WO2008028888A2 - Use of cripto-1 or cripto-3 for the treatment of skeletal muscle wasting - Google Patents

Abstract

The present invention relates to the field of muscle pathologies, more particularly to the field of diseases where skeletal muscle wasting occurs. The invention provides the use of cripto and functional fragments thereof for the regeneration of skeletal muscle.

Description

Means and methods for the stimulation of skeletal muscle regeneration

Field of the invention The present invention relates to the field of muscle pathologies, more particularly to the field of diseases where skeletal muscle wasting occurs. The invention provides the use of cripto and functional fragments thereof for the regeneration of skeletal muscle.

Background of the invention Skeletal muscles are composed of bundles of highly oriented and dense muscle fibers, each a multinucleoated cell derived from myoblasts. The muscle fibers in native skeletal muscle are closely packed together in an extracellular three-dimensional matrix to form an organized tissue with high cell density and cellular orientation to generate longitudinal contraction. After muscle injuries, myofibers become necrotic and are removed by macrophages. A specialized myoblast sub-population called satellite cells scattered below the basal lamina of myofibers are capable of regeneration. The incidence of satellite cells in skeletal muscle is very low (1%-5%) and depends on age and muscle fiber composition. These cells remain in a quiescent and undifferentiated state and can enter the mitotic circle in response to specific local factors. This induces proliferation and fusion of myoblasts to form multinucleated and elongated myotubes, which self-assemble to form a more organized structure, namely muscle fiber. Besides satellite cells migrate and proliferate in the injured area and can form a connective tissue network (muscle fibrosis). This process is called "scar tissue formation" and leads to loss of functionality. Several diseases often result in significant loss of skeletal muscle tissue. Examples include skeletal myopathies such as muscular dystrophy and spinal muscular atrophy. In addition traumatic injury, aggressive tumor ablation and prolonged denervation lead to skeletal muscle loss. Until now, few alternatives exist to provide functional and aesthetic restoration of lost muscle tissues aside from transfer of muscle from local or distant sites. However, the tissue engineering of skeletal muscle tissue still remains a challenge. In addition, a number of extracellular factors are known to be involved in the muscle regeneration that is triggered in response to muscle damage. Some of them, such as insulin-like growth factors (IGFs), fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), transforming growth factor (TGF)-like molecules, leukemia inhibitor factor (LIF) or platelet-derived growth factors (PDGFs), are involved in the activation of cell proliferation that operates before muscle differentiation. In addition, factors such as IGFs, neuregulins (NRGs), sonic hedgehog (Shh) or Wnt promote muscle differentiation. However up till now none of these molecules have proven to be useful for muscle regeneration therapy and it is clear that a more detailed understanding of the signalling pathways triggered by these factors is needed. A different molecule which we have been studying is human cripto. Crypto is a M_r 36,000 molecule, classified in the epidermal growth factor-Cripto-FRL-Criptic (EGF-CFC) family, caused by the conservation of six cysteines in the central region (amino acids 77-1 13); however, there is little resemblance to EGF, and Cripto does not bind to any EGF receptors. Cripto contains a signal sequence, a characteristic EGF-like domain, a second cysteine-rich region motif (CFC domain), and a hydrophobic COOH-terminus. Additionally, Cripto is a membrane-bound protein, anchored in the lipid bilayer of cell surfaces by a glycosyl-phosphatidylinositol, and acts as both cell surface co-receptors and has activity when expressed as a soluble protein. Cripto plays an important role in embryonic development in zebrafish and Xenopus; in the mouse, a germ line knockout of the mouse Cripto gene was lethal, and in zebrafish, injection of recombinant Cripto protein into late blastulae rescued a mutant phenotype. Cripto is also an oncogenic growth factor because it enhances cancer cell migration in vitro and branching morphogenesis of mammary epithelial cells that may contribute to cancer metastasis. Cripto is absent or in low levels on normal tissues and is expressed in most malignant tumors, including colon, breast, lung, ovarian, and pancreatic cancers. Hence, WO02088170 and WO02077033 claim the use of antibodies against cripto for cancer treatment. In addition, WO2004083375 discloses the use of cripto for the induction of stem cell differentiation into cardiomyocytes. Given the physiological role of Cripto in the initial instructive events leading to the commitment of mesodermal cells, both in the embryo and in Embryonic Stem (ES) cells we have evaluated a role of Cripto in pathological conditions using a mouse model of hind limb ischemia. We have found that cripto induces the regeneration of skeletal muscle cells. There is no suggestion in the art that cripto can be used for the stimulation of skeletal muscle cell differentiation. It is described that cripto is necessary for the differentiation of ES cells into cardiomyocytes but is not required for the differentiation into skeletal muscle cells (see Xu C. et al (1998) Dev. Biol. 196(2):237). With the present finding we have shown that cripto is useful for the manufacture of a medicine to treat muscle wasting diseases.

Figures

Figure 1. Effect of CR-1 on mpc proliferation. (A) CR-1 induces mpc proliferation in a dose- dependent manner. Both commercially available (R&D) and in house preparation (HM) of recombinant CR-1 proteins, were used, showing comparable results. (B) Mpc treatment with anti-CR-1 antibodies reduced proliferation and abolished the proliferative effect exerted by exogenous CR-1. Activity was expressed as percentage of mpc proliferation over control. bFGF was used as control. (C) lmmunostaining showing increasing expression of MyoD in mpc treated with recombinant CR-1 (100 ng/ml) compared to control cells. (D) Mpc treatment with specific siRNA targeting CR-1 also reduced proliferation, indicating that endogenous CR-1 is involved in the mechanism. Activity was expressed as percentage of mpc proliferation over control. bFGF was used as positive control and siRNA Cy3 as negative control.

Figure 2. Effect of CR-1 on mpc migration. (A) Recombinant CR-1 induces chemoattraction of mpc in a dose-dependent manner. Numbers of migrating cells/field are indicated. (B) Incubation with anti-CR-1 antibodies abolishing the pro-migratory effect exerted by exogenous CR-1. Activity was expressed as percentage of cell migration over control. Complete Growth Medium (GM) was used as positive control.

Figure 3. CR-1 counteracts TGF-β effects on myoblast proliferation and signals through Alk4 for migration. (A) RT-PCR products of mRNA extracted from myoblasts (Mb), Myotubes (MT) and control cells (CtI). (B) Incubation with anti-Alk4 antibodies abolishing the pro-migratory effect exerted by exogenous CR-1. Complete Growth Medium (GM) was used as positive control. (C) Incubation with anti-Alk4 antibodies did not interfere with the proliferative effect exerted by exogenous CR-1. (D) Incubation with anti-TGF-β pathway antibodies (TGF-b; TbRI) or pre-incubation of TGF-β with CR-1 abolishing the anti-proliferative effect exerted by exogenous TGF-β.

