WO2010129406A2 - Methods of promoting muscle growth - Google Patents

Abstract

The present invention relates to use of the interaction of myostatin with the activin type II receptors, and more specifically to use of soluble ACVR2 to increase muscle growth in a subject. As such, methods of increasing muscle mass in a subject are provided. In addition, methods of ameliorating the severity of a pathologic condition characterized, at least in part, by a decreased amount, development or metabolic activity of muscle are provided.

Description

METHODS OF PROMOTING MUSCLE GROWTH

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0001] The invention relates generally to the interaction of myostatin with the activin type II receptors, and more specifically to use of soluble ACVR2 to increase muscle growth in a subject.

BACKGROUND INFORMATION

[0002] Growth and differentiation factor-8 (GDF-8), also known as myostatin, is a member of the transforming growth factor-beta (TGF-β) superfamily of structurally related growth factors, all of which possess important physiological growth-regulatory and morphogenetic properties. GDF-8 is a negative regulator of skeletal muscle mass, and there is considerable interest in identifying factors which regulate its biological activity. For example, GDF-8 is highly expressed in the developing and adult skeletal muscle. The GDF-8 null mutation in transgenic mice is characterized by a marked hypertrophy and hyperplasia of the skeletal muscle. Similar increases in skeletal muscle mass are evident in naturally occurring mutations of GDF-8 in cattle.

[0003] The proteins of the TGF-β family are initially synthesized as a large precursor protein which subsequently undergoes proteolytic cleavage at a cluster of basic residues approximately 110-140 amino acids from the C-terminus. The C-terminal regions, or mature regions, of the proteins are all structurally related and the different family members can be classified into distinct subgroups based on the extent of their homology. Although the homologies within particular subgroups range from 70% to 90% amino acid sequence identity, the homologies between subgroups are significantly lower, generally ranging from only 20% to 50%. In each case, the active species appears to be a disulfϊde-linked dimer of C-terminal fragments. Studies have shown that when the pro-region of a member of the TGF-β family is co-expressed with a mature region of another member of the TGF-β family, intracellular dimerization and secretion of biologically active homodimers occur.

[0004] A number of human and animal disorders are associated with loss of or functionally impaired muscle tissue. Recent studies have also shown that muscle wasting associated with HIV-infection in humans is accompanied by increases in GDF-8 protein expression. To date, very few reliable or effective therapies exist for these disorders. However, the terrible symptoms associated with these disorders may be substantially reduced by employing therapies that increase the amount of muscle tissue in patients suffering from the disorders. While not curing the conditions, such therapies would significantly improve the quality of life for these patients and could ameliorate some of the effects of these diseases. Thus, there is a need in the art to identify new therapies that may contribute to an overall increase in muscle tissue in patients suffering from these disorders.

SUMMARY OF THE INVENTION

[0005] The present invention is based on the seminal discovery that myostatin (GDF-8) may not be the sole regulator of muscle mass, and thus the capacity for increasing muscle growth by manipulating TGF-β signaling pathways may be more extensive than previously appreciated. Accordingly, the present invention relates to methods of increasing muscle tissue growth in a subject by administering to the subject a therapeutically effective amount of a soluble Activin Type II Receptor (ACVR2) polypeptide. The method may further include comparing an increase in muscle tissue growth in the subject to muscle tissue growth of a corresponding subject treated with a specific inhibitor of myostatin expression or activity.

[0006] In another aspect, the invention provides a method of increasing the growth of muscle tissue in a subject by administering to the subject a therapeutically effective amount of an inhibitor of myostatin expression or activity in combination with a therapeutically effective amount of a soluble Activin Type II Receptor (ACVR2) polypeptide either prior to, simultaneously with or following the inhibitor of myostatin expression or activity, thereby increasing muscle tissue growth in the subject.

[0007] In another aspect, the invention provides a method of ameliorating the severity of a pathologic condition characterized, at least in part, by a decreased amount, development or metabolic activity of muscle. The method includes contacting a muscle cell of a subject in need thereof with a soluble Activin Type II Receptor (ACVR2) polypeptide or a polynucleotide encoding the ACVR2. In one embodiment, the method further includes comparing an increase in muscle tissue growth in the subject to muscle tissue growth of a corresponding subject treated with a specific inhibitor of myostatin expression or activity. In one embodiment, the pathological condition is a wasting disorder such as cachexia, anorexia, muscular dystrophy or a neuromuscular disease. In another embodiment, the pathological condition is a metabolic disorder such as obesity or type II diabetes.

[0008] In another aspect, the invention provides a method of ameliorating the severity of a pathologic condition characterized, at least in part, by a decreased amount, development or metabolic activity of muscle in a subject. The method includes contacting a muscle cell of a subject in need thereof with an inhibitor of myostatin expression or activity in combination with a soluble Activin Type II Receptor (ACVR2) polypeptide either prior to, simultaneous with or following the inhibitor of myostatin expression or activity. In one embodiment, the method further includes comparing an increase in muscle tissue growth in the subject to muscle tissue growth of a corresponding subject treated with a specific inhibitor of myostatin expression or activity. In one embodiment, the pathological condition is a wasting disorder such as cachexia, anorexia, muscular dystrophy or a neuromuscular disease. In another embodiment, the pathological condition is a metabolic disorder such as obesity or type II diabetes.

