound to a target cell, e.g. a muscle cell. Such a targeting ligand can be e.g. an antibody, an antibody fragment comprising the epitope recognition site or an other recognition molecule. The fusion component of the invention may also be another active compound, which may be any agent that is desirable to deliver to a pre-selected site for therapeutic purposes.

The dosage regimen will be determined by the attending physician considering various factors which modify the action of the GDF-3 protein, e.g. amount of tissue desired to be formed, the site of tissue damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue, the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and the types of GDF3 compound. Generally, systemic or injectable administration, such as intravenous (IV), intraperitoneal (IP), intramuscular (IM) or subcutaneous (Sub-Q) injection are considered or used.

Administration will generally be initiated at a dose which is minimally effective, and the dose will be increased over a preselected time course until a positive effect is observed.

Subsequently, incremental increases in dosage will be made limiting such incremental increases to such levels that produce a corresponding increase in effect, while taking into account any adverse affects that may appear. The addition of other medicaments, in particular other known growth factors, such as IGF I (insulin like growth factor 1), or growth hormones which may aid in increasing muscle mass, to the final composition, may also affect the dosage.

Progress can be monitored by periodic assessment of tissue growth and/or repair as well as any techniques described herein [see for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding].

Routes of administration and pharmaceutical preparations

Methods known in the art for the therapeutic delivery of agents such as proteins or nucleic acids can be used for the therapeutic delivery of a GDF3 compound of the invention or a nucleic acid encoding it.

A nucleic acid can be delivered e.g. by cellular transfection, gene therapy, direct administration with a delivery vehicle or pharmaceutically acceptable carrier, indirect delivery by providing recombinant cells comprising a nucleic acid encoding said polypeptide of the invention.

Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, parenteral and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

Various further delivery systems are known and can be used to administer the fusion polypeptide of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis [see, e.g., Wu and Wu, (1987), J. Biol. Chem. 262:4429-4432],

In particular, the GDF3 compound may be administered into the blood stream, e.g. by intravenous injection. This administration route is preferred in case of muscle disorders, in particular in case of systemic diseases e.g. those listed herein.

Intraperitoneal (IP) administration is usual in case of animals, like mammals.

Oral administration is also a preferred route of systemic administration from the point of view of ease and complience of the subject.

Methods for oral administration of GDF3 are disclosed e.g. in US 20070172481A1

Briefly oral preparations may be in the form of capsules, cachets, pills, tablets, lozenges, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles, each containing a predetermined amount of a GDF3 compound as an active ingredient.

The GDF3 compound may be mixed with one or more pharmaceutically acceptable carriers or excipients, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol etc. which may even stabilize the protein; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; absorbents, such as kaolin and bentonite clay; lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring and flavoring agents.

As a preferred concept the protein should be protected from decomposition e.g. proteolytic cleavage in the gastrointestinal tract wherein a protective coating may be applied. Such coatings are well known in the art.

A further helpful method may be engineering GDF3 against proteolytic cleavage by proteases of the gastrointestinal tract.

A review on modern methods regarding administration of proteins and other biopharmaceuticals are disclosed in by Mitragotri, S. et al. [Mitragotri, S. et al. Nature Reviews Drug Discovery 13, 655-672 (2014)].

Oral formulations may be prepared in analogy of oral formulations for IGF1. For example, more specifically, oral formulation of GDF3 may be prepared e.g. as disclosed by Burrin, D. G. et al. for a formulation of IGF-1 [Burrin, D. G. et al., American Journal of Physiology (1996) 270(5) R1085- R1091]. The present invention also provides pharmaceutical compositions comprising a protein of the invention and a pharmaceutically acceptable carrier. Formulation should follow the guidelines provided in e.g. a regulatory agency of the US Federal or a state government or listed in the U.S. Pharmacopeia or the European Pharmacopoeia 8th Edition (with e.g. supplements 8.6, 8.7 and 8.8) or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Pharmaceutical carriers can be any carried uses in this field of art. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of injections, solutions, suspensions, emulsion, gels, powders, sustained-release formulations and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

For example GDF3 can be prepared in the form of an injection. Expediently, GDF3 is provided in a lyophilized form which is reconstituted freshly before administration. Any usual physiological carrier solution may be applied. The injection may be e.g. local administration, like intramuscular or subcutaneous administration. Alternatively, systemic administration like intravenous injection may be applied.

As an other example GDF3 can be prepared in a spray formulation. As probably GDF3 has a limited storage time stabilizer agents can be added.

GDF3 can be included into lyposomes which formulation can be applied for example in sprays, ointments and gels. Gels may be hydrogels, like gelatin hydrogen, methylcellulose gels etc.

Sustained release local administration are also known. For example, GDF3 can be included in a hydrogel which can be laid on or temporarily implanted into the injured muscle site. Example for such formulation is described e.g. by K.M. Lorentz et al. [Biomaterials 33 (2012) 494-503].

Co-administration with other muscle regenerating compounds

Based on the disclosure provided herein a skilled person will understand that GDF3 may be coadministered with other muscle regenerating agents. Such muscle regenerating agents may be, without limitation, myoblast proliferation agents, like myostatin (GDF8) inhibitors, Mox2 agonists, Myf5 agonists, MyoD agonists and IGF1 as well as, without limitation other myoblast differentiation agents like myogenin (Myf4 agonists), MRF4 or Herculin (Myf6 agonists) to mention a few type of candidates or agents as examples. Co-administration can be effected by administering these agents in separate dosage units or even via different administration routes or, if appropriate in the same dosage unit.

Increasing muscle mass

It is contemplated that promoting differentiation of newly formed muscle fibers leads to increased muscle mass. This feature can be utilized e.g. in livestock animals for producing animals with increased quantity and possibly increased quantity of meat. It is known in the art that an increase in muscle fibers or myotubes which is associated with muscle differentiation contributes to the increase of muscle mass, in particular in mammals.

In this embodiment the GDF3 compound can be provided to the animal by any means disclosed herein, specifically by any of the following administration means.

By oral administration as disclosed above. Oral formulation can be added for example to the feed of the animals. The animals can also be treated intravenously or by any other means appropriate for targeting the muscle. By cells expressing the protein e.g. by macrophages.

By preparing a transgenic animal wherein GDF3 is expressed possibly in muscle cells or sells of the blood stream e.g. macrophages.

The animal is preferably a vertebrate animal, more preferably mammalian (including a nonprimate and a primate) including, but not limited to, murines, simians, mammalian farm animals (e.g., bovine, porcine, ovine), mammalian sport animals (e.g., equine), and mammalian pets (e.g., canine and feline). The term also refers to avian species, including, but not limited to, chickens and turkeys.

Health compositions, dietary supplements and neutraceuticals.

The present invention also provides GDF3 compounds in the form of dietary supplements or health composition other similar composition which are formulated for regular oral consumption by humans or animals, preferably mammals. Such compositions are similar and analogous to oral compositions as disclosed above.

The health claims comprise statements about a relationship between food and health.

For example in Europe Commission Regulation (EU) No 432/2012 established the list of permitted health claims i.e. to which new health claims may be added. The European Food Safety Authority (EFSA) is responsible for evaluating the scientific evidence supporting health claims, for example 'Function Health Claims'(or Article 13 claims) e.g. relating to the growth, development and functions of the body.

The dosage is set to a level wherein regular daily consumption provides a level of GDF3 in the blood and muscles appropriate for maintaining differentiated muscle fiber level or increasing it to an appropriate level to maintaining or increasing muscle strength and homeostasis.

Cell therapies

An alternative way of administration is cell therapy wherein cells expressing GDF3 are administered to the site of muscle to be repaired. Typically cells expressing GDF3 at a high level are applied to target the muscle needing treatment or regeneration.

In case of a local injury the cells can be administered locally e.g. topically as in a patch or emplastrum. In this embodiment the cells overexpressing GDF3 may be in principle any type of cells which are pharmaceutically acceptable or tolerable. For example cells compatible with the surrounding tissue like cells of the connective tissue, adipocytes, skin cells like keratinocytes or mesenchymal stem cells or macrophages may be applied.

Expediently the cells may be provided in an appropriate medium, e.g. in a matrix e.g. gel-like matrix, e.g. a hydrogel or alginate gel or a matrix comprising hidrocolloid, which is laid on or formed on the surface of the injury. The matrix should comprise appropriate medium appropriate to ensure survival or even growth of the cells for a sufficient time. The gel may be attached to or form part of the patch or emplastrum thereby forming a composite dressing. Techniques for preparing such devices are well known in the art.

Such technologies are described e.g. in the following publications: [Bryant, R., & Nix, D. (2007). Acute and Chronic Wounds (3rd ed.). Mosby.; Baranoski, S., & Ayello, E. A. (2004). Wound Care Essentials: Practice Principles. Lippincott Williams & Wilkins.; Sussman, C, & Bates-Jensen, B. M. (2007). Wound Care: A Collaborative Practice Manual. Lippincott Williams & Wilkins.] and publications cited therein. Surgical dressings are described e.g. in Diane Krasner: Surgical Dressings and Wound Management 2nd Edition: A Reference on the Development, Properties and Clinical Use of Surgical Dressings and Modern Wound Management Materials Kestrel Health Information, 2012 ISBN 0972841407, 9780972841405.

The cells are preferably cells of the same species as the patient in order to avoid rejection.

In an embodiment the cells are separated from the injured muscle tissue e.g. by being embedded into the matrix. Said matrix may have some barrier which prevent the cells from being contacted with the injured muscle tissue but which allows the GDF3 protein to be transported through the barrier. If the cells used for medical treatment are cells of a different species this embodiment should be preferably used.

Alternatively, the cells are applied intravenously and the blood of the patient carries the cells to the target cite.

In case of a systemic disease systemic e.g. intravenous administration is preferred. In this case blood cells, preferably macrophages are used.

In an embodiment in case of muscle injuries the GDF3 compound is administered on day 1, 2, 3, 4, 5 or 6 after the injury, preferably on day 3, 4 or 5 after the injury, highly preferably on day 4 after the injury. Alternatively the macrophage are administered when the inflammatory phase of healing starts to decline or after it has started to decline.

Alternatively, in case of systemic diseases the GDF3 compound is administered continuously for the time required, e.g. typically for 1, 2, 3 or 3 weeks, from 2 to 12 months or for years.