Figure 4. Therapeutic effect of CR-1 on skeletal muscle regeneration. (A) CR-1 overexpression increased muscle regeneration in the gastrocnemius muscle at 4 days after CTX injection; R (regeneration), V (viable), O (other). (B) CR-1 overexpression increased muscle regeneration in the gastrocnemius muscle at 7 days after femoral artery ligation. Areas are percentage of total muscle area. ^*P < 0.05 (A-B). (C) CR-1 overexpression increased cell proliferation in the gastrocnemius muscle of CTX mouse model (p<0.02). lmmunostaining revealing colocalization of BrdU and CR-1 (D). Therapeutic revascularization with CR-1 in injured limb. CR-1 overexpression resulted in increased angiogenesis (E), capillary density (F) enhanced total muscle perfusion (G).

Figure 5. CR-1 reduced fibrosis at 15 days after CTX injection (A, left panel) and 7 days after hind limb ischemia model (A, right panel). Quantification and analysis on Sirius red stained sections as described in Materials and Methods.

Aims and detailed description of the invention

The EGF-CFC family includes a group of structurally related proteins that have been identified only in vertebrates. This family consists of human cripto-1 , human cryptic and criptin, mouse criptin-1 and cryptic, chicken cripto, Xenopus FRL1 and zebrafish Oneyed pinehead. Human criptin-1 , originally known as teratocarcinoma-derived growth fact or (TDGF-1 ), is the founding member of this family of proteins. Multiple copies of cripto-related genes are present in the human and mouse genomes (Scognamiglio et a/ (1999) Cytogenet. Cell Genet. 84, 220-224). The EGF-CFC gene family encodes for proteins that contain two important functional domains: a variant epidermal growth factor (EGF)-like domain and a unique cysteine-rich motif (CFC). It has been shown in the art that the EGF domain is necessary for binding to Nodal and the CFC domain is responsible for binding to the ALK4 receptor. The carboxy terminus contains in some cases a consensus sequence for a glycosylphosphatidylinositol (GPI) anchorage site that serves to attach the protein to the cell membrane. Whereas the overall sequence identity between these proteins is only 22-32%, within the EGF-like domain and the CFC domain the sequence similarity is nearly 60-70% and 35-48%, respectively. Biochemical characterization by peptide mapping, mass spectrometry and glycosidase treatment have identified Asn-79 as an N-linked glycosylation site with >90% occupancy, and Ser-40 and Ser-161 as O-linked glycosylation sites with 80 and 40% occupancy, respectively. In addition, a biologically significant O-fucosylation has been identified within the second and third cysteines of the EGF- like domain of all EGF-CFC proteins and localized to Thr-88 in human cripto-1.

The invention provides the use of cripto-1 or cripto-3 or a functional fragment or functional variant derived thereof or a homologue with at least 90% identity with cripto-1 or cripto-3 for the manufacture of a medicament to treat skeletal muscle wasting disorders. In other words cripto-1 or cripto-3 or a functional fragment or functional variant derived thereof or a homologue with at least 90% identity with cripto-1 or cripto-3 can be used for the manufacture of a medicament to enhance skeletal muscle regeneration. The wording "skeletal muscle wasting disorder" is equivalent with the wording "skeletal muscle degeneration disorder" and with the term "myopathies". The word 'enhance' means also to 'stimulate' or to 'induce'. It is understood that the enhancement of skeletal muscle regeneration is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more with respect to the tissue which has not been treated with cripto-1 or cripto-3 or a functional fragment or functional variant derived thereof or a homologue with at least 90% identity with cripto-1 or cripto-3. Cripto-1 or cripto-3 or a functional fragment or functional variant derived thereof or a homologue with at least 90% identity with cripto-1 or cripto-3 can also be used to stimulate skeletal muscle growth by increasing the proliferation of muscle precursor cells. The amino acid sequence of human cripto-1 is depicted in SEQ ID NO: 2 and the nucleotide sequence of human cripto-1 is depicted in SEQ ID NO: 1. The amino acid sequence of human cripto-3 is depicted in SEQ ID NO: 4 and the nucleotide sequence of human cripto-3 is depicted in SEQ ID NO: 3. In one embodiment cripto-1 or cripto-3 is used as a protein or a functional homologue or a functional fragment (a functional peptide) derived thereof. In another embodiment cripto-1 or cripto-3 is used as a nucleic acid or a functional homologue or a functional fragment (a functional portion) derived thereof. Cripto-1 , also known as teratocarcinoma derived growth factor-1 , is described in US5792616, US5,256,643 and US5,654,140 while cripto-3 is described in US5264557, 5620866 and 5650285. Cripto-1 and cripto-3 are herein further commonly designated as cripto. Thus it is an object of the present invention that cripto can be used to stimulate the regeneration of skeletal muscle cells.

Functional fragments, functional variants and homologues of cripto

To improve or alter the characteristics of cripto polypeptides, protein engineering may be employed. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions. For instance, for many proteins, including the mature form(s) of a secreted protein, it is known in the art that one or more amino acids may be deleted from the N-terminus or C-terminus without substantial loss of biological function. For instance, Ron and colleagues (J. Biol. Chem., 268:2984-2988 (1993)) reported modified KGF protein that had heparin binding activity even if 3, 8, or 27 N-terminal amino acid residues were missing. In the present case, deletion of 30 N-terminal amino acids from SEQ ID NO: 2, which corresponds with the signal sequence, still retains full biological activity (see Foley SF et al (2003) Eur. J. Biochem. 270, 3610), in particular the mature form of human cripto starts at amino acid 31 in SEQ ID NO: 2. Accordingly, the present invention further provides polypeptides having 30 or more residues deleted from the amino terminus of the amino acid sequence of the cripto shown in SEQ ID NO: 2 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues 31-188, 32- 188, 33-188, 34-188, 35-188, 36-188, 37-188, 38-188, 39-188, 40-188, 41-188, 42-188, 43- 188, 44-188, 45-188, 46-188, 47-188, 48-188, 49-188, 50-188, 51-188, 52-188, 53-188, 54- 188, 55-188, 56-188, 57-188, 58-188, 59-188, 60-188, 61-188, 62-188, 63-188, 64-188, 65- 188, 66-188, 67-188, 68-188, 69-188 of SEQ ID NO: 2 and polynucleotides encoding such polypeptides which can be used in the present invention.