[0009] In various embodiments, the subject is a mammal such as ovine, porcine, bovine, murine or human. As such, the invention further provides a transgenic non-human mammal whose genome contains a nucleic acid sequence comprising a soluble Activin Type II Receptor (ACVR2) polypeptide and a regulatory element. In one embodiment, the regulatory element includes a muscle-specific promoter operably linked and integrated into the genome of the mammal, wherein the nucleic acid sequence is expressed so as to result in elevated levels of ACVR2 and an increase in muscle mass in the mammal as compared to a corresponding mammal whose genome contains a myostatin-null mutation or a decreased level of myostatin as compared with normal basal levels.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is a graphical diagram showing the effects of heterozygous loss of Fst on muscle mass and function. Percent change in muscle weights due to heterozygous loss of Fst. Bottom panel shows percent decrease in muscle weights in Fst ^+/~ mice compared to wild type mice. Middle panel shows percent decrease in muscle weights in Fst ^+/~, Mstn ^+A mice compared to Fst ^+/+, Mstn ^+/~ mice. Top panel shows percent decrease in muscle weights in Fst ^+/', Mstn ^"A mice compared to Fst ^+/+, Mstn ^~f~ mice. All calculations were made from the data shown in Table 1. Muscles analyzed were: pectoralis (P), triceps (T), quadriceps (Q), and gastrocnemius (G).

[0011] Figure 2 is a graphical diagram showing the effects of mutations in genes encoding inhibin-β subunits. Numbers represent percent increase or decrease in muscle mass relative to wild type mice and were calculated from the data shown in Table 1. Data from Mstn ^+/~ mice (Lee, 2007) are shown for comparison. In the graphs shown in (b) and (d), muscles analyzed were: pectoralis (P), triceps (T), quadriceps (Q), and gastrocnemius (G).

[0012] Figure 3 is a graphical diagram showing the effect of administering purified ACVR2/Fc to wild type adult mice. Mice were given weekly injections at the indicated doses for a period of 4 weeks. Percent increases in muscle weights relative to vehicle (PBS) are shown for four muscles: pectoralis (P), triceps (T), quadriceps (Q), and gastrocnemius (G).

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention is based on the discovery that myostatin (GDF-8) may not be the sole regulator of muscle mass, and thus the capacity for increasing muscle growth by manipulating TGF-β signaling pathways may be more extensive than previously appreciated.

[0014] Myostatin is a secreted protein that normally acts to suppress muscle growth (for review, see Lee, 2004). Myostatin is expressed almost exclusively in the skeletal muscle lineage both during embryonic development and in adult animals, and mice engineered to lack myostatin exhibit dramatic and widespread increases in muscle mass, with individual muscles weighing approximately twice as much as those of control animals. All skeletal muscles throughout the body appear to be affected by the mutation, and the increase in muscle mass in these mice results from a combination of both increased fiber number and increased fiber size. These findings suggested that myostatin normally functions as a negative regulator of muscle growth and raised the possibility that inhibition of myostatin activity may be useful for increasing muscle mass for both human therapeutic and agricultural applications. In this respect, the myostatin gene has been extraordinarily well conserved through evolution, with the sequence of the active portion of the molecule being identical among most mammalian species (McPherron and Lee, 1997). The function of myostatin also appears to have been conserved, as naturally-occurring mutations in the myostatin gene have been shown to be responsible for increased muscling in cattle (Grobet et al, 1997, 1998; Kambadur et al, 1997; McPherron and Lee, 1997), sheep (Clop et al, 2006), dogs (Mosher et al, 2007), and humans (Schuelke et al, 2004).

[0015] Like other TGF-β family members, myostatin is synthesized as a precursor protein that undergoes proteolytic processing to generate an N-terminal propeptide and a 12.5 kDa C- terminal fragment. It is a disulfide-linked dimer of C-terminal fragments that is the biologically active species (see U.S. Pat. No. 5,827,733, incorporated herein by reference). Following proteolytic processing, the propeptide and C-terminal dimer remains bound together non-covalently, and in this complex, the propeptide maintains the C-terminal dimer in an inactive, latent state (Lee & McPherron, 2001; Thies et al, 2001; Wolfman et al, 2003). The inhibitory activity of the propeptide on the myostatin C-terminal dimer has been documented extensively both in vitro and in vivo (see U.S. Pat. No. 6,891,082, incorporated herein by reference). Specifically, the purified propeptide can block the activity of the purified myostatin C-terminal dimer in both receptor binding and reporter gene assays (Lee & McPherron, 2001; Thies et al, 2001), and transgenic mice overexpressing the propeptide in muscle phenocopy Mstn knockout mice in terms of increased muscle mass (Lee & McPherron, 2001; Yang et al, 2001). The latent complex of the propeptide and the C- terminal dimer also appears to exist in vivo. Myostatin has been shown to circulate in the blood in a latent form that can be activated by acid treatment (Zimmers et al, 2002) and biochemical studies have shown that at least one protein normally complexed to myostatin in the blood is the propeptide (Hill et a/., 2002).