Therapy with macrophages

Macrophages have long been known as cellular vehicles in gene therapy. For example macrophages have been used as system to deliver gene therapy to tumors and pathological hypoxia, for example via adenoviral and lentiviral gene delivery [Griffiths L et al. "The macrophage - a novel system to deliver gene therapy to pathological hypoxia" Gene Therapy (2000) 7, 255-262; Escobar J et al. "Oncolmmunology Engineered tumor-infiltrating macrophages as gene delivery vehicles for interferon-a activates immunity and inhibits breast cancer progression" 3, e28696; 2014].

Burke B. et al. already in 2002 reviewed the efficacy of various gene transfer methods in transfecting macrophages in vitro, and the results obtained when transfected macrophages are used as gene delivery vehicles [Journal of Leukocyte Biology (2002) vol. 72 no. 3 417-428. The authors also discussed the use of various viral and nonviral methods to transfer genes to macrophages in vivo. Furthermore, Zhang, Xia et al. [Methods Mol Biol. 2009 ; 531: 123.] discusses the state of the art in 2009 regarding the expression of exogenous genes in macrophages and concludes that relatively high efficiency gene expression in primary human or murine macrophages is becoming more routine. Among others, adenoviruses, Antiviruses, adeno-associated viruses, and poxviruses, as well as a wide array of nonviral methods, have been used for gene delivery into macrophages. Viral methods generally give higher trans fection efficiencies, however, non-viral methods allow a more rapid functional testing of constructs. Typically, transformation of macrophages may be effected by electroporation, nucleofection, viral transduction by adenoviruses, adeno-associated viruses, retroviruses or lentiviruses. Viral vectors useful in the present invention typically include all the sequences for nucleic acid replication, encapsidation and host cell integration. Vectors comprising the GDF3 gene and appropriate regulation sequences can be prepared using techniques known in the art. Preferred viral administration routes, in particular adenoviral administration are administration into the muscle or into the liver.

In somewhat more detail, adenoviruses are a preferred method of gene transfer to primary macrophages due to their ability to infect nondividing cells with high efficiency and reasonable longevity (up to several weeks) of transgene expression (see above). The advantage of adeno- associated viruses (AAV) is e.g. their ability to integrate into the genome of host cells and mediate long-term expression of transgenes. Poxviruses and herpes simplex virus have also been used to transfer transgenes to macrophage cell lines. The relatively low size limit of the length of foreign DNA should not be a problem in the present invention as the gene coding for GDF3, in particular mature GDF3 is smaller.

Among non-viral methods receptor-mediated gene transfer provided an appropriate solution as ligands such as mannose and transferrin incorporated into gene transfer vehicles have been shown to increase the efficacy of transfection for primary macrophages in vitro. Microorganisms as vehicles for transfection of macrophages provide a further option.

Electroporation and lipofection, once ineffective methods, have become more efficientrecently.

Macrophages manipulated ex vivo have been shown to be capable of homing to experimentally damaged muscle. Nevertheless, as manipulated macrophages might be trapped in other organs an administration into the injured muscle is a preferred route. In this regard e.g. an intramuscular injection can be applied. Alternatively the cells may be provided in a gel matrix, e.g. a hydrogel. In this case a topical administration is applicable.

In an embodiment the macrophages are manipulated to express a higher level of GDF3 preferably natural GDF3. For example, a higher expression level of GDF3 is achieved by administration of a PPARg agonist to the macrophages. Such agonists are e.g. rosiglitazone (RSG) or any other pharmaceutically acceptable or tolerable PPARg agonist.

Without any limitation and among other as culture media RPMI medium (like RPMI-1640-10 medium), DMEM/F12-10 medium etc. can be used.

Macrophage populations typically self-maintain independently of haematological progenitors which advises for their use as therapeutic cells. As examples for use of macrophages in muscle, Novak et al. [J Pathol. 2014 February ; 232(3): 344-355.] disclosed cell therapy with exogenous Ml macrophages which reduced fibrosis and enhanced muscle fiber regeneration in lacerated muscles. Rybalko, Viktoriya et al. [PLoS One. 2015; 10(12): e0145550] disclose the development of macrophage-mediated cell therapy to improve skeletal muscle function after injury. The authors used bone-marrow derived in vitro LPS/IFN-y-induced Ml macrophages to enhance functional muscle recovery after tourniquet-induced ischemia/reperfusion injury. Interestingly, they found that MO bone marrow-derived unpolarized macrophages significantly impaired muscle function highlighting the complexity of temporally coordinated skeletal muscle regenerative program.

For example, Bencze M. et al. [Bencze M. et al. Molecular Therapy vol. 20 no. 11 nov. (2012) 2168] teach a strategy for the improvement of cell-based therapy for muscular dystrophies based on their finding that proinflammatory macrophages play a supportive role in the regulation of myoblast behavior after transplantation into preinjured muscle, and could thus potentially optimize transplantation of myogenic progenitors in the context of cell therapy. The authors describe that "the availability of blood-derived monocytes from patients that can be further differentiated and activated toward proinflammatory or anti-inflammatory macrophages, ... are in favor of this."

In the present invention similar protocols may be applied, however, manipulated macrophages are used which overexpress GDF3. It is thought that GDF3 is secreted in a mature form from the macrophages which suggest that expression of the full length form is preferred which is processed by the macrophage.

In a preferred invention autologous gene therepy is used. In this case the first step is the isolation of macrophages from muscle. Muscles were dissociated in appropriate medium. CD45+ cells are isolated e.g. using magnetic sorting (Miltenyi Biotec). CD45+ cells are the further sorted as necessary, e.g. into Ly6C+ F4/801ow macrophages, Ly6C- F4/80+ macrophages and Ly6Cmid F4/80- neutrophils. Typically Ly6C- F4/80+ macrophages are used in the present invention for gene delivery.

Alternatively bone marrow macrophages are used for gene delivery and then macrophages are differentiated. At appropriate level of differentiation CD45+ cells, and in particular CD45+ Ly6C- or preferably CD45+ Ly6C- F4/80+ macrophages are obtained.

In a preferred embodiment the macrophages are augologous macrophages. In a preferred embodiment GDF3 is recombinantly expressed in the macrophages. In a preferred embodiment the macrophages are M<t s differentiated into Ly6C- cells. Preferably, the macrophages are CD45+ Ly6C- F4/80+ macrophages.

In an embodiment in case of muscle injuries the macrophages are administered on day 1, 2, 3, 4, 5 or 6 after the injury, preferably on day 3, 4 or 5 after the injury, highly preferably on day 4 after the injury. Alternatively the macrophage are administered when the inflammatory phase of healing starts to decline or after it has started to decline.

Gene and cell therapy solutions in clinical medicine are disclosed in the comprehensive book of Nancy Smyth Templeton (Ed): Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Fourth Edition, CRC Press, 2015. The book discloses state of the art technologies and concepts, vector design and construction, delivery systems, therapeutic strategies, gene expression, regulation and detection, disease targets, clinical applications and trials, cell-based therapies, novel imaging systems, gene regulation, and regulatory affairs.

Transgenic animals and cells

Transgenic animals may be laboratory animals or animal models as disclosed herein. Such animals may have a silenced or knocked down or knocked out GDF3 gene and thereby the animal can be used as a model of a muscle disease.

Alternatively, transgenic animals may comprise additional recombinant expression of GDF3.

Genetic Engineering of livestock animals are well known in the art [Wheeler, M. B. (2013) Transgenic Animals in Agriculture. Nature Education Knowledge 4(11):1 and references cited therein].

Below the invention and creation thereof are described in more detail by specific non-limiting experimental examples.

RESULTS

PPAR expression in macrophages of the cardiotoxin induced skeletal muscle injury model To characterize the immune derived regulators of skeletal muscle injury, we triggered skeletal muscle damage in the tibialis anterior (TA) muscle of mice by intramuscular injection of the snake venom, CTX, to induce a homogenous muscle damage that is repaired with the active contribution of infiltrating immune cells. This model provides a very reproducible kinetics of a series of sequential events: myofiber necrosis, infiltration by inflammatory cells and phagocytosis of damaged fibres by ΜΦΒ, regeneration of myofibers, which first appear as small basophilic structures that will eventually grow as centrally nucleated myofibres (Mounier et al., 2013). To map the regenerative mechanisms provided by infiltrating ΜΦ populations, we isolated representative ΜΦ populations from injured muscle and interrogated their gene expression profiles by microarray analysis. In injured muscles, an initial wave of Ly6C+ ΜΦ infiltration is replaced by the emergence of Ly6C- ΜΦΒ from day 2 on. The simplified, but widely accepted paradigm about these two main ΜΦ populations posits that the Ly6C+ ΜΦ5 are inflammatory, while Ly6C- ΜΦ5 are repairing. Surprisingly, when the expression profiles of Ly6C+ inflammatory and Ly6C- repair ΜΦ5 derived from injured muscle at day 2 CTX injury were compared, GO categories belonging to lipid and carbohydrate metabolism dominated the biological processes that were the most robustly upregulated with the transition to the Ly6C- (repair) ΜΦ5 (Fig S1A). This finding is in line with recent reports that suggested cell metabolism as a defining factor in ΜΦ identity and functional status (Lavin et al., 2014; Odegaard and Chawla, 2011; Okabe and Medzhitov, 2014; Vats et al., 2006). When analyzing the expression data for metabolic regulators that could account for such a predilection for metabolic genes, we found that a master regulator of metabolism, Pparg, was expressed at a high level in these ΜΦ5. While the in vitro antiinflammatory effects of ΜΦ PPARy are well documented, the in vivo functions of PPARy in ΜΦ5 remained relatively unexplored (Gautier et al., 2012; Varga et al., 2011). We have previously shown that both the Ly6C+ and Ly6C- ΜΦ subtypes in injured muscle are likely derived from a single blood monocyte population (Ly6C+ blood monocytes) (Varga et al., 2013). The common origin of the ΜΦ subtypes allowed us to compare the gene expression profile of ΜΦΒ, that infiltrate the muscle upon injury, to that of their likely precursor (Ly6C+) blood monocytes and also other ΜΦ and dendritic cell (DC) populations, using the publicly available gene expression data generated within the Immunological Genome Project (Fig. SIB). We found that the expression of Pparg was induced in infiltrating M<t s compared to their precursor monocytes and that its expression was further induced as inflammatory Ly6C+ muscle M<t s differentiated into Ly6C- repair M<t s. We also found that Pparg in muscle M<t s was highly expressed, and that only two in vivo ΜΦ subtypes, alveolar ΜΦΒ and splenic red pulp ΜΦΒ expressed Pparg at a higher level.

