WO2016120647A2 - Régénération musculaire - Google Patents

Régénération musculaire Download PDF

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WO2016120647A2
WO2016120647A2 PCT/HU2015/050019 HU2015050019W WO2016120647A2 WO 2016120647 A2 WO2016120647 A2 WO 2016120647A2 HU 2015050019 W HU2015050019 W HU 2015050019W WO 2016120647 A2 WO2016120647 A2 WO 2016120647A2
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gdf3
muscle
ppary
mos
differentiation
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PCT/HU2015/050019
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WO2016120647A4 (fr
WO2016120647A3 (fr
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László NAGY
Tamás VARGA
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Debreceni Egyetem
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11013—Protein kinase C (2.7.11.13)

Definitions

  • the present invention relates to the field of muscle regeneration.
  • 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).
  • immune cells and in particular MOs have a dual role during damage and regeneration.
  • 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.
  • 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.
  • Third, many aspects of skeletal muscle development and regeneration are well understood and can be studied in vitro (Yin et al., 2013). Several attempts have been made to treat muscle related disorders.
  • growth and differentiation factor-8 also known as myostatin
  • 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.
  • WO2014000042A1 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- ⁇ 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.
  • active TGF- ⁇ family ligands chosen from activin B, activin A,
  • the invention relates to a GDF3 compound for use in the treatment of a patient having a condition/disease associated with impaired muscle (def) and said patient being in need of muscle differentiation (def).
  • the muscle differentiation comprises the differentiation of myoblasts (def) to myotubes (myotube differentiation (def)).
  • the muscle differentiation is myotube differentiation.
  • impaired muscle includes impaired muscle structure and/or impaired muscle function.
  • impaired muscle in particular, in the impaired muscle there is a need of myotube formation.
  • undifferentiated myoblasts are present (detection).
  • the invention relates to a GDF3 compound for use in improving regeneration or differentiation of muscle of a patient in need thereof.
  • the invention relates to a use of GDF3 for improving muscle regeneration in a condition/disease that is characterized by a failure in muscle regeneration.
  • the invention relates to GDF3 for use in improving regeneration in muscle.
  • the muscle is skeletal muscle.
  • the muscle comprises undifferentiated myoblasts.
  • the patient is a mammal, optionally a human.
  • said impaired muscle is due to or associated with a condition/disease 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,
  • muscle injury selected from an injury due to a toxin (def), impaired supply of the muscle, such as hypoxia or hypoglycaemia or impaired blood supply, 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), etc.
  • a toxin def
  • impaired supply of the muscle such as hypoxia or hypoglycaemia or impaired blood supply
  • 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
  • a GDF3 compound is a compound comprising or consisting essentially of GDF3.
  • the GDF3 compound is a recombinant GDF3 compound.
  • the GDF3 compound is a wild type GDF3 compound.
  • the recombinant or wild type GDF3 is a mammalian GDF3, optionally a human GDF3.
  • the origin of GDF3 is the same species as that of the patient.
  • the GDF3 compound is a mutant GDF3 compound (a mutant variant of a wild type GDF3).
  • 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).
  • 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.
  • 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.
  • the recombinant or mutant or fragment maintains the fold of a wild type GDF3 compound.
  • the invention relates to a pharmaceutical composition for any use as defined above said composition comprising a GDF3 compound as an active ingredient.
  • 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.
  • the invention relates to a pharmaceutical composition for any use as defined above said composition comprising a GDF3 expression construct as an active ingredient.
  • the GDF3 expression construct is useful for expressing GDF3 in mammalian immune cells.
  • the pharmaceutical composition comprises a manipulated macrophage overexpressing GDF3.
  • 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.
  • the GDF3 compound is administered systemically.
  • the GDF3 compound is administered locally to the impaired muscle.
  • a macrophage overexpressing a GDF3 compound is administered locally to the impaired muscle.
  • the present application also relates to methods for gene therapy.
  • GDF3 as a natural molecule, is a member of the bone morphogenetic protein (BMP) family and the TGF- beta superfamily.