Similarly, many examples of biologically functional C-terminal deletion muteins are known. For instance, Interferon gamma shows up to ten times higher activities by deleting 8-10 amino acid residues from the carboxy terminus of the protein (Dobeli, et al., J. Biolechnology 7:199-216 (1988)). Accordingly, the present invention further provides polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of the cripto shown in SEQ ID NO:2 and polynucleotides encoding such polypeptides. More in particular, the invention provides polypeptides having the amino acid sequence of residues 31-150, 31-151 , 31-152, 31-153, 31-154, 31-155, 31-156, 31-157, 31-158, 31-159, 31-160, 31-161 , 31-162, 31-163, 31- 164, 31-165, 31-166, 31-167, 31-168, 31-169, 31-170, 31-171 , 31-172, 31-173, 31-174, 31- 175, 31-176, 31-177, 31-178, 31-179, 31-180, 31-181 , 31-182, 31-183, 31-184, 31-185, 31- 186, 31-187, 31-188 of SEQ ID NO:2 and polynucleotides encoding said polypeptides. In a preferred embodiment a polypeptide of residues 1-169 of SEQ ID NO: 2 and a polynucleotide encoding such a polypeptide is provided. Such a polypeptide (C-terminally deleted for the signal peptide for GPI-anchorage) was made by Foley SF et al (2003) Eur. J. Biochem. 270, 3610. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini as described above. In addition, the present invention also includes similar variants and fragments of SEQ ID NO: 4. Also included are nucleotide sequences encoding a polypeptide consisting of a portion of the complete cripto amino acid sequence encoded by SEQ ID NO: 1 or 3. Polynucleotides encoding all of the above deletion mutant polypeptide forms also are provided. As mentioned above, even if deletion of one or more amino acids from the N-terminus or C-terminus or N- and C-terminus of cripto results in modification of loss of one or more biological functions of cripto, other biological activities such as the ability to induce skeletal muscle regeneration may still be retained. Such cripto muteins or functional fragments or functional derivatives can be tested for their ability to induce skeletal muscle regeneration in for example a hind limb ischaemia assay or in a myogenic precursor cell proliferation assay as described herein further in the examples. Functional fragments and functional derivatives of cripto can also be tested the presence of biological activity in several biological assays. One example of such an assay is the ability of a functional fragment to rescue the MZoep phenotype in zebrafish embryos (Minchiotti G. et al (2001 ) Development 128, 4501-10). Yet another example of such an assay is the ability of a functional fragment to rescue the cardiac differentiation of embryonic stem cells wherein cripto is deleted (Parisi et al (2003) J. Cell. Biol. 163, 303-14). Functional fragments and functional variants of cripto are herein defined as nucleotide sequences or peptide sequences that have to ability to induce regeneration of skeletal muscle cell regeneration. A particular preferred fragment of cripto is a fragment consisting of the EGF-CFC domain. Minchiotti G. et al (2001 ) Development 128, 4501-10 and Parisi et al (2003) J. Cell. Biol. 163, 303-14 have shown that the EGF-CFC- domain is sufficient for the biological activity of cripto. Based on the homology between the EGF-CFC domain of mouse and human cripto we calculate that the EGF-CFC domain of cripto-1 and cripto-3 are between amino acids 69-173 in SEQ ID NO: 2 and SEQ ID NO: 4. Such an EGF-CFC - fragment is conveniently made by for example fusing it to the genetic information encoding the signal sequence (amino acids 1-30 of SEQ ID NO 2 or 4). The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl temini of a cripto polypeptide, which may be made by combinations as described herein above.

In addition to N- and C- or combined N- and C- terminal deletion forms of cripto as discussed above, it also will be recognized by one of ordinary skill in the art that some amino acid sequences of the cripto polypeptide can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity for skeletal muscle cell regeneration. Thus, the invention further includes variants of cripto, thus polypeptides which show substantial cripto polypeptide activity for skeletal muscle cell regeneration or which include regions of cripto protein with skeletal muscle cell regeneration activity. Such mutants include deletions, insertions, inversions, repeats, and type substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality. Proteins are surprisingly tolerant of amino acid substitutions. Most buried amino acid residues require non-polar side chains, whereas few features of surface side chains are generally conserved. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, VaI, Leu and lie; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and GIu, substitution between the amide residues Asn and GIn, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr. Thus, the fragment, variant or homologue of the polypeptide of SEQ ID NO:2, or that encoded by SEQ ID NO: 1 may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the above form of the polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the polypeptide (e.g. a histidine tag) or a pro-protein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein. Thus, cripto of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation. As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein. Embodiments of the invention are directed to cripto polypeptides which comprise the amino acid sequence of a cripto polypeptide of SEQ ID NO: 2, but having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 30 conservative amino acid substitutions, even more preferably not more than 20 conservative amino acid substitutions, still more preferably not more than 10 conservative amino acid substitutions, when compared with the cripto polypeptide sequence described herein. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of a cripto polypeptide, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions. To improve or alter the characteristics of cripto polypeptides, protein engineering may be employed. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions. Thus, the invention also encompasses cripto derivatives (variants) and homologues that have one or more amino acid residues deleted, added, or substituted to generate cripto polypeptides that are better suited for expression, scale up, etc., in the host cells chosen. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges. In SEQ ID NO: 2 the cysteine-residues are at position 115, 133, 149, 131 and 140. According to Foley SF et al (2003) Eur. J. Biochem. 270, 3610; Cys1 15 forms a disulfide bond with Cys133, Cys128 with Cys149 and Cys131 with Cys140. N- linked glycosylation sites could also be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites. To this end, a variety of amino acid substitutions at one or both of the first or third amino acid positions on any one or more of the glycosylation recognitions sequences in the cripto polypeptides of the invention, and/or an amino acid deletion at the second position of any one or more such recognition sequences will prevent glycosylation of the cripto polypeptide at the modified tripeptide sequence. Amino acids in the cripto protein of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for skeletal muscle cell regenerative activity. Of special interest are substitutions of charged amino acids with other charged or neutral amino acids which may produce proteins with highly desirable improved characteristics, such as less aggregation. Aggregation may not only reduce activity but also be problematic when preparing pharmaceutical formulations, because aggregates can be immunogenic. The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinant form of the cripto polypeptide can be substantially purified by recombinant expression in E. coli (cripto expression and purification in E. coli is described by Seno M et al (1998) Growth Factors 15(3): 215). Expression and purification of cripto in CHO cells is described in Brandt R. et al (1994) J. Biol. Chem. 269(25): 17320. Polypeptides of the invention also can be purified from natural or other recombinant sources using for example anti-cripto antibodies in methods which are well known in the art of protein purification.

By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a cripto polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the cripto polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

By "% similarity" for two polypeptides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence

Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park,

575 Science Drive, Madison, Wis. 5371 1 ) and the default settings for determining similarity.

Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied

Mathematics 2:482-489, 1991 ) to find the best segment between two sequences. The invention also encompasses fusion proteins in which the full-length cripto polypeptide or fragment, variant, derivative, or homologue thereof is fused to an unrelated protein. These fusion proteins can be routinely designed on the basis of the cripto nucleotide and polypeptide sequences disclosed herein. For example, as one of skill in the art will appreciate, cripto polypeptides and fragments (including epitope-bearing fragments) thereof described herein can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric (fusion) polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. An example of such a fusion protein is a soluble form of the CFC domain, comprised of the signal peptide and amino acids 112-169, fused to the hinge and Fc region of human IgGI which still binds to the ALK4 receptor (Foley SF et al (2003) Eur. J. Biochem. 270, 3610. Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric cripto polypeptide or polypeptide fragments alone (Fountoulakis, et al., J. Biochem. 270:3958-3964 (1995)). Examples of cripto fusion proteins that are encompassed by the invention include, but are not limited to, fusion of the cripto polypeptide sequences to any amino acid sequence that allows the fusion proteins to be displayed on the cell surface (e.g. the IgG Fc domain); or fusions to an enzyme, fluorescent protein, or luminescent protein which provides a marker function.