Based on these findings we hypothesized that ΜΦ PPARy is a regulator of skeletal muscle regeneration. To test this hypothesis, we made use of the LysM Cre mouse strain, to generate mice that were deficient in PPARy specifically in their myeloid lineages [Pparg^l^, LysMCre⁺ mice, herein referred to as PPARg MacKO) (Clausen et al., 1999). When CD45+ cells, which comprise all infiltrating hematopoietic cells, or sorted ΜΦ5 were isolated from injured skeletal muscle, we could easily detect the expression of Pparg in these cells by RT-qPCR (Fig SIC and ID) in wild type (WT) animals. Furthermore, the expression of Pparg was greatly diminished in corresponding CD45+ and ΜΦ cells isolated from PPARg MacKO animals (with an average deletion efficiency of 95 % in the CD45+ and 85.5 and 87 % in the Ly6C+ and Ly6C- ΜΦ compartments at day 2, respectively) (Fig SIC and ID), validating the suitability of this genetic model for these experiments.

Macrophage PPARyregulates skeletal muscle regeneration

WT and PPARg MacKO animals were injected with CTX to induce TA muscle injury and then regeneration was analyzed by a combination of morphometric and FACS analysis. We found that PPARg MacKO animals showed a pronounced delay in their TA muscle regeneration. First, cross- sectional area (CSA) of the regenerating muscle fibers in the PPARg MacKO animals were significantly smaller than in WT mice at day 8 and day 21 following CTX injury (Fig 1A, 1C, ID and Fig 9A). The distribution of CSA in the PPARg MacKO animals exhibited a characteristic shift to the left, indicating an increased number of small and fewer large regenerating myofibers (Fig 1C and Fig SI). Second, there were a significantly higher number of phagocytic/necrotic fibers present (FiglA and IB) at day 8 post CTX in PPARg MacKO mice, indicating either a delayed clearance of dying myofibers or an altered dynamics of muscle fiber death in KO animals. Third, regenerative areas with increased inflammatory infiltrations persisted in PPARg MacKO muscles (Fig 1A), suggesting that the resolution of inflammation was delayed. As PPARy is expressed in muscle at a low but detectable level, we wanted to ascertain whether the PPARy deficiency in the hematopoietic compartment was the major contributor to the observed phenotype. To prove this, we elected to use a second genetic model, in which bone marrow from the specific and total epiblastic conditional KO of PPARy [Pparg^l-, Sox2Cre⁺) (Nadra et al., 2010) or WT animals were used to reconstitute the hematopoietic compartment in irradiated WT animals (bone marrow transplanted or BMT animals). TA muscles of recipient BMT animals were injected with CTX 12 weeks after BMT and histological analysis of muscle regeneration was carried out 22 days post injury. When compared to animals that received WT bone marrow (WT BMT), mice that received bone marrow deficient in PPARy (PPARg KO BMT) exhibited a profound delay in regeneration. Similar to the delayed regeneration seen in the PPARg MacKO animals, muscle sections of PPARg KO BMT mice contained significantly more small myofibers, as demonstrated by the lower CSA (Fig IE and IF). Further underlying the importance of PPARy in muscle regeneration, full body Pparg^l- Sox2Cre⁺ animals displayed a drastic impairment in their skeletal muscle regeneration (Fig. 9B). Altogether, the results from these distinct genetic models clearly indicated that PPARy activity in muscle infiltrative M<t s critically contributed to the timely resolution of inflammation and to regeneration.

PPARy deficiency does not alter macrophage infiltration or differentiation in injured muscle Several possible reasons could explain why M<t s PPARy deficiency leads to such a critical impairment in muscle regeneration. First, if PPARy deficiency leads to a decreased ΜΦ infiltration and/or altered cellular differentiation, then the underlying reason behind our observations would be the inability of a smaller infiltrating ΜΦ compartment to support muscle regeneration. To monitor the cellular dynamics of immune infiltration in CTX injured muscle, we treated WT and PPARg MacKO animals with CTX injection and on days 1, 2 or 4 all hematopoietic cells were isolated from the injured muscles using CD45+ magnetic bead selection. We found no difference between the numbers of infiltrating immune cells in WT vs. PPARy deficient animals (Fig. 2A) at any time point. This observation excluded the scenario of a greatly diminished ΜΦ infiltration in PPARy deficient animals, but did not exclude the possibility of a change in the cellular composition of immune infiltration. Therefore, we examined the dynamics and composition of immune infiltration in injured muscle in WT and PPARg MacKO animals (Fig 10). At day 1 the predominant immune cells within injured muscle are neutrophils and Ly6C+ ΜΦΒ. We determined the proportion of the Ly6C^mid F4/80- neutrophils and Ly6C⁺ F4/80^low ΜΦ≤ within the CD45+ cells isolated from injured muscle at day 1 (Fig 2B). We found that fewer neutrophils infiltrated the injured muscle in PPARg MacKO animals (68.05 % vs. 64 %), which also meant that the ratio of Ly6C+ ΜΦ≤ were slightly higher in PPARy deficient animals (19.4 % vs. 22.7 %). Although the observed alteration in the proportion of cells was significant, it must be noted that the absolute number of infiltrating neutrophils or Ly6C+ ΜΦΒ were very high, therefore when we calculated the absolute number of infiltrating neutrophils and Ly6C+ ΜΦΒ (Fig 2C), we concluded that very similar absolute numbers of immune cells infiltrated the injured muscle in WT and PPARg MacKO animals (1.19 vs. 1.15 (X10⁶)). Due to the fact that PPARy is expressed in ΜΦΒ but not in neutrophils, we decided to follow on the differentiation dynamics of ΜΦΒ from day 2 onward, as the observed effect of PPARy deficiency must derive from the ΜΦ subsets. We determined the proportion of Ly6C+ F4/80^low and Ly6C^" F4/80^hi§h ΜΦΒ in injured muscle at day 2, and found no differences in the cellular composition of ΜΦ subsets between WT and PPARg MacKO animals (Fig. 2D). Similarly, no differences in the subset composition of infiltrating M<t s were detected at day 4 after CTX injury (Fig 2E). Collectively, these results show no major effect of PPARy deficiency in myeloid cells on ΜΦ infiltration or fate within the regenerating muscle. Therefore the observed delay in muscle regeneration is likely due to alterations in the functions of PPARg MacKO M<t s.

Macrophage PPARy regulates myoblast differentiation in a paracrine manner in vitro

Next, we wanted to explore which ΜΦ functions might be relevant to muscle regeneration and regulated by PPARy activity in M<t s. M<t s have traditionally been considered to be primarily phagocytic cells. Therefore, one plausible explanation is that PPARy activity in M<t s is required for the clearance of dying muscle tissue, and the failure of regeneration in PPARg MacKO animals is due to improper clearance of debris. While the importance of M<t s in phagocytic clearance is beyond doubt, it recently became widely accepted that M<t s could promote regeneration via other important functions apart from phagocytosis, such as the production of biologically active molecules [e.g. IGF1) that regulate muscle growth or differentiation (Tonkin et al., 2015). Therefore, ΜΦ PPARy could plausibly improve the regenerative capacity of skeletal muscle by affecting MPC expansion or differentiation, independent of its phagocytic activity. We elected to use in vitro experimental systems to test whether either an altered phagocytic capacity or a pro-regenerative PPARy activity in ΜΦ≤ was behind the beneficial effect of ΜΦ PPARy in muscle regeneration. Since the number of ΜΦ≤ isolated from injured muscle and the fickle nature of ΜΦ phenotypes make the development of in vitro experimental systems using muscle derived ΜΦ≤ extremely difficult, we decided to use bone marrow derived ΜΦ≤ (BMDMs) isolated from WT, PPARg MacKO or WT BMT and PPARg KO BMT animals to test the impact of on the phagocytic activity of in ΜΦ≤ (Fig 3A and B). We set up a phagocytosis assay in which fluorescently labeled, necrotic C2C12 myoblasts were co-incubated with BMDMs labeled with a different fluorescent dye. The uptake of necrotic C2C12 cells by BMDMs could be monitored by measuring the ratio of the fluorescence intensities of BMDMs emitting both fluorescent signals. PPARg MacKO BMDMs showed a slight, but not significant increase in the number of BMDMs that phagocytosed C2C12 cells when compared to WT BMDMs, indicating that PPARg KO BMDMs had a borderline increased propensity to engage in phagocytosis. The median fluorescence intensity of the BMDMs that took up C2C12 cell debris was not different between double positive BMDMs of WT or PPARg MacKO genotypes, indicating that once phagocytosis commenced, both cells were able to take up the same load of foreign material. Due to the fact that the efficiency and timing of the LysM-Cre driven deletion of floxed target genes in BMDMs is unclear, we repeated this experiment using BMDMs derived from BMT WT or PPARg KO BMT animals, which provides a better reporter system to test the effect of complete absence of PPARy expression in ΜΦ≤. We found a similar, not significant increase in the number of BMDMs that actively participate in phagocytosis. Unlike the case of PPARg MacKO cells, however, PPARg KO BMT BMDMs were able to phagocytose a greater load (Median fluorescence intensity, or MFI=122.8 vs. 192.8 in WT BMT vs. PPARg KO BMT, respectively) once phagocytosis commenced. Our original model was that PPARg MacKO animals suffered a delay in muscle regeneration due to an incomplete clearance of debris. However, the results from the in vitro experiments indicated that such an inadequate phagocytic clearance is not responsible for the observed phenotype, as PPARy deficient BMDMs showed a slightly improved phagocytic activity, if any.