[0016] Promyostatin polypeptides have been identified in mammalian, avian and piscine species, and myostatin is active in various other species, including vertebrates and invertebrates. During embryonic development and in adult animals, myostatin, for example, is expressed specifically by cells in the myogenic lineage (McPherron et al., Nature 387:83- 90, 1997, which is incorporated herein by reference). During early embryogenesis, myostatin is expressed by cells in the myotome compartment of developing somites. At later embryonic stages and in adult animals, myostatin is expressed widely in skeletal muscle tissue, although the levels of expression vary considerably from muscle to muscle. Myostatin expression also is detected in adipose tissue, although at lower levels than in muscle. [0017] Based on a variety of studies, it appears that the primary mechanism by which this latent complex is activated in vivo is by proteolytic cleavage of the propeptide by members of the BMP-1/tolloid family of metalloproteases (see U.S. Pat. No. 7,572,599, incorporated herein by reference). Each of the four proteases in this family (BMP-I, TLD, TLL-I, and TLL-2) is capable of cleaving the myostatin propeptide immediately N-terminal to aspartate residue 76 in vitro, thereby activating the MSTN latent complex (Wolfman at al, 2003). Moreover, mice either carrying a point mutation (D76A) rendering the propeptide resistant to proteolysis or carrying inactivating mutations in the TlU gene exhibit significant increases in muscle mass, consistent with a critical role for these proteases in activating latent myostatin in vivo (Lee, 2008).

[0018] In addition to the propeptide, several other proteins have also been shown to be capable of binding and inhibiting the activity of the myostatin C-terminal dimer. One of these is follistatin, which is known to be capable of binding a number of other TGF-β family members as well (Nakamura et al, 1990; Yamashita et al, 1995; de Winter et al, 1996; Fainsod et al, 1997; Iemura et al, 1998; and U.S. Pat. No. 6,004,937, incorporated herein by reference). A number of studies suggest that follistatin can function as a potent myostatin antagonist and plays an important role in modulating myostatin activity in vivo. First, follistatin has been shown to be capable of blocking myostatin activity in both receptor binding and reporter gene assays in vitro (Lee & McPherron, 2001; Zimmers et al, 2002) as well as in nude mice implanted with myostatin expressing cells in vivo (Zimmers at al, 2002). Second, transgenic mice overexpressing follistatin in muscle exhibit dramatic increases in muscle growth, consistent with inhibition of myostatin activity (Lee & McPherron, 2001), and, conversely, mice homozygous for a deletion of follistatin gene exhibit reduced muscle mass at birth (Matzuk et al, 1995b), which is what one might expect for excess myostatin signaling. Three other proteins, follistatin-like related gene (FLRG), GDF-associated serum protein- 1 (GASP-I)₃ and GDF-associated serum protein-2 (GASP-2), also appear to be involved in regulating the activity of the myostatin C-terminal dimer extracellularly (Hill et al, 2002, 2003; Kondas et al, 2008; and WO2008/109779, incorporated herein by reference). Both FLRG and GASP-I have been shown to be complexed to myostatin in the blood of both mice and humans, and studies with recombinant proteins have shown that both of these proteins can bind with high affinity to the myostatin C-terminal dimer and inhibit its activity, as assessed by reporter gene assays. FLRG was originally identified as a follistatin-related gene present at a chromosomal translocation breakpoint in a patient with B-cell chronic lymphocytic leukemia and was shown to be capable of blocking the activity of the TGF-β family members' activin and BMP-2 in in vitro assays (Hayette et al, 1998; Tsuchida et al, 2000). GASP-I and GASP-2 are novel proteins that also contain a follistatin-related domain but in addition contain multiple domains found in proteinase inhibitors.

[0019] Once activated from the latent complex, the myostatin C-terminal dimer is capable of signaling to target cells. Like other TGF-β family members (for review, see Massague, 1998), myostatin is believed to signal by binding to both type I and type II serine/threonine kinase receptors, thereby activating Smad proteins. A variety of evidence suggests that the initial signaling event is the binding of myostatin to the activin type II receptors, ACVR2 and ACVR2B (see, for example, U.S. Patent No. 5,885,794, which is incorporated herein by reference). Specifically, the myostatin C-terminal dimer is capable of binding both ACVR2 and ACVR2B in vitro, and transgenic mice expressing a dominant negative form of ACVR2B in muscle have dramatic increases in muscle mass comparable to those seen in myostatin knockout mice (Lee and McPherron, 2001). Furthermore, studies with mice carrying inactivating mutations Acvr2 and Acvr2b genes have shown that loss of either receptor leads to increases in muscle mass and that these two receptors are functionally redundant with respect to regulating muscle growth (Lee et al, 2005). Taken together, these data provide strong evidence that both of these type II receptors mediate myostatin signaling in vivo.

[0020] There is less information regarding the identity of the type I receptors that mediate myostatin signaling, although cross-linking studies with cells co-transfected with ACVR2B and individual type I receptor expression constructs have shown that myostatin is capable of binding two type I receptors, ALK-4 and ALK-5 (Rebbapragada, et al, 2003). Following receptor binding, intracellular signaling appears to involve activation of Smad proteins. Treatment of cells in culture with purified myostatin protein has been shown to cause increased levels of both phospho-Smad2 and phospho-Smad3 as well as activation of Smad2/Smad3 -responsive reporter genes (Thies, et al. 2001; Langley, et al. 2002; Rebbapragada, et al. 2003). Moreover, there are considerable genetic data showing that the nuclear protein c-ski, which is capable of interacting with and blocking the activity of Smad 2, 3, and 4 (Luo, et al. 1999; Stroschein, et al. 1999; Sun, et al. 1999a and b; Akiyoshi, et al. 1999), is a potent regulator of muscle growth. Mice lacking c-ski have been shown to have a severe reduction in skeletal muscle mass (Berk, et al. 1997), and transgenic mice overexpressing c-ski in muscle have been shown to have dramatic muscle hypertrophy (Sutrave, et al. 1990). The simplest interpretation of these data is that c-ski normally functions to block myostatin signaling in vivo by blocking functions of Smad proteins activated by myostatin.