Use of cripto, functional fragments, functional derivatives and homologues thereof as a nucleic acid

In a specific embodiment, nucleic acids comprising sequences encoding cripto, homologues or functional derivatives thereof, are administered to stimulate skeletal muscle cell regeneration by way of gene therapy. Thus in a particular embodiment SEQ ID NO: 1 or 3 or a variant sequence encoding a functional derivative of SEQ ID NO: 2 or 4 or a fragment thereof encoding a functional fragment of SEQ ID NO: 2 or 4 or a homologue encoding a sequence with at least 90% identity with SEQ ID NO: 2 or 4 can be used for the manufacture of a medicament to treat skeletal muscle wasting disorders. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce cripto, a functional fragment, a functional variant or homologue thereof mediates skeletal muscle cell regeneration. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

In case a nucleic acid sequence or a portion thereof, capable of encoding cripto or a fragment thereof, is used for the manufacture of a medicament to treat skeletal muscle disorders, said medicament is preferably intended for delivery of said nucleic acid sequence into the cell, in a gene therapy treatment. A large number of delivery methods are well known to those of skill in the art. Preferably, the nucleic acids are administered for in vivo or ex vivo gene therapy uses. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. A preferred method of non-viral delivery is via direct electroporation in muscle cells. Lipofection is described in, e.g., US Pat. No. 5,049,386, US Pat No. 4,946,787; and US Pat. No. 4,897,355 and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Flegner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or directly to target tissues such as muscle fibers (in vivo administration). The preparation of lipid: nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, 1995; Blaese et al., 1995; Behr, 1994; Remy et al., 1994; Gao and Huang, 1995; U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871 , 4,261 ,975, 4,485,054, 4,501 ,728, 4,774,085, 4,837,028, and 4,946,787). The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo). Conventional viral based systems for the delivery of nucleic acids include amongst others retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

In cases where transient expression of the nucleic acid is preferred, adenoviral based systems, including replication deficient adenoviral vectors may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors, including recombinant adeno-associated virus vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., U.S. Patent No. 4,797,368; WO 93/24641 ; Kotin, 1994; The construction of recombinant AAV vectors is described in a number of publications, including U.S. Pat. No. 5,173,414; Hermonat & Muzyczka, 1984; Samulski et al., 1989). Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intratracheal, subdermal, or intracranial infusion) or topical application. In a particular embodiment intramuscular administration is preferred. In another embodiment the invention also envisages the use of a hydrodynamic gene therapeutic method. Hydrodynamic gene therapy is disclosed in US6627616 (Mirus Corporation, Madison) and involves the intravascular delivery of non-viral nucleic acids encoding cripto or a functional fragment or a variant thereof whereby the permeability of vessels is increased through for example the application of an increased pressure inside said vessel or through the co-administration of vessel permeability increasing compounds such as for example papaverine.

Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy, myoblasts) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transfected cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from the subject organism, transfected with a nucleic acid (gene or cDNA), and re- infused back into the subject organism (e.g., patient). Various cell types suitable for ex vivo transfection are well known to those of skill in the art. In ex vivo transfection, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny. The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art. Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc. In a preferred embodiment, the cell used for gene therapy is autologous to the patient. In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding cripto are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for a therapeutic effect in skeletal muscle regeneration. In a specific embodiment, stem or progenitor cells (e.g. myoblasts) are used. In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the cripto coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.

Skeletal muscle cell diseases:

The wording 'diseased skeletal muscle cells' refers to skeletal muscle cells that have been exposed for example to an ischemic insult, or for example skeletal muscle cells that possess a reduced glycolytic rate, or for example skeletal muscle cells that have been exposed to serum deprivation. Molecules that can be used are molecules that are able to induce the activity of cripto by inducing its synthesis or translation. By molecules it is meant peptides, tetrameric peptides, proteins, organic molecules and zinc finger proteins. By synthesis it is meant trancription of cripto. Small organic molecules can bind on the promoter region of cripto and strengthen the binding of a transcription factor or said molecules can bind said transcription factor and stimulate binding to the cripto promoter. The activity of cripto can be calculated by measuring the % stimulation of skeletal muscle cell regeneration. Preferably undifferentiated myoblast cultures are used for measuring cripto activity. 'Degeneration' is herein equivalent to the terms necrotic skeletal muscle cell death, apoptotic muscle skeletal cell death, muscle skeletal cell atrophy, skeletal fiber injury and skeletal muscle wasting.

The term 'skeletal muscle degenerative diseases' or 'skeletal muscle wasting diseases' comprises any of a group of diseases where degeneration (atrophy) occurs of skeletal muscle cells or diseases where structural changes or functional impairment occur in skeletal muscle. A serious indication where skeletal muscle degeneration takes place is due to ischemic insults. For example it has become increasingly recognized that skeletal muscle atrophy is common in patients with chronic pulmonary disease (COPD). Another example where skeletal muscle atrophy occurs is critical limb ischemia (CLI) which is a disease manifested by sharply diminished blood flow to the legs. Up to 10 million people in the US alone suffer from severe leg pain (claudication) and non-healing-ulcers (peripheral vascular disease), both of which can ultimately lead to CLI. The most common causes that can lead to CLI are atherosclerosis and embolization (e.g. a clot that has been ejected from a failing heart, or from an aneurysm in the aorta, into the leg). Yet another class of skeletal muscle degenerative diseases are muscle pathologies associated with a reduced glycolytic rate such as McArdle's disease and phosphofructokinase disease (PFKD). Yet another class comprises muscle atrophy which occurs due to muscle denervation. In such denervation atrophy, there occurs a lack of tonic stimuli and muscle cells become atrophic. Causes of denervation atrophy include localized loss of nerve function (neuritis) or generalized loss of the entire motor unit. After denervation, muscles become rapidly atrophic and 50% of muscle mass could be lost in just a few weeks. Examples are peripheral motor neuropathies and motoneuron disorders such as amyotrophic lateral sclerosis, Guillain-Barre syndrome and diabetic neuropathy. Another class of such diseases comprises muscle degeneration which occurs due to immobilization. 'Immobilization' means here that the skeletal muscle system is unloaded because of for example prolonged space flight (an astronauts disease), during conservative treatment after sports injuries or by a plaster cast after orthopedic surgery. This immobilization causes a serious atrophy of muscle mass leading to a decrease in physical performance and high power output capacity. Yet another class of such diseases where muscle degeneration takes place comprises muscular dystrophies. These disorders include a progressive wasting of skeletal muscle. The most common examples are Duchenne and Becker muscular dystrophy. Yet another class of conditions were muscle degeneration takes place comprises critical illness. Critical illness (e.g. burns, sepsis) is associated with a serious muscle wasting and muscle weakness. In yet another embodiment cripto, functional variants, homologues and functional fragments can be used for the treatment of muscle wasting in cachexia (e.g. which frequently occurs in cancer patients), in aging related sarcopenia, in spinal muscle atrophy, in arthritis, in stroke, in steroid therapy, in poliomyelitis, in spinal cord injury and in myotonia congenita.