These conclusions led us to test if ΜΦ PPARy activity confers a yet unidentified muscle growth, or differentiation-promoting phenotype to M<t s, which could explain the observed delayed muscle regeneration in animals deficient in PPARy in M<t s. To test this hypothesis, we elected to use the well-established in vitro primary muscle progenitor proliferation or differentiation assays that utilize primary myoblasts isolated from young WT mice (Fig 3C and 3D). These myoblasts can be maintained in serum rich medium in their progenitor status for a limited number of cell divisions, but when cultured in serum-restricted conditions, they are able to form myotubes that are generated by fusion of individual differentiating myocytes. In the first assay we cultured primary myoblasts in conditions favoring cell proliferation and measured the proliferation index by detecting Ki67⁺ cells by immunofluorescence (IF). It is known that cell culture supernatant derived from classically polarized M<t s can promote myoblast proliferation (Mounier et al., 2013). Indeed, in the presence of conditioned medium derived from classically activated WT BMDMs the myoblast proliferation index increased substantially. Surprisingly, conditioned medium from non-treated PPARg MacKO BMDMs phenocopied the proliferation enhancing effect of inflammatory WT BMDMs on myoblasts (Fig 3C). The increased myoblast proliferation in PPARg MacKO BMDM supernatant was also detected even when BMDMs were treated with IL4. These results indicate that PPARy in M<t s modulates an unknown signaling system that could influence myoblast proliferation in a paracrine manner. Next, we tested the effect of BMDM derived conditioned media on the differentiation of myoblasts. For the differentiation assay we counted the number of cell nuclei within freshly formed desmin positive (by IF) myotubes cultured in differentiation medium. We tested the differentiation of primary myoblasts that were cultured in conditioned medium with reduced serum content harvested from non-treated or IL4-treated WT and PPARg MacKO BMDMs (Fig 3D). In line with earlier reports, we observed a large increase in differentiation when myoblasts were grown in conditioned medium derived from IL4-treated WT BMDMs (Fig 3D), as compared to medium derived from non-treated M<t s. Importantly, this increase in differentiation was abrogated when conditioned medium from IL4-treated PPARg MacKO BMDMs was added to differentiating myoblasts. This effect was seen when several, independently isolated primary myoblast cell lines were used for the experiments (Fig. IOC). Together, these results strongly support a model in which PPARy regulated signaling systems in BMDMs act on primary myoblast proliferation and/or differentiation in a paracrine manner and in which the activity of PPARy in BMDMs favored myoblast differentiation over proliferation. The in vitro assays used in these experiments served as a model for the in vivo events occurring upon sterile muscle injury. Therefore, our results raised the possibility that similar signaling events took place in situ, where the cellular equivalents of our model system, namely muscle infiltrative M<t s and MPCs might interact to achieve a synchronized and timely regeneration. PPAR regulates cell type specific genes in muscle infiltrating M0s

As only few paracrine signaling pathways between M<t s and tissue progenitors have been described, we decided to pursue the identification of the putative regulatory circuit that might connect muscle infiltrating M<t s to myotube differentiation in a PPARy dependent manner. As PPARy is a transcription factor, we presumed that a critically relevant change in the gene expression in muscle M<t s must shed light on the regulatory circuit that is abrogated in PPARy KO ΜΦΒ. TO identify PPARy dependent gene expression changes in muscle infiltrating ΜΦΒ, we isolated representative populations of M<t s from injured and regenerating muscle from WT and PPARg MacKO animals and analyzed their gene expression profiles by microarray analysis (Fig. 11). We selected Ly6C+ M<t s at day 1 and 2 and Ly6C- M<t s at day 2 and 4 post CTX injury and compared their gene expression by 2 way ANOVA test. The central objective of our analyses was to identify the cell type in which PPARy was active and the genes that were regulated by PPARy. To present the gene expression changes observed, we created separate heat maps for each examined ΜΦ subset, that show all genes that were differentially expressed in the relevant subsets but that also show the gene expression pattern of these same genes in all the other ΜΦ subsets. We found that surprisingly little difference in gene expression was detectable between WT and PPARg MacKO Ly6C+ ΜΦΒ at day 1 (Fig 4A). Altogether, only 5 genes were upregulated and 6 genes were downregulated in PPARy deficient ΜΦΒ. It was also evident that although these genes were differentially expressed between WT and PPARg MacKO Ly6C+ ΜΦΒ at day 1, they showed surprisingly dynamic expression changes when different ΜΦ subsets were compared, indicating that these genes were most likely under complex regulation and PPARy was only one of the potential regulators affecting their expression. In Ly6C+ ΜΦΒ isolated at day 2 after injury, many more genes were differently expressed when comparing WT and PPARg MacKO ΜΦΒ (Fig 4A), albeit the fold change difference in their expression was still modest. Remarkably, PPARy deficiency in day 2 and 4 ΜΦΒ predominantly led to increased gene expression, rather than to repression/lack of induction. The top 5 genes that were most differentially regulated in WT vs. PPARg MacKO cells are shown in Fig. 4B. The number of genes that were concordantly regulated in a PPARy mediated manner in more than one ΜΦ subtypes is shown in Fig. 4C. We analyzed the differentially expressed genes for two reasons. First, we wanted to find those genes that were regulated by PPARy in ΜΦΒ. Our knowledge about the transcriptomic activity of PPARy in ΜΦΒ is surprisingly limited, partly because of the scarcity of ex vivo ΜΦ subtypes in which PPARy is expressed at high levels and transcriptionally active. More importantly, we wanted to identify those PPARy dependent genes whose differential expression might account for the observed muscle regeneration delay in PPARy deficient animals.

We used two parallel approaches to identify PPARy regulated genes in muscle infiltrating ΜΦΒ. First, we hypothesized those genes could be under regulation by PPARy that were expressed differently in more than one subtype of muscle ΜΦΒ. Accordingly, we combined the lists of upregulated genes reported by the ANOVA analysis of WT vs. PPARg MacKO comparisons. Although many genes were differentially regulated in a single type of muscle ΜΦΒ, only 5 genes [Saa3, Hebpl, Plxndl, Apoldl, Tsgl Ol) were upregulated in all 4 investigated subtypes of PPARy deficient muscle ΜΦΒ (Fig. 4C and table SI). Similarly, only 2, 2, 1 and 0 genes were upregulated in 3 subtypes of PPARg MacKO ΜΦΒ, depending of the combination of subtypes. When we relaxed our criteria to identify those genes that showed PPARy dependency in two subtypes, Ly6C+ and Ly6C- M<t s isolated on day 2 showed far the largest overlap by far (46 PPARy dependent genes). Next, we analyzed the gene sets that were reproducibly downregulated in PPARg MacKO M<t s. Strikingly, there was only 1 gene, namely Growth differentiation factor 3 (Gd/3), that was consistently downregulated in all 4 investigated ΜΦ subtypes (Fig. 4B and C). Even when we relaxed our criteria to find PPARy dependent downregulated genes in any combination of two different ΜΦ subtypes, we found very few genes that were similarly regulated in different ΜΦ populations, with day 1 and day 2 Ly6C+ ΜΦΒ showing the greatest overlap (20 genes). In summary, we identified several putative PPARy target genes that showed consistent PPARy dependency in several muscle ΜΦ subsets. To ascertain the PPARy dependent regulation of some representative genes, we measured mRNA levels of Gd/3, Apoldl, Hebpl and Plxndl by RT-qPCR in ΜΦ subsets sorted from injured muscle (Fig. 12).

Interestingly, the genes we identified as PPARy dependent by the above strategy did not belong to the group of canonical PPARy regulated genes described in various myeloid cells in earlier studies (such as Plin2 (AdrpJ, Cd36, Angptl4 or Fabp4) (Szanto et al, 2010; Welch et al, 2003). One possible reason for this discrepancy could be that most in vitro studies apply synthetic or natural ligands of PPARy to study the transcriptional activity of the receptor upon ligand activation. Therefore, we wanted to see if synthetic PPARy ligand activation of infiltrating ΜΦ5 gave rise to transcriptional changes that are more reminiscent of the list of previously identified PPARy target genes. For this reason we treated WT animals with rosiglitazone (RSG) via gavage daily prior to CTX injury and throughout the first two days of regeneration. Next, we isolated Ly6C+ and Ly6C- ΜΦ5 at day 2 from RSG treated mice and compared their gene expression profiles to those of animals untreated with RSG (Fig 4D and Table SI). Several unexpected features of the gene expression changes were evident. First, many more genes were regulated by RSG treatment in Ly6C+ cells than in Ly6C- ΜΦ5. Again, the genes that showed differential expression upon RSG treatment in Ly6C+ cells did not contain established PPARy regulated genes. The RSG regulated gene list did not contain the 6 differently regulated genes that appeared to be very consistently under PPARy regulation in all ΜΦ subsets in untreated animals. Second, although RSG treatment caused the differential regulation of fewer genes in Ly6C- ΜΦ5, the most robustly upregulated gene was Angptl4, one of the best-characterized PPARy target genes. This suggests that not only do Ly6C- ΜΦ5 at day 2 express PPARy , but the receptor is also sensitive to the activating effect of an exogenous ligand in Ly6C- cells. It is important to note that Gd/3, the gene that was found to be consistently downregulated in PPARg MacKO ΜΦ subsets, was also regulated by RSG treatment in Ly6C- ΜΦΒ. HOW can one reconcile the facts that only a few genes were differently expressed in Ly6C- cells, yet from a biased point of view based on literature data, this gene list seemed far the most likely reporter of the transcriptional response of synthetic ligand activation of PPARy in infiltrating ΜΦ subsets? To resolve this apparent contradiction, we took the list of 43 genes that showed ligand dependence in Ly6C- ΜΦΒ upon RSG treatment and then we created a heat map representation to see how these genes were regulated in the absence of RSG treatment (Fig 4D and 11). Even without RSG treatment, most of the otherwise RSG dependent genes showed a characteristic induction as Ly6C+ ΜΦΒ differentiated into Ly6C- cells. This observation raised the intriguing possibility that the underlying reason behind the limited number of PPARy ligand regulated genes in Ly6C- M<t s was that most of these genes were already induced during muscle regeneration, even in the absence of exogenous synthetic ligand treatment.

GDF3 is a ΜΦ derived PPARy dependent member of the TGFfi family

To focus on putative PPARy regulated genes whose activity could promote muscle regeneration, we pursued a biased screening approach where we interrogated the list of differently expressed genes based on a set of criteria designed to maximize our chance to find such factors. We were looking for genes that were (1) PPARy dependent in more than one ΜΦ subset. (2) coded a secreted factor and (3) whose activity might be linked to muscle differentiation based on existing data. Strikingly and surprisingly, one gene. Gtf/3 (Levine and Brivanlou. 2006: Levine et al. 2009: Shen et al. 2009). fit all these criteria. Gtf/3 was statistically significantly downregulated in PPARg MacKO ΜΦΒ in all four investigated ΜΦ subsets. In fact, Gd/3 was the top ranked gene (ranked by fold change difference) in 3 out of 4 ΜΦ subsets (Fig 4B). Second, GDF3 belongs to the TGFft family, whose members are secreted factors acting in a paracrine manner. Finally, several members of the TGFft family are known regulators of muscle regeneration, including GDF8 (also known as Myostatin) (Massague et al., 1986; McPherron et al., 1997). Therefore, we selected Gd/3 as the most likely PPARy dependent gene that contributes to muscle regeneration for further analysis.