[0021] Based on what is known about how myostatin activity is regulated and how myostatin signals to target cells, a number of myostatin inhibitors have been developed that are capable of inducing significant muscle growth when administered to wild type adult mice. One of these is a myostatin neutralizing monoclonal antibody (JA16). Weekly injections of this antibody over a period of 12 weeks to wild type mice have been shown to be capable of increasing muscle mass by about 25% (Whittemore et al., 2003).

[0022] A second myostatin inhibitor capable of causing muscle growth in vivo is a mutant form of the myostatin propeptide resistant to proteolysis by members of the BMP-1/tolloid family. This mutant propeptide appears to be more potent than the JA 16 neutralizing antibody, as comparable effects on muscle mass can be achieved at lower doses administered over a shorter time period (Wolfman, et al., 2003).

[0023] Finally, an even more potent inhibitor, namely a soluble form of the ACVR2B receptor (AC VR2B/Fc), has been shown to capable of inducing muscle growth by 40-60% following just two injections of the purified protein over a span of two weeks (Lee et al, 2005). Taken together, these studies examining the effects of administering myostatin inhibitors to mice demonstrate conclusively that MSTN plays a crucial role in regulating muscle growth in adult mice and suggest that targeting this signaling pathway could be an effective strategy for increasing muscle growth in a variety of clinical settings. Indeed, myostatin inhibitors have been shown to have beneficial effects when administered to dystrophic mice (Bogdanovich et al., 2002, 2005), and in this regard, at least two myostatin inhibitors, namely, a humanized monoclonal antibody (MYO-029) directed against myostatin and the soluble form of the ACVR2B receptor, either have been (Wagner et al, 2008) or are currently being tested in clinical trials in patients with muscular dystrophy. [0024] In the course of conducting these experiments examining the effects of various myostatin inhibitors in vivo, it has been noted in two sets of experiments that the increases in muscle mass that were observed seemed to be greater than could be accounted for simply as a result of inhibition of myostatin activity.

[0025] One set of experiments was the analysis of transgenic mice overexpressing follistatin. Follistatin (FST) is a protein capable of acting as a potent myostatin antagonist. Specifically, follistatin has been shown to be capable of binding directly to myostatin and inhibiting its activity in receptor binding and reporter gene assays in vitro (Lee and McPherron, 2001; Zimmers et al, 2002; Amthor et al, 2004). Moreover, follistatin also appears to be capable of blocking endogenous myostatin activity in vivo, as transgenic mice overexpressing follistatin specifically in skeletal muscle have been shown to exhibit dramatic increases in muscle growth comparable to those seen in Mstn knockout mice (Lee and McPherron, 2001; Lee, 2007; Haidet et al, 2008). Finally, mice homozygous for a targeted mutation in the Fst gene have reduced muscle mass at birth (Matzuk et al, 1995b), consistent with a role for follistatin in inhibiting myostatin activity during embryonic development. Because mice homozygous for a deletion of Fst gene die immediately after birth and because many components of the myostatin regulatory system have shown dose-dependent effects when manipulated in vivo, the possibility that the Fst mutation might exhibit haplo- insufficiency with respect to muscle growth and function was investigated. The Fst loss-of- function mutation was backcrossed at least 10 times onto a C57BL/6 background and then muscle weights in Fst^+/~ mice were analyzed at 10 weeks of age. As shown in Table 1 and Figure 1 (bottom panel), Fst^+/~ mice exhibited a clear muscle phenotype, with muscle weights in Fst^{+ '} mice being reduced by about 15-20% compared to those of wild type mice. These reductions in weights were highly statistically significant (p values ranged from 10^" to 10^" ) and were seen in all four muscles that were analyzed (pectoralis, triceps, quadriceps, and gastrocnemius) as well as in both males and females.

[0026] In order to determine whether the effects seen in Fst^+/~ mice resulted from loss of inhibition of myostatin activity, the genetic interactions between Fst and Mstn were examined. The rationale for these studies was that two lines of investigation had demonstrated that other members of the TGF-β family, in addition to myostatin, seem to play important roles in limiting muscle growth. In particular, both overexpression of follistatin as a muscle-specific transgene and systemic administration of a soluble form of one of the known myostatin receptors (ACVR2B) had been shown to cause increases in muscle mass not only in wild type mice but also in Mstri ^' mice, implying that these inhibitors were exerting their effects by targeting other TGF-β family members in addition to myostatin (Lee and McPherron, 2001; Lee et al, 2005; Lee, 2007). Hence, an object of the experimentation was to determine whether the reductions in muscle weights seen in Fst^{+ '} mice resulted entirely from increased levels of myostatin signaling.

[0027] The approach was to look for genetic interactions between Fst and Mstn by examining the effect of introducing the Fst mutation onto a Mstn mutant background. If the sole role for follistatin in regulating muscle mass in vivo is to block myostatin signaling, then the Fst mutation would be predicted to have no effect in the complete absence of myostatin. If, on the other hand, follistatin normally acts to block multiple ligands to regulate muscle mass, then the Fst mutation might be expected to have at least some effect on muscle mass even in a Mstn mutant background. As shown in Table 1 and Figure 1, it was found that the latter is the case. Specifically, heterozygous loss of Fst caused reductions in muscle mass in both Mstn^+/~ and Mstn^A mutant backgrounds in both male and female mice. The effects of the Fst mutation were somewhat attenuated in the complete Mstn null background, implying that part of the effect of follistatin loss in Mstn^+/+ mice likely results from loss of inhibition of myostatin signaling. Nevertheless, the fact that the Fst mutation had at least some effect on muscle weights even in the Mstn null background implies that this residual effect resulted from loss of inhibition of other TGF-β family members in these mutant mice. Hence, these studies suggest that follistatin normally acts in vivo to inhibit multiple TGF-β family members, including myostatin, that function to limit muscle mass.