  • BMP bone morphogenetic protein
  • Mammalian, including human variants of the gene product (full length GDF3) comprise a signal peptide 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.
  • the members of this family are regulators of cell growth and differentiation in both embryonic and adult tissues.
  • 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.
  • 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.
  • GDF3 or "GDF3 protein” refer herein to a protein encoded by the gdf3 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.
  • 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.
  • the length of the mature peptide is about 112, 113, 114, 115, 116, 117 or 118 amino acids.
  • 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.
  • 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.
  • variant GDF3 molecule 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.
  • 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.
  • Micromature 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.
  • 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.
  • isolated refers to a molecule that is substantially free of its natural environment.
  • an isolated protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it is derived.
  • 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.
  • treating 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).
  • 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.
  • muscle disorder refers herein in particular to disorders of muscle, in particular disorders associated with muscle differentiation.
  • 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 (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 (including but not limited to severe or benign X-linked muscular dystrophy, limb-girdle dystrophy, facio
  • P117750 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.
  • ALS amyotrophic lateral sclerosis
  • terapéuticaally 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.
  • subject used interchangeably herein, refer 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.
  • mammalian farm animals e.g., bovine, porcine, ovine
  • mammalian sport animals e.g., equine
  • mammalian pets e.g., canine and feline
  • avian species including, but not limited to, chickens and turkeys.
  • a "patient” is a subject who is a target of therapy.
  • 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 or more amino acids or nucleobases than the other sequence.
  • 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.
  • the sequence of interest 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.
  • Fig. Number and fate of infiltrating cells in injured muscle. For gating strategy, see Fig S3.
  • 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+ MOs and the (C) calculated neutrophil and ⁇ numbers (derived from the % of the total numbers) at day 1 in injured muscles.
  • D and E Percentage of Ly6C+ and Ly6C- MOs in injured muscles at day 2 and day 4.
  • FIG. 1 Impact of PPARy on ⁇ 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.
  • Fig 4. Transcriptional analysis of Ly6C+ and Ly6C- ⁇ populations derived from WT and PPARg MacKO animals. For schematics of comparisons, see Fig S4.
  • (A) Heatmap representation of genes that show differential (p 0.05, min. 1.5X FC) expression in the four sorted WT vs PPARg MacKO MOs 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.
  • 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 MOs in the four analyzed MO 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 MO subtypes. Different Hist2h3 isoforms are labeled as histone genes.
  • Gdf3 is a PPARy target gene in BMDMs
  • B Identification of possible enhancers around the Gd/3 locus. The enhancer identification strategy around AngptI4 is shown in Fig S6A.
  • 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 S7A and S7B. Specificity of the anti-GDF3 antibody is shown in Fig S7C.
  • FIG. 1 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.
  • 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- MOs isolated from CTX injured muscle.
  • FIGS. 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 Pparg n l + , Sox2Cre- (controls) and Pparg ⁇ l-, Sox2Cre+ (KO) animals isolated at day 8 post CTX.
  • Fig S5. mRNA expression of PPARy dependent genes in muscle derived sterile inflammatory MOs detected by RT-qPCR.
  • FIG S6 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 S7 Additional analysis of the expression of TGF 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.
  • CX cardiotoxin
  • PPARy Peroxisome Proliferator-Activated Receptor gamma
  • GDF3 a secreted factor
  • our data reveal a novel integrated pathway with sensory, gene regulatory and effector components in which PPARy in repair MOs responds to signals and supports the timely promotion of tissue repair during muscle regeneration.
  • our data also point to GDF3 as a repair ⁇ -derived novel pro-differentiation factor in muscle regeneration.
  • the GDF3 ["growth differentiation factor 3"; also known as Vg-related gene 2 (Vgr-2), KFS3, MC0P7 or MC0PCB6] gene provides instructions for making a protein that 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.
  • TGFp transforming growth factor beta
  • the GDF3 protein belongs to the bone morphogenetic protein family, which is involved in regulating the growth and maturation (differentiation) of bone and cartilage.
  • Cartilage is a tough but flexible tissue that makes up much of the skeleton during early development.