The present invention not only aims at using cripto, functional variants, homologues and functional fragments for treatment of humans but also aims at using these molecules for veterinary diseases and conditions. Common causes of myopathies (degenerative diseases of muscle) in animals which can also be treated with cripto are: (1 ) metabolic myopathies (e.g. porcine stress syndrome, malignant hyperthermia and pale soft exudative pork), (2) exertional myopathies which comprise a group of diseases which result in severe muscle degeneration following strenuous exercise (e.g. azoturia and tying-up in horses, greyhound myopathy in dogs, capture myopathy in wild animals and compartment syndrome in poultry), (3) traumatic myopathies (e.g. Downer syndrome which is an ischemic necrosis of ventral and limb muscles following prolonged recumbency (disease/anesthesia) and Crush syndrome).

Use of cripto, functional fragments, functional derivatives and homologues thereof as a polypeptide for the manufacture of a medicament for treatment of skeletal muscle wasting disorders

The term 'medicament to treat' relates to a composition comprising molecules as described above and a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably) to treat skeletal muscle wasting diseases. Suitable carriers or excipients known to the skilled man are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers. The 'medicament' may be administered by any suitable method within the knowledge of the skilled man. The preferred route of administration is parenterally. In parental administration, the medicament of this invention will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with the pharmaceutically acceptable excipients as defined above. However, the dosage and mode of administration will depend on the individual. Generally, the medicament is administered so that the protein, polypeptide, peptide of the present invention is given at a dose between 1 μg/kg and 10 mg/kg, more preferably between 10 μg/kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg. Preferably, it is given as a bolus dose. Continuous infusion may also be used and includes continuous subcutaneous delivery via an osmotic minipump. If so, the medicament may be infused at a dose between 5 and 20 μg/kg/minute, more preferably between 7 and 15 μg/kg/minute. It is clear to the person skilled in the art that the use of a therapeutic composition comprising for example cripto or a fragment thereof for the manufacture of a medicament to treat skeletal muscle wasting diseases can be administered by any suitable means, including but not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In addition, the therapeutic composition is suitably administered by pulse infusion, particularly with declining doses of the antibody. Preferably the therapeutic composition is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

Use of enhancers of cripto expression for the manufacture of a medicament to treat skeletal muscle wasting disorders

The present invention also envisages the use of zinc finger proteins (ZFPs) to enhance the expression of cripto. Said ZFPs can also be used for the manufacture of a medicament to induce skeletal muscle cell regeneration and to treat skeletal muscle wasting disorders. The general use of zinc finger proteins to regulate endogenous gene expression is disclosed in patent application US20030087817 (Sangamo Biosciences) of which paragraphs [0049] until [0217] from the detailed description are herein specifically incorporated by reference. Usually ZFPs are linked to regulatory domains, creating chimeric transcription factors to activate transcription. Preferably, ZFPs have a K_d of less than about 25 nM to activate gene transcription. Generally ZFPs can be used to activate transcription of an endogenous cellular gene by about 1.5 fold or more. For transcription factors it is known that simple binding and sufficient proximity to the cripto promoter are all that is generally needed. Exact positioning relative to the cripto promoter, orientation, and within limits, distance do not matter greatly. This feature allows considerable flexibility in choosing sites for constructing artificial transcription factors. The target site recognized by the ZFP therefore can be any suitable site in the cripto gene that will allow activation of cripto expression by a ZFP, optionally linked to a regulatory domain. Preferred target sites include regions adjacent to, downstream, or upstream of the cripto transcription start site. In addition, target sites that are located in enhancer regions, repressor sites, RNA polymerase pause sites, and specific regulatory sites (e.g., SP-1 sites, hypoxia response elements, nuclear receptor recognition elements, p53 binding sites), sites in the cDNA encoding region or in an expressed sequence tag (EST) coding region. Typically each finger recognizes 2-4 base pairs, with a two finger ZFP binding to a 4 to 7 bp target site, a three finger ZFP binding to a 6 to 10 base pair site, and a six finger ZFP binding to two adjacent target sites, each target site having from 6-10 base pairs. Preferably the ZFP is linked to at least one or more regulatory domains, described below. Preferred regulatory domains include transcription factor activator domains such as VP16, co-activator domains, DNA methyl transferases, histone acetyltransferases, histone deacetylases, and endonucleases such as Fokl. For activation of gene expression, typically expression is activated by about 1.5 fold (i.e., 150% of non-ZFP modulated expression), preferably 2 fold (i.e., 200% of non-ZFP modulated expression), more preferably 5-10 fold (i.e., 500-1000% of non-ZFP modulated expression), up to at least 100 fold or more. The term "zinc finger protein" or "ZFP" refers to a protein having DNA binding domains that are stabilized by zinc. The individual DNA binding domains are typically referred to as "fingers" A ZFP has least one finger, typically two fingers, three fingers, or six fingers. Each finger binds from two to four base pairs of DNA, typically three or four base pairs of DNA. A ZFP binds to a nucleic acid sequence called a target site or target segment. Each finger typically comprises an approximately 30 amino acid, zinc-chelating, DNA-binding subdomain. A "target site" is the nucleic acid sequence recognized by a ZFP. A single target site typically has about four to about ten base pairs. Typically, a two-fingered ZFP recognizes a four to seven base pair target site, a three-fingered ZFP recognizes a six to ten base pair target site, and a six fingered ZFP recognizes two adjacent nine to ten base pair target sites. In yet another particular embodiment cripto-1 or cripto-3 or a functional derivative or a functional fragment or a homologue with at least 90% amino acid identity can be used for the in vitro preconditioning of donor's myoblasts for improving the transplantation success in compatible patients suffering of muscle wasting diseases. In yet another particular embodiment a nucleic acid sequence encoding cripto-1 or cripto-3 or a nucleic acid sequence encoding a functional derivative or a functional fragment or a homologue with at least 90% amino acid identity can be used for the in vivo preconditioning of donor's myoblasts. It is understood that said in vitro preconditioning also includes the stimulation of proliferation or other properties of cripto which are described in the examples.

Examples

1. CR-1 is expressed in vivo during skeletal muscle regeneration

The expression of cripto-1 (CR-1 ) has been systematically studied during normal development and in cancer progression. In the present study we investigated whether the expression of CR- 1 was re-activated in adult life during for example regeneration processes. To directly address this issue, we performed immunostainings with anti-CR-1 antibodies on both normal and regenerative muscle. We were unable to detect CR-1 expression in normal living muscle fibers whereas we found a strong expression in the regenerative area and newly formed myotubes. Moreover, by double staining with either desmin or MHC specific antibodies, we showed that CR-1 expressing cells are mainly myogenic cells. Interestingly, among the other cell types expressing CR-1 we found, a subpopulations of both vascular endothelial cells and macrophages as assessed by double staining of CR-1 with CD31 and F4/80, respectively.

2. Myogenic precursor cells express CR-1

Having shown that activated myogenic cells expressed CR-1 during muscle regeneration process, we went on to evaluate CR-1 expression in primary myogenic precursor cells (mpc) in culture. Indeed, immunostaining performed with anti-CR-1 antibodies from different sources, both in house preparation and commercially available, showed that CR-1 was expressed at the membrane of isolated precursor cells. Double staining with myogenic markers such as MyoD, Desmin, CD34, mCadherin was performed to demonstrate that those cells were indeed myogenic cells. Expression was confirmed by RT-PCR.