[0028] As discussed above, administration of ACVR2B/Fc to wild type mice was shown to have greater effects on muscle growth than was observed with other myostatin inhibitors, such as the JA 16 neutralizing monoclonal antibody or the mutant form of the propeptide resistant to proteolysis. The effect of administering ACVR2B/Fc to myostatin knockout mice was therefore examined to determine whether the effect of ACVR2B/Fc in terms of inducing muscle growth resulted entirely from inhibition of myostatin. It was observed that the effects of ACVR2B/Fc were attenuated but not eliminated in mice lacking myostatin; These results further suggest that at least one of the reasons for the enhanced effects observed with ACVR2B/Fc is that this inhibitor is capable of targeting additional ligands besides myostatin.

[0029] These studies demonstrate that myostatin is not the sole ligand that normally acts to suppress muscle growth and that the capacity for enhancing muscle growth by targeting this general signaling pathway is substantially greater than previously appreciated. Accordingly, the invention provides methods of increasing growth of muscle tissue in a subject. In one embodiment, the invention includes administering to the subject a therapeutically effective amount of AVCR2. A greater increase in muscle tissue growth, as compared to the muscle tissue growth of a corresponding subject treated with a specific inhibitor of myostatin (GDF-8) expression or activity is indicative of increasing growth of muscle tissue in the subject as a result of AVCR2 administration. In one embodiment, the ACVR2 is administered as a fusion protein (ACVR2/Fc).

[0030] The term "subject" as used herein refers to any individual or patient to which the subject methods are performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.

[0031] The term "growth" as used herein is used in a relative sense in referring to the mass of muscle tissue or mass of adipose tissue in a subject that has been subjected to a method of the invention as compared to a corresponding subject that has not been subjected to a method of the invention. Thus, where a method of the invention is performed such that AVCR2 has been expressed, it will be recognized that the growth of muscle tissue in the organism would result in an increased muscle mass in the subject as compared to the muscle mass of a corresponding subject in which myostatin signal transduction has been inhibited or reduced.

[0032] Thus, as used herein, the term "modulate," when used in reference to an effect of a ligand that suppresses muscle growth, means that signal transduction in the cell either is increased or is reduced or inhibited. The terms "increase" and "reduce or inhibit" are used in reference to a baseline or basal level of signal transduction activity, which can be the level of activity of the signal transduction pathway in the absence of AVCR2, or the level of activity in a normal cell in the presence of AVCR2. The terms "reduce or inhibit" are used together herein because it is recognized that, in some cases, the level of signal transduction can be reduced below a level that can be detected by a particular assay. As such, it may not be determinable using such an assay as to whether a low level of myostatin signal transduction remains, or whether the signal transduction is completely inhibited.

[0033] The term "therapeutically effective amount" or "effective amount" means the amount of a compound or pharmaceutical composition that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.

[0034] The dosage regimen will be determined by the attending physician considering various factors which modify the activities of the ligands suppressing muscle growth, for example, 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. Generally, systemic or injectable administration, such as intravenous, intramuscular or subcutaneous injection is performed. Administration generally is initiated at a dose which is minimally effective, and the dose may be increased over a preselected time course until a positive effect is observed. Subsequently, incremental increases in dosage are made to such levels that produce a corresponding increase in effect, while taking into account any adverse affects that can appear. The addition of other known growth factors, such as IGF I (insulin like growth factor I), human, bovine, or chicken growth hormone, or other myostatin inhibitors, which can aid in increasing muscle mass, to the final composition, can also affect the dosage. In one embodiment, the soluble ACVR2 is generally administered within a dose range of about 0.1 μg/kg to about 100 mg/kg. In another embodiment, the soluble ACVR2 is administered within a dose range of about 10 mg/kg to 50 mg/kg.

[0035] The total amount of an agent to be administered in practicing a method of the invention can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a prolonged period of time. One skilled in the art would know that the amount of the pharmaceutical composition to treat a pathologic condition in a subject depends on many factors including the age and general health of the subject as well as the route of administration and the number of treatments to be administered. In view of these factors, the skilled artisan would adjust the particular dose as necessary. In general, the formulation of the pharmaceutical composition and the routes and frequency of administration are determined, initially, using Phase I and Phase II clinical trials.