  • the proteins in this family are regulators of cell growth and differentiation in both embryonic and adult tissue.
  • GDF3 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]
  • GDF3 a BMP inhibitor, regulates cell fate in stem cells and early embryos. Development 133 (2): 209-16.
  • GDF3 gene mutations that cause Klippel-Feil syndrome lead to a change in single protein building blocks (amino acids) in the GDF3 protein. These mutations likely lead to a reduction in functional protein. While the GDF3 protein is involved in bone growth, it is unclear how a shortage in these proteins leads to incomplete separation of the vertebrae, specifically the cervical vertebrae, in people with Klippel-Feil syndrome.
  • 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.
  • Rhinopithecus roxellana Rhinopithecus roxellana
  • the sequence of human GDF3 is as follows (SEQ ID NO: 1):
  • Tissue regeneration is an indispensable part of life that ensures survival.
  • the involvement of MOs in tissue regeneration appears to be an evolutionary conserved process, as MOs provide critical functions in regeneration of zebrafish tail fin, salamander limb or neonatal heart as well as adult liver and skeletal muscle in mammals, among others (Chazaud, 2014; Godwin et al., 2013; Petrie et al., 2014; Porrello et al., 2011).
  • MOs 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.
  • MOs 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 MOs 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 MOs instruct myoblasts to form muscle fibers. We have identified the ligand activated transcription factor, PPARy, as a regulator of this process.
  • PPARy ligand activated transcription factor
  • MO PPARy-deficient animals As skeletal muscle possesses excellent regenerative capacity, it was especially interesting that the delay in muscle regeneration in MO 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 MOs in muscle regeneration is the MO 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 MO PPARy deficient mice was comparable in its extent to the delay reported in MO AMPK deficient animals, thus appearing to be among the most dramatic reported deficiencies caused by impairments in MO functions. This suggests that PPARy is likely to play an important and integrated sensory and regulatory function to synchronize cellular interactions during regeneration.
  • P117750 as PPARy has been reported to be a regulator of ⁇ polarization (Odegaard et al., 2007). Either the Ly6C+ to Ly6C- ⁇ transition in this system does not correspond to canonical polarized ⁇ populations, or PPARy is not a critical component of alternatively polarized MOs in this setting. Phagocytosis was normal in PPARg MacKO MOs, 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 MOs.
  • GDF3 is a strong candidate as a MO derived paracrine factor with muscle regenerative functions, whose diminished MO
  • 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 MOs are the predominant, if not the only source of GDF3 within the injured tissue.
  • ⁇ derived GDF3 is a tissue regeneration factor that regulates MPC expansion/differentiation within the injured tissue.
  • recombinant GDF3 enhanced myotube fusion and in vitro differentiation of established primary myoblasts.
  • GDF3 was added to independently isolated primary myoblast cell lines, excluding a cell line dependent effect.
  • 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.
  • GDF3 which is expressed in and secreted by muscle infiltrating MOs 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.
  • This regulatory axis consists of PPARy, a ligand activated transcription factor that appears to have distinct, but overlapping functions in the inflammatory and repair MOs within the injured tissue. PPARy then regulates muscle regeneration through the action of GDF3, a secreted factor that regulates MPC differentiation.
  • GDF3 a secreted factor that regulates MPC differentiation.
  • GDF3 is only one of the TGF family members that are active during regeneration and that it has effects on other cell types such as fibro/adipogenic progenitors as well (Hidestrand et al., 2008; Joe et al., 2010; Lemos et al., 2015). It is remarkable, though, that the key elements of the complex delay phenotype can be modeled in vitro using macrophages and myoblasts only, arguing that these two cell types and their interactions are critical to supporting regeneration. Irrespective of the answers to the questions above, our findings also carry potential implications for pathological circumstances in which recurrent muscle damage and asynchrony in repair due
  • GDF3 is also a regulator of muscle regeneration in DMD or other types of myopathies, which are most of the time associated with the permanent presence of inflammatory cells, especially MOs.
  • 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.
  • 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.