PPARy occupies a complex set of active enhancers around the Gd/3 locus

Next, we wanted to characterize the genomic events that are responsible for the regulation of Gd/3 by PPARy. We elected to use BMDMs, a readily available in vitro model system that allowed us to employ high-throughput genomic and epigenetic methods to interrogate the regulatory mechanism exerted by PPARy on the Gd/3 locus. We established that WT and PPARy LysMCre BMDMs provided a platform with good correlation to study the PPARy dependent regulation of Gd/3, as PPARy deficiency in BMDMs abrogated the expression of both the canonical PPARy target gene Angptl4 and that of Gd/3 (Fig 5A). Then, we compiled an extensive range of epigenetic and genomic data to identify the relevant transcriptional regulatory unit and enhancers that were active and possibly under PPARy regulation in BMDMs (Fig 5B). We included CTCF as a binding factor of insulator regions and RAD21, as a component of the cohesin complex to determine the genomic regulatory region and boundaries of potential chromatin loops/topological domains, PU.l as a key lineage determining and proposed pioneering factor in ΜΦΒ, RXR (the obligate heterodimeric partner of PPARy), and PPARy ChlP-seq data derived from thioglycolate elicited peritoneal ΜΦΒ and adipocytes. We combined these data with active epigenetic marks from H3K4me3 ChlP-seq experiments from BMDMs. In addition, we also merged these data from our recent GRO-seq data from BMDM showing sites of nascent RNA production. Based on the common CTCF/RAD21 binding sites, which were defined by us and others (Daniel et al., 2014; Merkenschlager and Odom, 2013) as boundaries of topological and sub-domains, the transcription unit of Gd/3 appeared to be approximately between approximately -50 kb to +50 kb. Therefore, we focused our efforts to identify putative enhancers within these boundaries. Our definition of putative, active enhancers included the following genomic/epigenomic features: (1) binding of PU.l, (2) presence of detectable enhancer transcript (GRO-seq signal) (3) RXR binding in M<t s or (4) PPARy binding in any of the listed cell types due to the relatively low IP efficiency of PPARy in the thioglycolate ΜΦ PPARy ChlP- seq. The appropriateness of this approach was validated by applying the same set of criteria to the AngptM locus, in which we easily identified its PPARy dependent enhancer (Fig 13A). Based on these criteria we nominated 14 putative active enhancers at a distance from +38 Kb to -47 Kb relative to the transcription start site of Gd/3 (Fig 5B and 13B). To see which enhancers show PPARy binding in BMDMs, these putative cistromic regions were tested in independent ChlP-PCR experiments on BMDMs from four mice. As we show in Fig 5C, binding of PPARy and RXR could be readily detected on 5 of these selected enhancers (at +7.3 kb, -21kb, -25kb, -44kb and -47kb) if we compared WT to PPARy KO M<t s. These data strongly suggested that Gdf3 was regulated by one or several of these PPARy:RXR binding sites. Notably, the binding sites at +7.3kb and -21kb contain bona fide DR-1 binding sites determined by the PPARy and RXR motif matrices of the HOMER database. The complexity of this locus and the number of binding sites detected call for more detailed analyses to uncover the contribution of the individual binding sites to the regulation.

GDF3 is a regulator of myoblast proliferation, differentiation and muscle regeneration

Having characterized the regulation of Gd/3 at the genomic level, we next wanted to establish the functional relevance of the induction and regulation of ΜΦ derived GDF3 during muscle regeneration. According to our model, the regeneration delay in ΜΦ PPARy deficient animals was, at least partly, attributable to a diminished ΜΦ derived GDF3 secretion within regenerating muscles. This model posits that GDF3 deficiency in ΜΦΒ should yield impairment in regeneration comparable to PPARg MacKOs. It was reported that the full body deletion of GDF3 showed incomplete penetrance as it resulted in early embryonic lethality in approximately 35% of Gfd3 l- embryos (Shen et al., 2009). On one hand, this observation highlights the vital importance of GDF3 in development, but also implies that possible compensatory mechanisms might exist and rescue the phenotypes caused by GDF3 deficiency potentially beyond embryonic development. To limit the involvement of such compensatory mechanisms and to ascertain the cellular source of GDF3 during muscle regeneration we decided to generate BMT animals reconstituted with Gfd3-/^~ bone marrow. When the GDF3 chimeric animals were challenged with CTX induced muscle injury, they exhibited a delay in regeneration (Fig 6A-C). When compared to WT BMT animals. GDF3 chimeras contained more regenerating myofibers with smaller CSA and the regenerating muscle was replete with lipid accumulations, which are hallmarks of defective muscle regeneration. These pathological features underlined the importance of ΜΦ derived GDF3 in skeletal muscle regeneration and validated our model regarding the existence of a PPARy-GDF3 axis responsible for the regulation of tissue regeneration.

Next, we analyzed the GDF3 protein expression in whole muscle lysates of WT and PPARg MacKO mice. The expression showed a pronounced induction and consistent PPARy dependency during regeneration. Protein expression of GDF3 closely followed the induction seen at the mRNA level in ΜΦΒ and was especially high at day 4 fFig. 6D). at the time when inflammation subsides and regenerative processes start to dominate within the injured muscle. Importantly, the induction of GDF3 expression was diminished at both mRNA and protein levels in PPARg MacKO animals (Fig. 6E-F. 14A-C), The experiment with BMT animals reported that GDF3 deficiency in the hematopoietic compartment was sufficient to cause impairment in tissue repair. However, we wanted to further test the hypothesis that infiltrating M<t s are the exclusive source of GDF3. First, we separated WT CD45+ and CD45- cells at various time points following muscle injuries and detected Gd/3 mRNA by RT-qPCR. CD45+ cells, which comprise all infiltrating hematopoietic cells within the injured tissue, expressed Gd/3 at much higher levels than CD45- (the non-hematopoietic cells in injured muscle) cells (Fig 6F). Accordingly, while the CD45+ compartments isolated from injured muscles showed robust GDF3 protein levels (Fig 6G), the expression was much lower in CD45- cells. To summarize. GDF3 is a ΜΦ derived protein whose expression is induced during muscle regeneration in a PPARy dependent manner.

All above data pointed to a hypothesis in which ΜΦ PPARy deficiency leads to delayed muscle regeneration due to insufficient paracrine GDF3 production during muscle regeneration. This hypothesis can explain the observed deficiencies in regeneration only if GDF3 possesses an effector function during regeneration. To search for a mechanism by which a paracrine effect of ΜΦ derived GDF3 could influence muscle regeneration, we took advantage of the in vitro primary muscle progenitor assays introduced in Fig. 3 C and D. We cultured primary myoblasts with or without recombinant (rec) GDF3 and measured myoblast proliferation by IF. We found that addition of GDF3 to the culture medium slightly but significantly decreased myoblast proliferation in all 4 independently established primary myoblast lines (Fig 7 A. left panel). We detected an even more robust effect of GDF3 on myotube fusion, as myoblast cultures showed a pronounced increase in their fusion index in the presence of GDF3 (Fig 7 A. right panel and 7B). These results suggested that GDF3. once released from ΜΦ5 within the injured /regenerating tissues, could regulate molecular pathways relevant to muscle differentiation in primary muscle cells and thereby could skew the balance between myoblast proliferation and differentiation. In search for the molecular changes triggered in muscle progenitors in the presence of GDF3, we differentiated in vitro primary myoblasts with or without GDF3 and interrogated the gene expression changes by RNA-Seq. First, we compared the profile of primary myoblasts to differentiating myotubes that were cultured in the presence or absence of GDF3. A preselected list of genes relevant to muscle differentiation (Fig 7C) showed that the gene expression changes upon commitment to differentiation was in line with muscle differentiation and thus validated our experimental system. Besides the anticipated regulation of transcription factors, structural muscle proteins and cell surface molecules, a surprisingly dynamic regulation of a selection of secreted factors (TGFp family and components of IGF1 signaling system) were detected that are relevant to muscle regeneration but whose origin in injured muscles is more often ascribed to infiltrating immune cells. Importantly, Gd/3 expression was undetectable in the muscle cells by RNA-Seq, which set GDF3 apart from the other investigated immune cell derived secreted factors in this analysis. Next, we compared the expression profile of differentiating myotubes cultured with or without GDF3. The list of the differently regulated genes (Fig 7D and Table S2) suggests that a limited set of transcripts are either induced or repressed in the presence of GDF3. While the fold changes of the induction/repression were modest, several of the differentially regulated genes have been implicated in muscle regeneration and/or muscle structure, raising the possibility that ΜΦ derived GDF3 could elicit biologically relevant changes during muscle regeneration. BEX1 and KLF15 are known regulators of muscle regeneration or differentiation, respectively. Besides these factors, genes for several proteins responsible for Ca²⁺ homeostasis [Camklg, Capns2, Cacngl, Casg2) or muscle structure/neuromuscular junctions [Sgca, Rampl, Ramp3, Tctex, Afapl, Tub2b) were differently regulated. Notably, deficiencies in CAPNS2 and SGCA, both differently regulated by GDF3, cause myopathies in human patients. If GDF3, a ΜΦ derived secreted factor can regulate in vitro and in situ muscle differentiation/regeneration, then we wanted to ask if GDF3 is the only TGFft family member that is relevant in the context of CTX induced muscle injury. Therefore, we reanalyzed the transcriptomic features of muscle infiltrative ΜΦΒ to chart the expression and dynamics of the TGFft family signaling system (Fig 7E) to identify additional factors that might regulate regeneration in a paracrine or autocrine manner. Three ligands [βάβ, Gdfl5 and Inhbd) showed notable gene expression dynamics in muscle infiltrative M<t s. GDF3 expression peaked in repair M<t s and showed definitive, consistent regulation by PPARy. The two other family members, Gdfl5 and Inhba, were also regulated during muscle regeneration. Importantly, both genes exhibited partial PPARy dependency, but while Gdfl5 showed a similar expression pattern to that of Gd/3, expression of Inhba (activin βΑ) showed a temporary boost in its expression at the earliest stage of injury. Importantly, the activin system, including activin βΑ, has also been identified as a regulator of muscle growth or regeneration (Lach-Trifilieff et al., 2014; Yaden et al., 2014). Three non-ligand members of the TGFft family signaling system, Tgfbr2, Eng and Bambi were also regulated in muscle M<t s. Twelve other members of the signaling system were also expressed but not regulated in these ΜΦΒ, while the remaining members (including myostatin) were not expressed at all (Fig 14D). The PPARy-GDF3 regulatory axis described in this study therefore identifies a sensory-regulatory-effector mechanism, by which ΜΦΒ are regulators of the tissue progenitor compartment, namely MPCs. This axis orchestrates tissue regeneration, possibly in unison with other members of the TGF family, leading to synchronous regeneration. MATERIALS AND METHODS

Below at first a brief description of materials and method are provided. For more detailed descriptions of experimental procedures, see SUPPLEMENTAL MATERIALS AND METHODS.