[0036] As used herein, the term "myostatin activity" refers to one or more of physiologically growth-regulatory or morphogenetic activities associated with active myostatin protein. For example, active myostatin is a negative regulator of skeletal muscle. Active myostatin can also modulate the production of muscle-specific enzymes (e.g., creatine kinase), stimulate myoblast cell proliferation, and modulate preadipocyte differentiation to adipocytes. Myostatin is also believed to increase sensitivity to insulin and glucose uptake in peripheral tissues, particularly in skeletal muscle or adipocytes. Accordingly, myostatin biological activities include, but are not limited to, inhibition of muscle formation, inhibition of muscle cell growth, inhibition of muscle development, decrease in muscle mass, regulation of muscle-specific enzymes, inhibition of myoblast cell proliferation, modulation of preadipocyte differentiation to adipocytes, increasing sensitivity to insulin, regulations of glucose uptake, glucose hemostasis, and modulate neuronal cell development and maintenance. As such, a specific inhibitor of myostatin activity refers to any agent that inhibits, reduces, or otherwise prevents such myostatin biological activities. Exemplary specific inhibitors of myostatin activity include, but are not limited to, a polynucleotide, a peptide such as a functional peptide portion of a myostatin prodomain, a peptidomimetic, a peptoid such as a vinylogous peptoid, a small organic molecule, and a chemical agent that mimics the action of the GDF prodomain.

[0037] The term "peptide" or "peptide portion" is used broadly herein to mean two or more amino acids linked by a peptide bond. The term "fragment" or "proteolytic fragment" also is used herein to refer to a product that can be produced by a proteolytic reaction on a polypeptide, i.e., a peptide produced upon cleavage of a peptide bond in the polypeptide. Although the term "proteolytic fragment" is used generally herein to refer to a peptide that can be produced by a proteolytic reaction, it should be recognized that the fragment need not necessarily be produced by a proteolytic reaction, but also can be produced using methods of chemical synthesis or methods of recombinant DNA technology, as discussed in greater detail below, to produce a synthetic peptide that is equivalent to a proteolytic fragment.

[0038] Generally, a peptide useful in the methods of the invention contains at least about six amino acids, usually contains about ten amino acids, and can contain fifteen or more amino acids, particularly twenty or more amino acids. It should be recognized that the term "peptide" is not used herein to suggest a particular size or number of amino acids comprising the molecule, and that a peptide of the invention can contain up to several amino acid residues or more. For example, a full length mature C-terminal myostatin peptide contains more than 100 amino acids and a full length prodomain peptide can contain more than 260 amino acids.

[0039] As used herein, the term "substantially purified" or "substantially pure" or "isolated" means that the molecule being referred to, for example, a peptide or a polynucleotide, is in a form that is relatively free of proteins, nucleic acids, lipids, carbohydrates or other materials with which it is naturally associated. Generally, a substantially pure peptide, polynucleotide, or other molecule constitutes at least twenty percent of a sample, generally constitutes at least about fifty percent of a sample, usually constitutes at least about eighty percent of a sample, and particularly constitutes about ninety percent or ninety-five percent or more of a sample. A determination that a peptide or a polynucleotide of the invention is substantially pure can be made using well known methods, for example, by performing electrophoresis and identifying the particular molecule as a relatively discrete band. A substantially pure polynucleotide, for example, can be obtained by cloning the polynucleotide, or by chemical or enzymatic synthesis. A substantially pure peptide can be obtained, for example, by a method of chemical synthesis, or using methods of protein purification, followed by proteolysis and, if desired, further purification by chromatographic or electrophoretic methods.

[0040] The sequence of a peptide inhibitor of the invention also can be modified in comparison to the corresponding wildtype sequence by incorporating a conservative amino acid substitution for one or a few amino acids in the peptide. Conservative amino acid substitutions include the replacement of one amino acid residue with another amino acid residue having relatively the same chemical characteristics, for example, the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, for example, substitution of arginine for lysine; or of glutamic for aspartic acid; or of glutamine for asparagine; or the like.

[0041] In another aspect, the soluble ACVR2B can be used in combination with an inhibitor of myostatin activity. Thus, the methods of the invention can be performed, for example, by contacting under suitable conditions a target cell and an agent that specifically inhibits myostatin expression or activity in the cell in combination with soluble ACVR2B. ACVR2B may be administered prior to, simultaneously with or following the agent, such that the resulting increase in muscle growth is due to ACVR2B. Suitable conditions can be provided by placing the cell, which can be an isolated cell or can be a component of a tissue or organ, in an appropriate culture medium, or by contacting the cell in situ in a subject. For example, a medium containing the cell can be contacted with ACVR2B and an agent the affects the ability of myostatin to specifically interact with a myostatin receptor expressed on the cell, or with an agent that affects a myostatin signal transduction pathway in the cell. In general, the cell is a component of a tissue or organ in a subject, in which case contacting the cell can comprise administering the agent and ACVR2B to the subject. However, the cell also can be manipulated in culture, then can be maintained in culture, administered to a subject, or used to produce a transgenic nonhuman animal.

[0042] An agent useful in a method of the invention can be any type of molecule, for example, a polynucleotide, a peptide, a peptidomimetic, peptoids such as vinylogous peptoids, a small organic molecule, or the like, and can act in any of various ways to affect myostatin signal transduction. The agent can act extracellularly by binding myostatin or a myostatin receptor such as an activin receptor, thereby altering the ability of myostatin to specifically interact with its receptor, or can act intracellularly to alter myostatin signal transduction in the cell. In addition, the agent can be an agonist, which mimics or enhances the effect of myostatin on a cell, for example, the ability of myostatin to specifically interact with its receptor, thereby increasing myostatin signal transduction in the cell; or can be an antagonist, which can reduces or inhibits the effect of myostatin on a cell, thereby reducing or inhibiting myostatin signal transduction in the cell.