  • systemic or injectable administration such as intravenous (IV), 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.
  • P117750 isolated representative ⁇ populations from injured muscle and interrogated their gene expression profiles by microarray analysis.
  • an initial wave of Ly6C+ ⁇ infiltration is replaced by the emergence of Ly6C- MOs from day 2 on.
  • the simplified, but widely accepted paradigm about these two main ⁇ populations posits that the Ly6C+ MOs are inflammatory, while Ly6C- MOs are repairing.
  • the expression profiles of Ly6C+ inflammatory and Ly6C- repair MOs 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) MOs (Fig S1A).
  • mice that received bone marrow deficient in PPARy 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 PEl 131313 n _ Sox2 Cr e + animals displayed a drastic impairment in their skeletal muscle regeneration (Fig. S2B). Altogether, the results from these distinct genetic models clearly indicated that PPARy activity in muscle infiltrative MOs critically contributed to the timely resolution of inflammation and to regeneration.
  • PPARy does not alter macrophage infiltration or differentiation in injured muscle
  • P117750 injured muscle are neutrophils and Ly6C+ MOs.
  • Ly6C mid F4/80 neutrophils and Ly6C + F4/80 low MOs within the CD45+ cells isolated from injured muscle at day 1 (Fig 2 B).
  • Fig 2 B fewer neutrophils infiltrated the injured muscle in PPARg MacKO animals (68.05 % vs. 64 %), which also meant that the ratio of Ly6C+ MOs were slightly higher in PPARy deficient animals (19.4 % vs. 22.7 %).
  • MOs have traditionally been considered to be primarily phagocytic cells. Therefore, one plausible explanation is that PPARy activity in MOs 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 MOs in phagocytic clearance is beyond doubt, it recently became widely accepted that MOs 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).
  • IGF1 biologically active molecules
  • MO PPARy could plausibly improve the regenerative capacity of skeletal muscle by affecting MPC expansion or differentiation, independent of its phagocytic activity.
  • BMDMs bone marrow derived MOs
  • P117750 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.
  • conditioned medium from non-treated PPARg MacKO BMDMs phenocopied the proliferation enhancing effect of inflammatory WT BMDMs on myoblasts (Fig 3C).
  • P117750 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 ⁇ subtypes is shown in Fig. 4C.
  • Ly6C- MOs at day 2 express PPARy , but the receptor is also sensitive to the activating effect of an exogenous ligand in Ly6C- cells.
  • Gd/3 the gene that was found to be consistently downregulated in PPARg MacKO MO subsets, was also regulated by RSG treatment in Ly6C- MOs. 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 MO subsets?
  • GDF3 is a ⁇ derived PPARy dependent member of the TGFfi family
  • Gd/3 was the top ranked gene (ranked by fold change difference) in 3 out of 4 MO subsets (Fig 4B).
  • GDF3 belongs to the TGF family, whose members are secreted factors acting in a paracrine manner.
  • GDF8 also known as Myostatin
  • Gd/3 we selected Gd/3 as the most likely PPARy dependent gene that contributes to muscle regeneration for further analysis.
  • 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 Gdf3 locus.
  • 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 AngptH and that of Gd/3 (Fig 5A).
  • 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 MOs
  • RXR the obligate heterodimeric partner of PPARy
  • PPARy ChlP-seq data derived from thioglycolate elicited peritoneal MOs and adipocytes.
  • Gdf3 was regulated by one or several of these PPARy:RXR binding sites.
  • 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.
  • P117750 GDF3 is a regulator of myoblast proliferation, differentiation and muscle regeneration
  • 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.
  • GDF3 is a ⁇ derived protein whose expression is induced during muscle regeneration in a PPARy dependent manner.
  • GDF3 a ⁇ derived secreted factor can regulate in vitro and in situ muscle differentiation/regeneration
  • Fig 7E the transcriptomic features of muscle infiltrative MOs to chart the expression and dynamics of the TGF family signaling system (Fig 7E) to identify additional factors that might regulate regeneration in a paracrine or autocrine manner.