Mice. Ppargfl/flLysMCre+ (refered to as PPARg MacKO) and wild type C57BL/6J controls, Ppargfl/-Sox2Cre+ and littermate control Ppargfl/+LysMCre- animals, and Gd/3 KO and littermate C57BL/6 albino controls were used in the experiments. All experimental procedure conducted on animals were carried out in accordance with institutional regulations.

Muscle injury. Mice were anaesthetized with isoflurane and 50 μΐ of cardiotoxin (12X10-6 mol/1 in PBS) was injected in the tibialis anterior (TA) muscle. Muscles were recovered for flow cytometry analysis at day 1, 2 or 4 post-injury or for muscle histology at day 8 post-injury.

Histological analysis of muscle regeneration. Muscles were removed and snap frozen in nitrogen-chilled isopentane (-160°C). 8 μηι thick cryosections were cut and stained with hematoxylin-eosin (HE). HE stained sections were analyzed for cross sectional area (CSA) or for the presence of phagocytic fibers. Day 8 post CTX slides were also IF stained for Desmin / F4/80 / DAPI.

Macrophage cell culture for conditioned medium generation. Macrophages were obtained from bone marrow (BM) precursor cells that were were cultured in DMEM medium containing 20% FBS and 30% conditioned medium of L929 cell line 37 (enriched in CSF-1) for 7 days. Macrophages were activated with IFNy (50 ng/ml) or IL4 (10 ng/ml) to obtain macrophage-conditioned medium.

Myogenic precursor cell (MPC) culture. Murine MPCs were obtained from TA muscle and cultured using standard conditions in DMEM/ F12 (Gibco Life Technologies) containing 20% FBS and 2% G/Ultroser (Pall Inc). For proliferation studies, MPCs were incubated for 1 day with conditioned medium + 2.5% FBS or with 2.5% FBS medium containing GDF3 mouse recombinant protein. Cells were then incubated with anti-ki67 antibodies (15580 Abeam), which were subsequently visualized using cy3-conjugated secondary antibodies (Jackson Immunoresearch Inc). For differentiation studies, MPCs were incubated for 3 days with conditioned medium containing 2% horse serum or with 2% horse serum medium containing GDF3. Cells were then incubated with anti-desmin antibodies (32362 Abeam), in combination with a cy3-conjugated secondary antibody (Jackson Immunoresearch Inc).

Phagocytosis assay: BMDM cells and C2C12 cells were stained with CellVue or PKH67 (Sigma), respectively. Heat killed stained C2C12 were used as phagocytic substrates for stained BMDMs and fluorescent intensity was measured with a FACScalibur instrument.

Image capture and analysis for myoblast cultures. Fusion index (for myogenic cells) was calculated as the number of nuclei within myotubes divided by the total number of nuclei, nuclei number being estimated using the Image J software.

Isolation of macrophages from muscle. CD45+ cells were isolated from CTX injected muscles using magnetic sorting (Miltenyi Biotec). CD45+ cells then were labeled with fluorescently labeled antibodies and Ly6C+ F4/801ow macrophages, Ly6C- F4/80+ macrophages and Ly6Cmid F4/80- neutrophils were analyzed and sorted with a BD FACSAria III sorter.

RNA isolation from sorted MFs. MF subsets were sorted from day 1, 2 and 4 post-injury muscles with a FACSAria III sorter and total RNA was isolated with TRIZOL reagent according to the manufacturer's recommendation.

Microarray analysis of muscle macrophages: Global expression pattern was analyzed on Affymetrix GeneChip Mouse Gene 1.0 ST arrays. The microarray data are publicly available (Data access: GSE71155).

ChIP (Chromatin immunoprecipitation): ChIP was carried out in BMDMs using antibodies against pre-immune IgG (Millipore, 12-370), (pan) RXR (sc-774 Santa Cruz Biotechnology) and PPARy (Perseus #PP-A3409A).

Bioinformatic analysis of the active enhancers around the Gdf3 and AngptI4 locus: The list of published and/or publicly available datasets used for visualization in IGV2 to identify active enhancers can be found in the SUPPLEMENTAL MATERIALS AND METHODS section.

Western Blotting: GDF3 protein expression was measured using Western Blot analysis. Samples from CTX injected TA muscles or CD45+ cells were lysed in RIPA buffer. GDF3 was targeted using rabbit monoclonal Anti-GDF3 primary antibody (abl09617, Abeam, Cambridge, MA) at 1:1,000 dilution in 5% BSA/TBS-T overnight at 4°C. Anti-GAPDH mouse monoclonal primary antibody (AM4300, Ambion, Carlsbad, CA) was used as a protein loading control at 1:10,000 - 1:20,000 dilution in 5% BSA/TBST overnight at 4°C.

RNA sequencing (RNA-Seq) library preparation for myoblast gene expression analysis: cDNA library for RNA-Seq was generated from l\\.g total RNA using TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer's protocol. The RNA-Seq data are publicly accessible (data access: PRJNA290560/SRR2136645).

General statistical analyses. All experiments were performed using at least three different samples. Student's t-tests and 2 way ANOVA analyses were performed and P<0.05 was considered significant (P<0.05=* P<0.01=**, P<0.001=***). Mean and SD values, or mean and SEM values are shown in graphs.

SUPPLEMENTAL MATERIALS AND METHODS

Mice. Genetically modified (floxed) PPARg conditional KO mice and wild type C57BL/6J controls were bred under SPF conditions and used for experiments in accordance with Hungarian (license no.: 21/2011/DE MAE) and European regulations. Experiments were conducted on adult (2-4 month old) male mice. Breeding of genetically modified Gd/3 KO and their control C57BL/6 albino animals, and the experiments with them were accepted and conducted with the permission of Sanford Burnham Prebys Medical Discovery Institute at Lake Nona IACUC approval (protocol No. 2014-0107). Ppar#fl/flLysMCre+ (refered to as PPARg MacKO) (maintained on C57BL/6J background) and wild type C57BL/6J mice were used in most experiments. They were generated in Ppargfl/flLysMCre+ X Ppargfl/flLysMCre+ and WT X WT crossings. In a separate experiment, a small cohort of Ppargfl/flLysMCre+ and littermate control Pparg+/+LysMCre+ animals were generated from Ppargfi/ +LysMCre+ X Ppargfi/ +LysMCre+ crossings. The animals from this latter cohort were CTX injected and HE stained slides generated 8 days post CTX injections were visually evaluated in a double blind fashion. This experiment detected a delay in PPARg MacKO animals (vs. controls) that was indistinguishable from the delay seen in the PPARg MacKO samples generated in the non-littermate crossings. Ppargfl/-Sox2Cre+ and littermate control Ppargfl/+LysMCre- animals were generated in (male) Pparg+/-Sox2Cre+ X (female) Ppargfl/flSox2Cre- crossings. Gd/3 KO and littermate C57BL/6 albino controls were generated in Gd/3+/- X Gd/3+/- crossings.

Bone marrow transplantation (BMT) : The C57BL/6 congenic BoyJ strain, carrying the CD45.1 cell surface marker, was used as recipients for BMT studies. Recipients were 7-10 weeks old at the time of irradiation and BMT. Recipients were irradiated with a dose of 1 X 9.5 Gy and 3 h later transplanted with 5xl0⁶ bone marrow cells/200 μΐ RPMI/mouse by retro-orbital injection under anasthesia. This protocol gave a chimerism of >98% when Ppargfl/fl LysMCre+ or controls were used as donors and >98% when Ppargfl/- Sox2Cre+ were the donors. Transplanted animals were used for experiments 3 months after receiving BMT.

Muscle injury. Mice were anaesthetized with isoflurane and 50 μΐ of cardiotoxin (12X10 ⁶ mol/1 in PBS) (from Latoxan) was injected in the tibialis anterior (TA) muscle. Muscles were recovered for flow cytometry analysis at day 1, 2 or 4 post-injury or for muscle histology at day 8 post-injury.

Histological analysis of muscle regeneration. Muscles were removed and snap frozen in nitrogen-chilled isopentane (-160°C). 8 μηι thick cryosections were cut and stained with hematoxylin-eosin (HE).

Picture capture and counting. For each histological analysis, at least 5 slides (per condition) were selected where the total regernerative region within the CTX injured TA muscle was at least 70%. For each TA, myofibers in at least 3 fields randomly chosen in the entire injured area were counted and measured. At least 5 muscles were analyzed for each condition. HE muscle sections for the day 0, day 8 and day 21 PPARg MacKO vs. WT comparisons were recorded with a Nikon E800 microscope at 20X magnification connected to a QIMAGING camera. Cross-sectional area (CSA) measurement of these samples was carried out using Metamorph software and the CSAs are reported in arbituary units. HE muscle sections for the day 16 GDF3 KO BMT vs. WT BMT and for the day 22 PPARg KO BMT vs. WT BMT were scanned with Mirax digital slide scanner and the CSA was measured with Panoramic Viewer software. The CSAs for these latter samples are reported in μηι. Quantitative analysis of necrotic/phagocytic vs. centrally nucleated myofibers was performed using the Image J software and was expressed as a percentage of the total number of myofibers. Necrotic myofibers were defined as pink pale patchy fibers and phagocyted myofibers were defined as pink pale fibers, which are invaded by basophil single cells (macrophages).