[0043] As used herein, the term "specific interaction" or "specifically binds" or the like means that two molecules form a complex that is relatively stable under physiologic conditions. The term is used herein in reference to various interactions, including, for example, the interaction of myostatin and a myostatin receptor, the interaction of the intracellular components of a myostatin signal transduction pathway, the interaction of an antibody and its antigen, and the interaction of soluble ACVR2B with its intracellular components. A specific interaction can be characterized by a dissociation constant of at least about 1 x 10^" M, generally at least about 1 x 10^" M, usually at least about 1 x 10^" M, and particularly at least about 1 x 10^"9 M or 1 x 10^"10 M or greater. A specific interaction generally is stable under physiological conditions, including, for example, conditions that occur in a living individual such as a human or other vertebrate or invertebrate, as well as conditions that occur in a cell culture such as used for maintaining mammalian cells or cells from another vertebrate organism or an invertebrate organism. Methods for determining whether two molecules interact specifically are well known and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0044] Receptor-ligand interaction studies have revealed a great deal of information as to how cells respond to external stimuli, and have led to the development of therapeutically important compounds such as erythropoietin, the colony stimulating factors, and PDGF. Thus, continual efforts have been made at identifying the receptors that mediate the action of the TGF-β family members for agricultural and human therapeutic purposes, for example, for treating in various pathological conditions such as obesity, type II diabetes, and cachexia.

[0045] Since the ACVR2B acts intracellularly, it can be contacted with the cell directly, or a polynucleotide encoding the ACVR2B can be introduced into the cell and the peptide can be expressed in the cell. The term "polynucleotide" is used broadly herein to mean a sequence of two or more deoxyribonucleotides or ribonucleotides that are linked together by a phosphodiester bond. As such, the term "polynucleotide" includes RNA and DNA, which can be a gene or a portion thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence, or the like, and can be single stranded or double stranded, as well as a DNA/RNA hybrid. Furthermore, the term "polynucleotide" as used herein includes naturally occurring nucleic acid molecules, which can be isolated from a cell, as well as synthetic molecules, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR). In various embodiments, a polynucleotide of the invention can contain nucleoside or nucleotide analogs, or a backbone bond other than a phosphodiester bond. [0046] It is recognized that some of the peptides useful in a method of the invention are relatively large and, therefore, may not readily traverse a cell membrane. However, various methods are known for introducing a peptide into a cell. The selection of a method for introducing such a peptide into a cell will depend, in part, on the characteristics of the target cell, into which the polypeptide is to be provided. For example, where the target cells, or a few cell types including the target cells, express a receptor, which, upon binding a particular ligand, is internalized into the cell, the peptide agent can be operatively associated with the ligand. Upon binding to the receptor, the peptide is translocated into the cell by receptor- mediated endocytosis. The peptide agent also can be encapsulated in a liposome or formulated in a lipid complex, which can facilitate entry of the peptide into the cell, and can be further modified to express a receptor (or ligand), as above. The peptide agent also can be introduced into a cell by engineering the peptide to contain a protein transduction domain such as the human immunodeficiency virus TAT protein transduction domain, which facilitates translocation of the peptide into the cell (see Schwarze et al., Science 285:1569- 1572 (1999), which is incorporated herein by reference; see, also, Derossi et al., J. Biol. Chem. 271 : 18188 (1996). The target cell also can be contacted with a polynucleotide encoding ACVR2B, which can be expressed in the cell.

[0047] In another aspect, the invention provides methods for ameliorating the severity of various pathologic conditions, including, for example, the cachexia associated with chronic diseases such as cancer (see Norton et al., Crit Rev. Oncol. Hematol. 7:289-327, 1987), as well as conditions such as type II diabetes, obesity, and other metabolic disorders. As used herein, the term "pathologic condition" refers to a disorder that is characterized, at least in part, by an abnormal amount, development or metabolic activity of muscle or adipose tissue. Such pathologic conditions, which include, for example, obesity; conditions associated with obesity, for example, atherosclerosis, hypertension, and myocardial infarction; muscle wasting disorders such as muscular dystrophy, neuromuscular diseases, cachexia, and anorexia; and metabolic disorders such as type II diabetes, which generally, but not necessarily, is associated with obesity, are particularly amenable to treatment using a method of the invention.

[0048] As used herein, the term "abnormal," when used in reference to the amount, development or metabolic activity of muscle or adipose tissue, is used in a relative sense in comparison to an amount, development or metabolic activity that a skilled clinician or other relevant artisan would recognize as being normal or ideal. Such normal or ideal values are known to the clinician and are based on average values generally observed or desired in a healthy individual in a corresponding population. For example, the clinician would know that obesity is associated with a body weight that is about twenty percent above an "ideal" weight range for a person of a particular height and body type. However, the clinician would recognize that a body builder is not necessarily obese simply by virtue of having a body weight that is twenty percent or more above the weight expected for a person of the same height and body type in an otherwise corresponding population. Similarly, the artisan would know that a patient presenting with what appears to abnormally decreased muscle activity could be identified as having abnormal muscle development, for example, by subjecting the patient to various strength tests and comparing the results with those expected for an average healthy individual in a corresponding population.

[0049] Thus, a method of the invention can ameliorate the severity of a pathologic condition that is characterized, at least in part, by an abnormal amount, development or metabolic activity in muscle or adipose tissue, by modulating a signal transduction in a muscle or adipose tissue cell associated with the etiology of the condition. As used herein, the term "ameliorate," when used in reference to the severity of a pathologic condition, means that signs or symptoms associated with the condition are lessened. The signs or symptoms to be monitored will be characteristic of a particular pathologic condition and will be well known to skilled clinician, as will the methods for monitoring the signs and conditions. For example, where the pathologic condition is type II diabetes, the skilled clinician can monitor the glucose levels, glucose clearance rates, and the like in the subject. Where the pathologic condition is obesity or cachexia, the clinician can simply monitor the subject's body weight.