  • P117750 ligands [Gdf3, Gdfl5 and Inhba) showed notable gene expression dynamics in muscle infiltrative MOs. GDF3 expression peaked in repair MOs 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 ⁇ ) showed a temporary boost in its expression at the earliest stage of injury.
  • the activin system 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 TGF family signaling system, Tgfbr2, Eng and Bambi were also regulated in muscle MOs. Twelve other members of the signaling system were also expressed but not regulated in these MOs, while the remaining members (including myostatin) were not expressed at all (Fig S7D).
  • the PPARy-GDF3 regulatory axis described in this study therefore identifies a sensory-regulatory-effector mechanism, by which MOs 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.
  • mice Ppar fl/flLysMCre+ (refered to as PPARg MacKO) and wild type C57BL/6J controls, Ppargfl/- Sox2Cre+ and littermate control PpargflZ+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 ⁇ 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.
  • 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.
  • BM bone marrow
  • 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.
  • MPC Myogenic precursor cell 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-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-
  • P117750 ki67 antibodies (15580 Abeam), which were subsequently visualized using cy3-conjugated secondary antibodies (Jackson Immunoresearch Inc).
  • 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.
  • 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.
  • 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 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 supplememntal method section.
  • 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.
  • Anti-GAPDH mouse monoclonal primary antibody AM4300, Ambion, Carlsbad, CA
  • 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. The RNA-Seq data are publicly accessible (data access: PRJNA290560/SRR2136645).
  • 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 MAB) 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 Ppar5 , fl/flLysMCre+ X Ppar5 , fl/flLysMCre+ and WT X WT crossings. In a separate experiment, a small cohort of Ppar5 , fl/flLysMCre+ and littermate control Ppar5 , +/+LysMCre+ animals were generated from Ppar5 , fl/+LysMCre+ X Ppar5 , fl/+LysMCre+ crossings.
  • Bone marrow transplantation 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 5x106 bone marrow cells/200 ⁇ 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 ⁇ 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.
  • 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 2 OX 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 ⁇ .
  • 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 ⁇ g/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.
  • BM bone marrow
  • MPC Myogenic precursor cell 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
  • P117750 expanded.
  • 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 GDF3 mouse recombinant protein (300 ng/ml; 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).
  • 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.
  • 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.
  • Ly6C+ F4/801ow macrophages, Ly6C- F4/80+ macrophages and Ly6Cmid F4/80- neutrophils were sorted. In each experiment, both genotypes were parallelly processed to minimize experimental variation. Cells were analyzed and/or sorted with a BD FACSAria III sorter.
  • RNA isolation from sorted MFs were sorted from day 1, 2 and 4 post-injury muscles with a
  • 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
  • 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 PrimeTime assays from IDT (Gd/3 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.
  • BM bone marrow
  • DMEM bone marrow
  • ChIP ChIP immunoprecipitation: Cells were double crosslinked with 0,002M 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.
  • ChIP Lysis Buffer 1% Triton x-100, 0.1% SDS, 150 mM NaCl, ImM EDTA, and 20 mM Tris, pH 8.0
  • 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).
  • 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:
  • Tibialis anterior was removed from mice injected intramuscularly with cardiotoxin (CTX) at experimental time points and homogenized in RIPA buffer.
  • CTX cardiotoxin
  • 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).
  • MPCs 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 GDF3 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.
  • RNA-Seq data are publicly accessible (data access: PRJNA290560/SRR2136645).
  • Tissue regeneration requires inflammatory and reparatory activity of macrophages (MOs).
  • MOs detect and eliminate the damaged tissue and subsequently promote regeneration. This dichotomy requires the switch of the effector functions of MOs coordinated with other cell types inside the injured tissue.
  • the gene regulatory events supporting the sensory and effector functions of MOs involved in tissue repair are not well understood.
  • PPARy the lipid activated transcription factor, PPARy is required for proper skeletal muscle regeneration acting in repair MOs.
  • PPARy controls the expression of the TGF0 family member, GDF3, which in turn regulates the restoration of skeletal muscle integrity by promoting muscle progenitor cell differentiation.