Immunofluorescent detection of muscle regeneration in day 8 CTX injected muscle: Tissue sections were fixed and permeabilized in ice cold acetone for 5 min and blocked for 30 minutes at 20 °C (room temperature) in PBS containing 2 % bovine serum albumin (BSA). Tissues were stained for 1 h at room temperature using a primary antibody diluted in 2 % BSA. The primary antibodies used for immunofluorescence are listed in Supplementary Table 1. In all cases, the primary antibody was detected using secondary antibodies conjugated to FITC (JIR 712-095-153) or Cy3 JIR (711-165-152). The nuclei were counter stained with 0.1-1 μg/ml Hoechst. Fluorescent microscopy was performed using Carl Zeiss Axio Imager Z2 microscope equipped with lasers at 488, 568 and 633 nm. Figures were analyzed and assembled using Fiji and Illustrator CS5 (Adobe). List of primary antibodies used in immunofluorescence:

Macrophage cell culture for conditioned medium generation. Macrophages were obtained from bone marrow (BM) precursor cells. Briefly, total BM was obtained from mice by flushing femurs and tibiae bone marrow with DMEM. Cells were cultured in DMEM medium containing 20% FBS and 30% conditioned medium of L929 cell line (enriched in CSF-1) for 7 days. Macrophages were seeded (at 50000 cell/cm2 for all experiments) and were activated with IFNy (50 ng/ml) and IL4 (10 ng/ml) to obtain Ml and M2 macrophages, respectively, in DMEM containing 10% FBS medium for 3 days. After washing steps, DMEM serum-free medium was added for 24 h, recovered and centrifugated to obtain macrophage-conditioned medium.

Myogenic precursor cell (MPC) culture. Murine MPCs were obtained from TA muscle and cultured using standard conditions in DMEM/ F12 (Gibco Life Technologies) containing 20% FBS and 2% G/Ultroser (Pall Inc). Briefly, TA muscles of young mice were opened and cleared of nerves/blood vessels/fascia etc. Muscle preparations were lightly digested with collagenase and the resulting cells were plated then serially expanded. For proliferation studies, MPCs were seeded at 10000 cell/cm2 on matrigel (1/10) and incubated for 1 day with macrophage-conditioned medium + 2.5% FBS or with 2.5% FBS medium containing mature GDF3 mouse recombinant protein (300 ng/ml; E. coli-derived, Ala253-Gly366 R&D 958-G3-010). Cells were then incubated with anti-ki67 antibodies (15580 Abeam), which were subsequently visualized using cy3-conjugated secondary antibodies (Jackson Immunoresearch Inc). For differentiation studies, MPCs were seeded at 30000 cell/cm2 on matrigel (1/10) and incubated for 3 days with macrophage-conditioned medium containing 2% horse serum or with 2% horse serum medium containing GDF3 mouse recombinant protein (300 ng/ml; R&D). Cells were then incubated with anti-desmin antibodies (32362 Abeam), in combination with a cy3-conjugated secondary antibody (Jackson Immunoresearch Inc).

Phagocytosis assay: BMDM cells were generated as described earlier in this section. BMDMs were harvested with trypsin and careful scraping, washed twice in PBS and then stained with the lipophilic fluorescent dye CellVue (Sigma) according to the manufacturer's recommendation. Stained BMDMs were replated and left to recuparate for one day in DMEM medium. C2C12 cells were cultured in DMEM containing 10% FBS. Cells were harvested, washed and stained with the lipophilic fluorescent dye PKH67 (Sigma). Stained C2C12 cells were washed extensively and then heat killed at 55°C for 60 min. Heat killed C2C12 cells were added to BMDM cultures at 2:1 ratio and phagocytosis was commence at 37°C or 4°C (controls). Cells were harvested by scraping after 1 h and fluorescent intensity was detected with a FACScalibur instrument.

Image capture and analysis for myoblast cultures. Fusion index (for myogenic cells) was calculated as the number of nuclei within myotubes divided by the total number of nuclei, nuclei number being estimated using the Image J software.

Isolation of macrophages from muscle. Fascia of the TA was removed. Muscles were dissociated in RPMI containing 0.2% collagenase B (Roche Diagnostics GmbH) at 37°C for 1 hour and filtered through a 100 μηι and a 40 μηι filter. CD45+ cells were isolated using magnetic sorting (Miltenyi Biotec). For cell sorting, macrophages were treated with Fey receptor blocking antibodies and with 10% normal rat serum: normal mouse serum 1:1 mix, then stained with a combination of PE-conjugated anti-Ly6C antibody (HK1.4, eBioscience) and APC-conjugated F4/80 antibody (BM8, eBioscience). Ly6C+ F4/801ow macrophages, Ly6C- F4/80+ macrophages and Ly6Cmid F4/80- neutrophils were sorted. In each experiment, both genotypes were paralelly processed to minimize experimental variation. Cells were analyzed and/or sorted with a BD FACSAria III sorter.

RNA isolation from sorted MFs. MF subsets were sorted from day 1, 2 and 4 post-injury muscles with a FACSAria III sorter and total RNA was isolated with TRIZOL reagent according to the manufacturer's recommendation. 20 ug glycogen (Ambion) was added as carrier for RNA precipitation.

Microarray analysis of muscle macrophages: Global expression pattern was analyzed on Affymetrix GeneChip Mouse Gene 1.0 ST arrays. Ambion WT Expression Kit (Life Technologies, Hungary) and GeneChip WT Terminal Labeling and Control Kit (Affymetrix) were used for amplifying and labeling 150 ng of total RNA. Samples (n=3, 4 or 5) were hybridized at 45 ²C for 16 h and then standard washing protocol was performed using Affymetrix GeneChip Fluidics Station 450. The arrays were scanned on GeneChip Scanner 7G (Affymetrix). Microarray data (data acess: GSE44057) were analyzed with GeneSpring 12 GX software (Agilent BioTechnologies). Affymetrix CEL files were normalized with Robust Multichip Analysis (RMA) algorithm and median normalization. The microarray data are publicly available (Data access: GSE71155).

Expression data processing and analysis: Data quality control and analysis was carried out following the recommendations put forward in the Imgen website

(http://www.immgen.org/Protocols/ImmGen%20QC%20Documentation_ALLDataGeneration_ 0612.pdf). Data were loaded into the Genespring GX software and RMA summarization was carried out. Next, a set of filtering steps was applied to the dataset. Briefly, data distribution curve was generated and the lowest 5% of the entities with detectable signals were filtered out as not expressed. Duplicate entities, not/poorly annotated transcripts and transcripts reporting inconsistent expression values were also discarded. Further analysis was carried out on the filtered dataset. Data was analyzed either based on the RAW expression values or after following a "per gene" normalization (individual gene expression data normalized to the median of the gene). Further analysis of gene expression and comparisons were made either within Genespring GX or using the R software package. 2-way anova tests were performed in R using functions aov and TukeyHSD of package MASS, Heatmaps were drawn with package pheatmap. Statistically significant difference was considered if p < 0.05.

Mmicroarray validation by RT-qPCR: Transcript quantification was performed by quantitative real-time RT (reverse transcriptase) PCR (polymerase chain reaction) using SYBR Green assays (Apoldl, Hebpl and Plxndl) or Prime Time assays from IDT [βάβ and Pparg). Primer sequences and Taqman probes or PrimeTime assay IDs used in transcript quantification are available upon request. RT-qPCR results were analyzed with the standard delta Ct method and results were normalized to the expression of ActB.

Macrophage cell culture for ChIP: Macrophages were obtained from bone marrow (BM) precursor cells. Briefly, total BM was obtained from mice by flushing femurs and tibiae bone marrow with DMEM. Cells were RBC lysed with ACK solution and then plated on non-tissue culture grade plates then cultured in DMEM medium containing 20% FBS and 30% conditioned medium of L929 cell line (enriched in CSF-1) for 6 days. Macrophages were harvested from the culture plates and ChIP was carried out.

ChIP (Chromatin immunoprecipitation): Cells were double crosslinked with 0,002 M DSG (Sigma) for 30 minutes and then with 1% formaldehyde (Sigma) for 10 minutes. Nuclei were isolated with ChIP Lysis Buffer (1% Triton x-100, 0.1% SDS, 150 mM NaCl, ImM EDTA, and 20 mM Tris, pH 8.0) then chromatin were sonicated (also in ChIP Lysis Buffer) with Diagenode Bioruptor to generate 200-1000 bp fragments. Chromatin was diluted in ChIP Lysis buffer and immunoprecipitated with antibodies against pre-immune IgG (Millipore, 12-370), (pan) RXR (sc- 774 Santa Cruz Biotechnology) and PPAR gamma (Perseus #PP-A3409A). Chromatin antibody complexes were precipitated with Protein A coated paramagnetic beads (Life Technologies). Chromatin antibody complexes were washed on the beads once in IP Wash Buffer 1 (1% Triton, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, and 0.1% NaDOC), twice in IP Wash Buffer 2 (1% Triton, 0.1% SDS, 500 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, and 0.1% NaDOC) and once in IP Wash Buffer 3 (0.25 M LiCl, 0.5% NP-40, ImM EDTA, 20 mM Tris, pH 8.0, 0.5% NaDOC) and IP Wash Buffer 4 (10 mM EDTA and 200 mM Tris, pH8.0). DNA fragments were then eluted and column purified (Qiagen, MinElute). DNA was applied for QPCR analysis. QPCR results were analyzed with the standard delta Ct method and results were normalized to input signals.

Bioinformatic analysis of the active enhancers around the Gdf3 and AngptI4 locus: Primary analysis of the raw sequence reads has been carried out using our ChlPseq analysis command line pipeline. Alignment to the mm9 assembly of the mouse genome was done by the Burrows-Wheeler Alignment (BWA) tool. Genome coverage (bedgraph) files were generated by makeTagDirectory and makeUCSCfile.pl (HOMER) and were used for visualization with IGV2. Putative DR1 elements (reaching score 9) were determined by annotatePeaks.pl (HOMER) using the RXR and PPARg motif matrices of HOMER. The following datasets were used for the identification of active enhancers: Sample name SRA GEO Cell/tissue type Sample

identifier identifier type

Antibody

BMDM_PU.l SRX651749 - bone marrow derived ChlP-seq PU.l macrophage

BMDM_RXPv SRX651739 - bone marrow derived ChlP-seq RXR macrophage

mac_PPARg SRX019134 GSM532739 peritoneal macrophage ChlP-seq PPARg eWAT_PPARg SRX193440 GSM1018066 epididymal white ChlP-seq PPARg adipose tissue