3. CR-1 induces myogenic precursor cell proliferation in vitro Myogenic precursor cells (mpc) were treated with recombinant cripto for 24 hours and proliferation was measured as BrdU incorporation in the DNA. CR-1 induced a dose dependent increase of mpc proliferation (from 50ng/ml to 300ng/ml) (Fig.1A). Comparable results were obtained using different sources of CR-1 , either commercially available or in house preparation. bFGF was used as control. Furthermore, treatment with anti-CR-1 antibodies almost abolished the proliferative effects exerted by exogenous CR-1 , thus confirming the specificity of the result (Fig. 1 B). Finally, treatment of mpc with anti-CR-1 antibodies slightly reduced proliferation of mpcells (Fig. 4B). Data were confirmed using CR-1- siRNA, which resulted in reduction of proliferation of about 15% compared to control cells (Fig. 1 D). Finally, according to a functional role of CR-1 on myogenic cell proliferation, mpc exhibited a higher level of MyoD expression, a marker of proliferating myogenic cells, upon treatment with recombinant CR-1 (100ng/ml) for 24h, as shown by immunofluorescence analysis (Fig 1 C).

4. CR-1 induces chemoattraction from myogenic precursor cells in vitro

To achieve efficient regeneration, satellite cells have to proliferate and migrate towards the fibers; this prompted us to evaluate whether CR-1 might also exert a pro-migratory effect on mpc. To this end, proliferating mpc were serum starved and migration assay was performed in Boyden chambers. A dose-dependent increase of mpc migration was observed using increasing amount of recombinant CR-1 (50-200 ng/ml; p< 0.05; Fig. 2A). Confirming the specificity of the pro-migratory effect of CR-1 , the chemoattractive effect of exogenous CR-1 was almost completely abolished upon addition of anti-CR-1 antibodies in the lower chamber (Fig.2B). Complete growth medium (see Materials and Methods) was used as control.

5. In vivo CR-1 over-expression enhances and accelerates skeletal muscle regeneration after injury The observation that CR-1 was re-expressed during regenerative process, as well as the ability of CR-1 to promote both mpc proliferation and migration, prompted us to investigate whether CR-1 might affect regeneration of skeletal muscles in vivo, as well as to determine its function in the regeneration process. To this end, Ad-CR-1-IRES-GFP encoding replication-deficient adenovirus was used to overexpress CR-1 in the skeletal muscles after both hind limb ischemia or snake venoms injections (cardiotoxin - CTX) in gastrocnemius (GC) and tibialis anterior (TA) muscles (Arsic N et a/ (2004) MoI Ther. 10, 844-854; Arnold L et al (2007) J Exp Med 204, 1057-1069; Yan Z et al (2003) The Journal of Biol. Chemistry 278, 8826-8836). Muscles analysis was performed after 4, 8, 15 days of regeneration. The virus encoded the secreted form of CR-1 (CR-1 -His). We first verified CR-1 protein expression by ELISA on plasma samples. We observed CR-1 expression (5ng/ml) in plasma from injured-muscles injected with Ad-CR-1 but not with the control Ad-RRV. This expression was present, as early as 6 hours after virus injection, which decreased to reach a minimal constant level of 1 ng/ml after 6 days.

Four days after CTX injection, an increased percentage of the regenerative area was observed compared to control Ad-RRV treated animals (respectively 24.29 ± 2.24 vs 8.6% ± 4.32 from total area, p<0005). Accordingly, CR-1-overexpressing muscles also exhibited a smaller damaged area (58.50% ± 6.66 vs 84.1% ± 4.5; p<0.02) as well as more viable fibers (17.2% ±

4.14 vs 5.52% ± 3.23; p=ns) (Fig. 4A). Comparable results were obtained using limb ischemia as model system; indeed a higher level of regeneration was observed in over-expressing muscles 7 days after limb ischemia (30.54% ± 4.09 vs 5.59% ± 4.08, p<0.05) (Fig. 4B). All together our data indicated that CR-1 over-expression improves the regeneration process probably promoting proliferation and migration of myoblast cells.

6. In vivo CR-1 over-expression induces cells proliferation

To assess whether CR-1 overexpression might induce proliferation in vivo, we used the CTX model (Arsic N et a/ (2004) MoI Ther. 10, 844-854). CR-1 overexpression resulted in increased cell proliferation, as assessed by BrdU staining; indeed, the numbers of BrdU positive cells was almost doubled in CR-1 overexpressing muscles compared to control muscles, 3229 cells ± 208.73 vs 1580.75 cells ± 334.67, respectively (p<0.02; Fig. 4C). Furthermore, we found that most of the BrdU positive cells were also CR-1 positive or closed to CR-1 positive cells, as assessed by double staining with anti-CR-1 and anti-BrdU antibodies (Fig. 4D).

7. In vivo, CR-1 overexpression enhances angiogenesis during skeletal muscle regeneration after injury and reduces fibrosis.

The success of an efficient regeneration actively depends from blood supply, and blood- originated cells as macrophages, which are helpful to remove necrotic debris and secrete anti- apoptotic factors for myogenic cells. We evaluated the effect of CR-1 overexpression on muscle vascularisation in both limb ischemia and CTX models. Interestingly, CR-1 overexpression resulted in increased angiogenesis as global surface occupied by capillaries was doubled in muscles treated with Ad-CR-1 , compared to control virus (0.66% ± 0.2 versus 0.31% ± 0.11 ) (Fig. 4E). This was due to an increased capillaries density in CR-1 treated muscles of 1.6 times (87.32 ± 4.5 vs 53.8± 7.98, p<0.02) (Fig. 4F) which, as a consequence, enhanced the total muscle perfusion (9018.7 ± 1138 vs 4652.75 ± 713.5, p<0.02) (Fig. 4G). Comparable results were obtained using both MLI and CTX model.

The main limitation from efficient regeneration notably in muscle diseases such as muscular dystrophies is the replacement after many cycles of degeneration-regeneration from functional muscle tissue by fat and/or fibrosis, mainly due to the maintenance in situ of inflammatory macrophages. Fifteen days after CTX injection, muscles treated with Ad-CRI showed a reduction of the surface occupied by fibrosis. Indeed, the area of fibrosis from muscles treated with the control adenovirus RRV represented 3.09% ± 0.48 from total area compared to 1.95% ± 0.27 for Ad-CR- 1 treated muscles (p<0.05) (Fig. 5). All together our data indicated that CR-1 overexpression contributed to enhance muscle regeneration via mechanisms gathering favorable conditions such as proliferation and migration of myogenic cells, better perfusion from the muscle tissue and importantly also the reduction of fibrosis.

8. Production of recombinant cripto protein

The recombinant cripto-His-tag protein can be purified from the conditioned medium of 293 cells which are stably transfected with a recombinant expression vector: pCDNA3-cripto-His- tag as previously described in Minchiotti G. et al (2001 ) Development 128, 4501-10. Briefly, conditioned medium from serum-starved 293 cripto-His cell line is collected and the cripto-His protein is purified using Nickel-affinity chromatography (the Qiaexpress system from Qiagen can be conveniently used). The purified protein is dialysed against 50 mM Sodium Phoshate buffer pH 8.0.