  • GDF3 the TGF0 family member
  • P117750 effector protein acting on myoblasts and serving as an exclusively macrophage-derived regeneration factor in tissue repair.
  • ⁇ 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 MOs.
  • the GDF3 locus has multiple PPAREkRXR heterodimer bound active enhancers. GDF3 is needed for proper muscle regeneration and enhances differentiation of primary myoblasts
  • Urokinase-type plasminogen activator plays essential roles in macrophage chemotaxis and skeletal muscle regeneration. Journal of immunology (Baltimore, Md : 1950) 180, 1179-1188.
  • Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Nature cell biology 12, 153- 163.
  • Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell 159, 1312. Lemos, D.R., Babaeijandaghi, F., Low, M., Chang, C.K., Lee, S.T., Fiore, D., Zhang, R.H., Natarajan, A, Nedospasov, S.A., and Rossi, F.M. (2015). Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors. Nature medicine 21, 786-794.
  • GDF3 a BMP inhibitor, regulates cell fate in stem cells and early embryos. Development 133, 209-216.
  • GDF3 is a BMP inhibitor that can activate Nodal signaling only at very high doses. Developmental biology 325, 43-48.
  • Type beta transforming growth factor is an inhibitor of myogenic differentiation. Proceedings of the National Academy of Sciences of the United States of America 83, 8206-8210.
  • CTCF and cohesin linking gene regulatory elements with their targets.
  • Macrophage-specific PPARgamma controls alternative activation and improves insulin resistance. Nature 447, 1116-1120.
  • Tissue-specific signals control reversible program of localization and functional polarization of macrophages. Cell 157, 832-844.
  • Latent enhancers activated by stimulation in differentiated cells. Cell 152, 157-171.
  • a SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma. Nature 437, 759-763.
  • Rapalino 0., Lazarov-Spiegler, 0., Agranov, E., Velan, G.J., Yoles, E., Fraidakis, M., Solomon, A., Gepstein, R., Katz, A., Belkin, M., et al (1998). Implantation of stimulated homologous macrophages results in partial recovery of paraplegic rats. Nature medicine 4, 814-821.
  • STAT6 transcription factor is a facilitator of the nuclear receptor PPARgamma-regulated gene expression in macrophages and dendritic cells. Immunity 33, 699-712.
  • Monocyte/Macrophage-derived IGF-1 Orchestrates Murine Skeletal Muscle Regeneration and Modulates Autocrine Polarization. Molecular therapy : the journal of the American Society of Gene Therapy 23, 1189-1200.
  • PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell 93, 241-252.
  • PPARs are a unique set of fatty acid regulated transcription factors controlling both lipid metabolism and inflammation.
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  • Tissue LyC6- macrophages are generated in the absence of circulating LyC6- monocytes and Nur77 in a model of muscle regeneration. Journal of immunology (Baltimore, Md : 1950) 191, 5695-5701.

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Abstract

La présente invention concerne la régulation par PPARγ de l'expression de GDF3, membre de la famille du TGFß, qui régule à son tour la restauration de l'intégrité des muscles squelettiques en favorisant la différenciation des cellules progénitrices musculaires. Ainsi, l'invention porte sur GDF3 utilisé en tant que protéine effectrice extrinsèque sécrétée agissant sur des myoblastes et servant exclusivement de facteur de régénération dérivé de macrophages dans la réparation des tissus. L'invention concerne également un composé GDF3 utilisé dans le traitement d'un patient présentant une affection ou une maladie associée à une déficience musculaire, ledit patient ayant besoin d'une différenciation musculaire, ou un composé GDF3 utilisé dans l'amélioration de la régénération ou de la différenciation musculaire d'un patient en ayant besoin, ainsi que des compositions pharmaceutiques destinées à être utilisées dans ces traitements.
PCT/HU2015/050019 2015-01-30 2015-11-30 Régénération musculaire WO2016120647A2 (fr)

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CN115386539A (zh) * 2021-05-18 2022-11-25 南京大学 胸腺细胞在培养肌肉干细胞中的用途

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