iWAT_PPARg SRX193441 GSM1018067 inguinal white adipose ChlP-seq PPARg tissue

BAT_PPARg SRX193442 GSM1018068 brown adipose tissue ChlP-seq PPARg

BMDM_CTCF SRX651751 - bone marrow derived ChlP-seq CTCF macrophage

BMDM_RAD21 bone marrow derived ChlP-seq RAD2 macrophage 1

BMDM_H3K4me3 SRX651747 - bone marrow derived ChlP-seq H3K4 macrophage me3

BMDM_GRO-seq SRX651735 - bone marrow derived GRO-seq - macrophage

Western Blotting: GDF3 protein expression was measured using Western Blot analysis. The Tibialis anterior (TA) was removed from mice injected intramuscularly with cardiotoxin (CTX) at experimental time points and homogenized in RIPA buffer. CD45+/- cell populations were isolated from whole TA muscle using MACS Micro Magnetic Bead Separation system (Bergisch Gladbach, Germany). Cell populations were collected and lysed in RIPA buffer. Protein concentrations were determined by Qubit 2.0 Fluorometer Protein Assay (Life Technologies, Carlsbad, CA). Protein samples were prepared for SDS-PAGE with 2X Laemmli Sample Buffer (Bio-Rad, Hercules, CA) at a 1 mg/ml concentration. SDS-PAGE was completed using 4-20% Mini Protean TGX gels (Bio-Rad, Hercules, CA) at 110 volts for 1 hour. The SDS-PAGE gel was then transferred onto PVDF membrane (Thermo Fisher, Waltham, MA) at 0.35 amps for 1-2 hours at 4°C. Membranes were blocked in 5% BSA in TBS-T at room temperature for >1 hour. GDF3 was targeted using rabbit monoclonal Anti- GDF3 primary antibody (abl09617, Abeam, Cambridge, MA) at 1:1,000 dilution in 5% BSA/TBS-T overnight at 4°C. Anti-GAPDH mouse monoclonal primary antibody (AM4300, Ambion, Carlsbad, CA) was used as a protein loading control at 1:10,000 - 1:20,000 dilution in 5% BSA/TBST overnight at 4°C. Membranes were washed 3X with TBS-T for 5 minutes each for a total of 15 minutes. Goat Anti-Rabbit HRP secondary antibody was used for the detection of GDF3 at 1:10,000 dilution in 5%BSA/TBS-T at room temperature for 1 hour. Anti-Mouse HRP secondary (Cell Signaling, 7076S) and Donkey Anti-Mouse Alexa Fluor 680 secondary (abl75774) antibodies were used for the detection of GAPDH at 1:40,000 dilution at room temperature for 1 hour. Membranes were washed 3X with TBS-T for 5 minutes each for a total of 15 minutes, followed by 2 washes in TBS for 5 minutes. Super Signal West Pico Kit allowed for ECL visualization of the blot on Hyblot CL Film (Denville, E3018).

Primary myoblast differentiation for RNA-Seq: MPCs were seeded at 30000 cell/cm2 on matrigel (1/10) in full medium. Medium was replaced with differentiation medium containing 2% horse serum 6h later and cells were cultured overnight. Next morning +/- 150ng/ml recombinant mature mouse GDF3 (R&D, 958-G3-010) was added to the cultures and differentiating cells were harvested in 24h (referred to as "day 1 cells" in the manuscript).

RNA sequencing (RNA-Seq) library preparation for myoblast gene expression analysis: cDNA library for RNA-Seq was generated from ^g total RNA using TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer's protocol. Briefly, poly-A tailed RNAs were purified by oligodT conjugated magnetic beads and fragmented on 94 C degree for 8 minutes, then 1st strand cDNA was transcribed using random primers and Superscript II reverse transcriptase (Lifetechnologies, Carslbad, CA, USA). Following this step, second strand cDNA were synthesized and then double stranded cDNA molecules were end repaired resulting blunt ends. The 3' ends of the dscDNA molecules were adenylated then Illumina TruSeq index adapters were ligated. After adapter ligation step, enrichment PCR was performed to amplify the adapter-ligated cDNA fragments. Fragment size distribution and molarity of the libraries were checked on Agilent BioAnalyzer DNA1000 chip (Agilent Technologies, Santa Clara, CA, USA). ΙΟρΜ of denatured libraries were used for cluster generation on cBot instrument, then single read 50bp sequencing run was performed on Illumina HiScan SQ instrument (Illumina, San Diego, CA, USA). The RNA-Seq data are publicly accessible (data access: PRJNA290560/SRR2136645).

RNA-seq bioinformatics analysis: The Top Hat-Cufflinks toolkit was used for mapping spliced reads, making transcript assemblies, getting and sorting gene expression data. Genes with RPKM>=1 (at least in one sample) were considered to be expressed. 2- way ANOVA tests were performed in R using functions aov and TukeyHSD of package MASS, Heatmaps were drawn with package pheatmap.

General statistical analyses. All experiments were performed using at least three different samples. Student's t-tests and 2 way ANOVA analyses were performed and P<0.05 was considered significant (P<0.05=* P<0.01=**, P<0.001=***). Mean and SD values, or mean and SEM values are shown in graphs.

Gene expression analysis of muscle infiltrative macrophages isolated from regenerating muscles of WT and Ppargfl/fl LysMCre+ animals. Both RAW data and "per gene normalized (i.e. normalized to the median expression value of respective gene") data were obtained (data are not shown but were recorded in excel worksheets), after preliminary data processing of microarray data. Worksheet "Day 1 to 4 WT vs PPARg KO DATA" contains all data derived from macrophages isolated 1, 2 or 4 days post CTX treatment. Worksheet "Day 2 WT NT vs RSG DATA" contains all data derived from day 2 post CTX mice that were treated +/- RSG. "COMP" worksheets contain the list of genes that are differently expressed when 2 macrophage populations are compared (based on a 2 way ANOVA carried out on the whole data set). Genes with p < 0.05 are listed in the worksheets.

Gene expression analysis of differentiating primary myoblast by RNA-Seq. Expression data from undifferentiated primary myoblasts, myoblasts differentiated for 1 day and myoblasts differentiated for 1 day in the presence of GDF3 were compared (data are not shown but were recorded in excel worksheets). Worksheet "MyB_all_expressed_genes_anova" lists expression values, p values and log2 fold change values for all genes that are differently expressed between any two conditions by 2 way ANOVA (p < 0.05). The list of genes that are differently expressed between the two relevant conditions were recorded on worksheet.

SUMMARY AND INDUSTRIAL APPLICABILITY

Tissue regeneration requires inflammatory and reparatory activity of macrophages (M<t s). First, M<t s detect and eliminate the damaged tissue and subsequently promote regeneration. This dichotomy requires the switch of the effector functions of M<t s coordinated with other cell types inside the injured tissue. The gene regulatory events supporting the sensory and effector functions of M<t s involved in tissue repair are not well understood. Here we show that the lipid activated transcription factor, PPARy is required for proper skeletal muscle regeneration acting in repair M<t s. PPARy controls the expression of the TGF family member, GDF3, which in turn regulates the restoration of skeletal muscle integrity by promoting muscle progenitor cell differentiation. This work establishes PPARy as a required metabolic sensor and transcriptional regulator of repair M<t s. Moreover, this work also establishes GDF3 as a secreted extrinsic effector protein acting on myoblasts and serving as an exclusively macrophage-derived regeneration factor in tissue repair.

ΜΦ PPARy is required for skeletal muscle regeneration and for primary myotube formation in vitro. PPARy regulates the expression of GDF3 primarily in muscle infiltrating LyC6- repair M<t s. The GDF3 locus has multiple PPARy:RXR heterodimer bound active enhancers. GDF3 is needed for proper muscle regeneration and enhances differentiation of primary myoblasts

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Claims

1. A GDF3 compound for use in the treatment of a subject having a condition or disease associated with impaired muscle, said subject being in need of muscle differentiation.

2. A GDF3 compound for use in improving differentiation of muscle of a subject in need thereof by differentiation of newly formed muscle fibers.

3. The GDF3 compound for use according to claim 1 or 2 wherein the muscle differentiation comprises the differentiation of myoblasts to myotubes (myotube differentiation).

4. The GDF3 compound for use according to claim 3 wherein he impaired muscle of said subject to be treated undifferentiated myoblasts are present and in the impaired muscle there is a need of myotube formation.

5. The GDF3 compound for use according to any of the previous claims wherein the muscle is skeletal muscle, said muscle preferably comprising undifferentiated myoblasts.

6. The GDF3 compound for use according to any of the previous claims wherein the subject is a mammal, preferably a human.

7. The GDF3 compound for use according to any of the previous claims for use in the treatment of a condition/disease selected from the group consisting of

myopathies, sarcopenia, muscular atrophia, muscular dystrophy, injuries.

8. The GDF3 compound for use according to any of the previous claims wherein the GDF3 compound is selected from the group consisting of recombinant GDF3 compound, a wild type GDF3 compound, a mutant variant of a wild type GDF3, preferably a mutant variant of a wild type GDF3 the sequence of which is at least 70%, 80% or at least 90% identical with a wild type GDF3, preferably a mammalian, optionally a human GDF3.

9. A composition for use as defined in any of the previous claims, said composition comprising a GDF3 compound as an active ingredient.

10. A composition for use as defined in any of the previous claims, said composition comprising a GDF3 expression construct as an active ingredient, wherein preferably the GDF3 expression construct is useful for expressing GDF3 in mammalian immune cells.

11. A composition for use as defined in any of the previous claims, said composition comprising a manipulated macrophage overexpressing GDF3.

12. The composition for use according to any of claims 9 to 11, said composition being formulated for local administration to the impaired muscle.

13. A composition for use according to any of claims 8 to 12 wherein the GDF3 compound is coadministered with a further agent for use in muscle regeneration, preferably a further agent for use in muscle differentiation preferably differentiation of newly formed muscle fibers, in particular myotube differentiation.

14. A composition for use according to any of claims 8 to 13 wherein said composition is a pharmaceutical composition for systemic or topical administration, preferably a pharmaceutical composition formulated for administration selected from the group consisting of intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, parenteral and oral administration.

15. A composition for use according to any of claims 8 to 13 wherein said composition is composition formulated as a dietary supplement, nutraceutical, functional food, or as a food composition with a health claim.

16. Use of a GDF3 compound for differentiation of myoblasts to myotubes ex vivo.

17. Use of a GDF3 compound for differentiation of myoblasts to myotubes in a model animal.

18. Use of a GDF3 compound for increasing muscle mass by differentiation of myoblasts to myotubes in an animal, preferably in a mammal.

19. An animal model of a condition/disease associated with impaired muscle wherein the gene of GDF3 is knocked out or knocked down.

20. A transgenic animal having increased muscle mass associated with an increased number of muscle fibers said transgenic animal having stable recombinant expression of GDF3 protein, wherein preferably recombinant expression of GDF3 protein is provided by macrophages and/or muscle cells of said animal.