9. Treatment of a murine model for human motoneuron disease with recombinant cripto

The progressive motor neuronopathy (pmn) mutant mouse is an accepted animal model in the art for human motoneuron disease (Schmalbruch H et al (1991 ) J Neuropathol Exp Neurol 50(3): 192-204. Mice that are homozygous for the pmn gene defect appear healthy at birth but develop progressive motoneuron disease, resulting in severe skeletal muscle weakness and respiratory failure by postnatal week 3. In one experiment recombinant cripto-His protein is systemically delivered using an osmotic mini-pump inserted subcutaneously and in another experiment 1 , 10 and 50 μg cripto is delivered by local injection directly into the muscles.

10. Treatment of a murine model for skeletal muscle dystrophy with recombinant cripto

A mutant mouse model with an X-chromosome-linked muscular dystrophy (mdx) is a reliable animal model that mimics the human Duchenne muscular dystrophy (Tanabe Y et al (1986) Acta Neuropathol (Berl) 69(1-2):91-5) mainly because of the similar histological features. This is a strain of mice arising from a spontaneous mutation (mdx) in inbred C57BL mice. This mutation is X-chromosome-linked and produces viable homozygous animals that lack the muscle protein dystrophin, have high serum levels of muscle enzymes, and possess histological lesions similar to human muscular dystrophy. In one experiment recombinant cripto-His protein is systemically delivered using an osmotic mini-pump inserted subcutaneously and in another experiment 1 , 10 and 50 μg cripto is delivered by local injection directly into the muscles. 1 1. Treatment of a murine model for progressive muscular dystrophy with recombinant cripto Alpha-sarcoglycan deficient mice develop progressive muscular dystrophy and, in contrast to other animal models for muscular dystrophy, show ongoing muscle necrosis with age, a hallmark of the human disease (Duclos F et al (1998) J. Cell Biol. 142(6):1461-71 ). In one experiment recombinant cripto-His protein is systemically delivered using an osmotic mini- pump inserted subcutaneously and in another experiment 1 , 10 and 50 μg cripto is delivered by local injection directly into the muscles.

MATERIAL AND METHODS 1. Muscle injury and muscle preparation

For CTX mouse models, 10μl cardiotoxin (10^"3M I in PBS; Latoxan) was injected in the tibialis anterior (TA) and 15μl in the gastrocnemius (GC). Hind limb ischemia mouse models, histology and morphometric quantification were all performed as described (Luttin A et al (2002) Nat. Med. 8, 831-840). Limb ischemia was induced by unilateral right or bilateral ligation of the femoral artery and vein, and of the cutaneous vessels branching from the caudal femoral artery, sparing the femoral nerve. Gastrocnemius muscles were harvested 7 days after femoral artery ligation, and sections were analyzed after H&E staining or immunostaining for the EC marker CD31 (rat anti- CD31 ; BD Biosciences — Pharmingen). Vessel densities and tissue necrosis/regeneration were morphometrically analyzed using KS300 image analysis software (Carl Zeiss Inc.). Fibrosis was evaluated by quantitative digital analysis of connective tissue staining. Collagen was revealed by picrosirius red staining as previously described (Morrison J. et al (2005) The American Journal of Pathology 166, 1701-1710). Briefly, sections were fixed in neutral buffered formalin for 10 minutes at room temperature. Sections were washed with distilled water and incubated with Fast Blue [0.15% Fast Blue RR Salt (no. F0500, Sigma) in magnesium borate buffer (0.17 g MgSO4.7 H2O and 0.38 g NaBO2.4H2O in 100 ml of distilled water) and filtered through a 22-just before use] for 10 minutes at room temperature. Sections were then rinsed in distilled water and incubated with picrosirius red stain [0.1% Sirius Red F3B (no C. I. 35780; Lamb) in saturated picric acid (no. 10192; BDH) for 10 minutes at room temperature. Sections were rinsed thoroughly in distilled water before a rinse for 1 minute at room temperature in picric-alcohol (20 ml of absolute alcohol, 70 ml of distilled water, and 10 ml of saturated picric acid). Sections were counterstained in hematoxylin for 5 minutes and subsequently washed with tap water for a further 5 minutes. Sections were dehydrated with alcohol and mounted with DPX. Fibrosis was analyzed using KS300 image analysis software.

2. Intramuscular administration of Adenovirus

In the hind-limb ischemia mouse model, the recombinant vector solution (30μl; 8 x 10⁹ Adeno- CR-1/His or AdenoRRV vector particles) was injected into the gastrocnemius muscles (3 x 10μl injections per muscle) and muscles were harvested 7 days after ligation. The normoperfused muscles or the muscles treated cardiotoxin were injected with Adeno-CR-1/His or Adeno-control in a total volume of 25 μl and in two distinct injection sites. Muscles were harvested 4, 8, 15 and 30 days after damage according to the different experiments, either embedded unfixed in tissue-tek and frozen in liquid nitrogen fro cryosection or fixed in 2% formaldehyde, and embedded in paraffin. All the experiments were performed in groups including six animals.

3. Preparation of Primary Cultures

The forelimbs and hindlimbs were removed from neonatal mice (2-5 d old) and the bones were dissected away. The remaining muscle mass was weighed. A few drops of PBS were added and the muscle was minced into a coarse slurry using razor blades. Cells were enzymatically dissociated by the addition of 2 ml per g of tissue of a solution of dispase (grade II, 2.4U/ml, Roche) and collagenase B (1 %; Roche), supplemented with CaC12 to a final concentration of 2.5 mM. The slurry, maintained at 37°C for 30-45min, was triturated every 15 min with a 5-ml plastic pipette, and then passed through 80 mm nylon mesh (Nitex; Tetko, Inc., Monterey Park, CA). The filtrate was spun at 350 g to sediment the dissociated cells, the pellet was resuspended in growth medium, and the suspension was plated on collagen-coated dishes. During the first several passages of the primary cultures, myoblasts were enriched using a modified version of a previously described preplate technique (Richler and Yaffe (1970) Dev. Biol. 23(1 ): 1-22. All experiments were done on cells from preplating step 6 (pp6 cells).

4. Cell Culture, Growth Factors and Antibodies Pp6 myogenic cells were grown in growth medium consisting of DMEM-Glutamax-I (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 10% Horse serum, 1% Chick embryo extract, 1% penicillin-streptomycin Insulin-Transferin-Selenium X100, bFGF 1 ng/ml, IGF-I, 5 ng/ml (all reagents purchased from GIBCO BRL). For all the experiments, pp6 were used between passage 5 and 15. A murin recombinant CR-1 protein containing an His- tag was either expressed in 293 cells and purified, as previously described in Minchiotti G et al (2001 ) Development 128, 4501-4510 or purchased from R&D. Basic fibroblast growth factor (bFGF), insulin-like growth factor-l (IGF-I) proteins were purchased from R&D Systems (Minneapolis, MN). The blocking antibodies anti-CR-1 , anti-ALK4 and TGFR1 were purchased from R&D system.

5. lmmunostainings For immunofluorescence procedure, mpc were cultured on glass coverslips and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS). Cells, either not permeabilized or treated with 0.1% Triton X-100 (Sigma) 5' at RT, were blocked with 3% BSA in PBS for 30 min at RT and incubated over night with primary antibodies in 1 % BSA in PBS, at the following working dilutions: rabbit anti-CR-1 (Minchiotti G et al (2001 ) Development 128, 4501-4510); 5 μg/ml), sheep anti-CR-1 (R&D; 5 μg/ml), CD34 (Hycult Biotechnology, HBT; 1 :20), MyoD1 (Dako; 1 :20), Desmin (ICN; 1 :50), M-Cadherin (BD Biosciences; 1 :50). After washing, cells were either incubated with AlexaFluor-conjugated secondary antibodies (Molecular Probes; 1 :200), in 1%BSA, 10% NGS in PBS or with the TSA Fluorescence Amplification System (PerkinElmer), following manufacturer instructions. Following PBS wash, coverslips were mounted on slides with Vectashield medium containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Labeling was visualized by epifluorescent illumination by using an Axiovision microscope, and images were acquired on an Axiocam camera (Zeiss). For the immunodetection of protein expression on muscles, staining was performed using the TSA (Tyramide Signal Amplification) Fluorescence System (PerkinElmer), following manufacturer instructions. Muscle sections were incubated overnight at 4°C in a humidified chamber in 10% normal serum in TNB Blocking buffer with primary antibodies at the following working dilutions: anti-CR-1 (Minchiotti et al., 2001 ; 7μg/ml), CD31 (Mec13.3 BD-Pharmingen; 1/500), F4/80 (Serotec; 1/50), Desmin (ICN; 1 :50). Sections incubated without primary antibodies served as controls for the non-specific binding of secondary antibodies.

6. Cell Proliferation Assay

The day before experimentation, pp6 cells were cultured at 5.10⁴ cells per well on 96-well microtiter plates in growth medium for few hours and then serum starved overnight in DMEM with 5% FBS. The day after, cells were washed twice with phosphate-buffered saline (PBS), and then cultured in DMEM-FBS-5% medium without supplements but containing recombinant mouse CR-1 (5, 50, 100, 250, 500, 1000 ng/mL, R&D system and home made by 293 cells production), human bFGF (10 ng/mL) or human IGF-I (50ng/ml, Promocell), human TGF-β (100ng/ml, R&D system) or neutralizing antibodies at 4ug/ml (anti-CR1 , MAB1538, R&D system; anti-Alk4, AF1477, R&D system; anti-TGF-β, MAB1835, R&D system, anti-T Rl, sc- 33933, Santa-Cruz). These concentrations of CR-1 and bFGF have been previously shown to stimulate proliferation of epithelial or endothelial cells and myogenic cells. In some experiments, antibodies were replaced by specific Silencer Pre-designed siRNA (Ambion Europe, Cambridge, UK) and used with lipid transfection agent, according to manufacturer instructions. To measure cell proliferation, we used the BrdU cell proliferation assay kit (Roche, Indianapolis, IN) based on incorporation of bromodeoxy-uridine in replicative viable cells. Cultures were incubated for 24 hours at 37°C with medium containing recombinant proteins and/or antibodies, BrdU (10 μl_ per well, dilution 1 :10) was added, and cultures were incubated for 24 hours at 37 ⁰C. The BrdU incorporation was revealed according to the manufacturer's instructions and measured by the absorbance of the samples in an ELISA reader at 370 nm (reference wavelength: approx. 492 nm). The experiment was repeated five times, and the samples were tested in triplicate.

7. Migration Assays

Cell migration assays, were performed in Boyden chambers (Cell Migration Assay Kit, Chemicon, Temecula, CA). Dulbecco's modified Eagle medium (DMEM) containing 20% fetal bovine serum was used in the lower Boyden chamber as positive control for migration. Mpc were serum starved in DMEM medium containing 0.5% bovine serum overnight, harvested by trypsinization, and resuspended in DMEM containing 0.5% bovine serum. We placed 6.5 10⁴ cells in the upper chamber with the components indicated at the following concentrations: recombinant CR-1 protein at 200ng/ml_, anti-CR-1 mAb 4μg/ml_. The Boyden chambers were incubated 4h to 12h at 37°C. Cells on the topside of the filter were removed, and cells that had migrated through the filter and attached to the bottom of the membrane were stained with crystal violet stain solution (Chemicon). The crystal violet stain solution was eluted with 10% acetic acid extraction buffer (Chemicon) and transferred to wells of a 96-multiwell plate, and the absorbance was read at 595nm in each well. Experiments were repeated three times with triplicate samples.

8. ELISA assay

96 wells plates were coated with 0.5ng/ml of in house preparation of anti-CR-1 antibodies in PBS 1x pH 7.5 overnight at 4°C and washed three times with PBS-Tween (PBT). Unbinding sites were blocked with PBS-BSA 1 % (180μl/well) for 2h RT. After washing three times, the mouse sera (100μl) were added and incubated overnight at 4°C. Plates were then incubated with 1 μg/ml of biotinylated anti-CR-1 antibodies (R&D) in PBE-T 1 hr at 37°C and 1 hr at RT. Following incubation with avidin/streptavidin complex horse-radish peroxidase -conjugated (Vectastain elite ABC kit) for 1 h at RT, plates were developed with OPD (o-phenylene-diamine- peroxidase) substrate and the absorbance was read at 490 nm on a Benchmark microplate reader.

9. RT-PCR

Total RNA from the cells was extracted with TRIZOL Kit (Life Technologies) according to the manufacturer's instructions and reverse transcribed to cDNA with QuantiTect Reverse

Transcription Kit (QIAGEN). cDNA samples synthesized from 1 μg of total RNA were subjected to PCR amplification with specific primers during 35 cycles. (CR-1 first PCR 33 cycles at 58°C, nested PCR 35 cycles at 50⁰C, ALK5, 35 cycles at 54°C; AIkI , TβRII, TGF-β Glypican-1 , 35 cycles at 60⁰C and Alk4, 33 cycles at 60⁰C) (Table 1 ).

Table 1. Primers sequences

Claims

Claims

1. Use of SEQ ID NO: 1 or 3 or a variant sequence thereof encoding a functional derivative of SEQ ID NO: 2 or 4 or a fragment thereof encoding a functional fragment of SEQ ID NO: 2 or 4 or a homologue encoding a sequence with at least 90% identity with

SEQ ID NO: 2 or 4 for the manufacture of a medicament to treat skeletal muscle wasting disorders.

2. Use of SEQ ID NO: 2 or 4 or a functional derivative or a functional fragment or a homologue with at least 90% identity with SEQ ID NO: 2 or 4 for the manufacture of a medicament to treat skeletal muscle wasting disorders.

3. Use according to claims 1-2 wherein said skeletal muscle wasting is due to an ischemic insult.

4. Use according to claims 1-2 wherein said skeletal muscle wasting is due to a prolonged immobilization.

5. Use according to claims 1-2 wherein said skeletal muscle wasting is due to muscle denervation.

6. Use according to claims 1-2 wherein said skeletal muscle wasting is due to a muscle dystrophy.

7. Use according to claims 1-2 wherein said skeletal muscle wasting is due a reduced glycolytic rate.