[0050] The agent or agents to be administered to the subject are administered under conditions that facilitate contact of the agents with the target cell and, if appropriate, entry into the cell. Entry of a polynucleotide agent into a cell, for example, can be facilitated by incorporating the polynucleotide into a viral vector that can infect the cells. If a viral vector specific for the cell type is not available, the vector can be modified to express a receptor (or ligand) specific for a ligand (or receptor) expressed on the target cell, or can be encapsulated within a liposome, which also can be modified to include such a ligand (or receptor). A peptide agent can be introduced into a cell by various methods, including, for example, by engineering the peptide to contain a protein transduction domain such as the human immunodeficiency virus TAT protein transduction domain, which can facilitate translocation of the peptide into the cell (see Schwarze et al., supra, 1999; Derossi et al., supra, 1996).

[0051] As used herein, the term "target cells" means muscle cells or adipocytes that are to be contacted with the agent. For administration to a living subject, the agent generally is formulated in a pharmaceutical composition suitable for administration to the subject. Thus, the invention provides pharmaceutical compositions containing an agent, which is useful for modulating myostatin signal transduction in a cell, in a pharmaceutically acceptable carrier. As such, the agents are useful as medicaments for treating a subject suffering from a pathological condition as defined herein.

[0052] Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of the conjugate. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the physico-chemical characteristics of the therapeutic agent and on the route of administration of the composition, which can be, for example, orally or parenterally such as intravenously, and by injection, intubation, or other such method known in the art. The pharmaceutical composition also can contain a second reagent such as a diagnostic reagent, nutritional substance, toxin, or therapeutic agent, for example, a cancer chemotherapeutic agent.

[0053] The agent can be incorporated within an encapsulating material such as into an oil- in-water emulsion, a microemulsion, micelle, mixed micelle, liposome, microsphere or other polymer matrix (see, for example, Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, FIa. 1984); Fraley, et al., Trends Biochem. Sci., 6:77 (1981), each of which is incorporated herein by reference). Liposomes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. "Stealth" liposomes (see, for example, U.S. Pat. Nos. 5,882,679; 5,395,619; and 5,225,212, each of which is incorporated herein by reference) are an example of such encapsulating materials particularly useful for preparing a pharmaceutical composition useful for practicing a method of the invention, and other "masked" liposomes similarly can be used, such liposomes extending the time that the therapeutic agent remain in the circulation. Cationic liposomes, for example, also can be modified with specific receptors or ligands (Morishita et al., J. Clin. Invest., 91:2580-2585 (1993), which is incorporated herein by reference), hi addition, a polynucleotide agent can be introduced into a cell using, for example, adenovirus-polylysine DNA complexes (see, for example, Michael et al., J. Biol. Chem. 268:6866-6869 (1993), which is incorporated herein by reference).

[0054] The pharmaceutical composition can be formulated for oral formulation, such as a tablet, or a solution or suspension form; or can comprise an admixture with an organic or inorganic carrier or excipient suitable for enteral or parenteral applications, and can be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, or other form suitable for use. The carriers, in addition to those disclosed above, can include glucose, lactose, mannose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form. In addition auxiliary, stabilizing, thickening or coloring agents and perfumes can be used, for example a stabilizing dry agent such as triulose (see, for example, U.S. Pat. No. 5,314,695).

[0055] In view of the present disclosure, it will be recognized that various animal model systems can be used as research tools to identify agents useful for practicing a method of the invention. For example, transgenic mice or other experimental animals can be prepared using the ligands (i.e., activin isoforms) that cooperate with myostatin to suppress muscle growth, and the transgenic non-human organism can be examined directly to determine the effect produced by expressing various levels of ACVR2 and/or a particular agent in the organism. Thus, in one embodiment, a transgenic non-human organism of the invention is a mammal whose genome contains a nucleic acid sequence encoding ACVR2 and a regulatory element comprising a muscle-specific promoter operably linked and integrated into the genome of the mammal. A detected increase in ACVR2 and/or observed increase in muscle mass as compared to a corresponding mammal whose genome contains a myostatin-null mutation or a decreased level of myostatin as compared with normal basal levels will be indicative of an agent that modulates the signal transduction pathway associated with muscle growth.

[0056] As used herein, the term "operably linked" or "operably associated" means that two or more molecules are positioned with respect to each other such that they act as a single unit and affect a function attributable to one or both molecules or a combination thereof. For example, a polynucleotide sequence encoding a peptide of the invention can be operably linked to a regulatory element, in which case the regulatory element confers its regulatory effect on the polynucleotide similarly to the way in which the regulatory element would affect a polynucleotide sequence with which it normally is associated with in a cell. A first polynucleotide coding sequence also can be operatively linked to a second (or more) coding sequence such that a chimeric polypeptide can be expressed from the operably linked coding sequences. The chimeric polypeptide can be a fusion polypeptide, in which the two (or more) encoded peptides are translated into a single polypeptide, i.e., are covalently bound through a peptide bond; or can be translated as two discrete peptides that, upon translation, can operatively associate with each other to form a stable complex.

[0057] A chimeric polypeptide generally demonstrates some or all of the characteristics of each of its peptide components. As such, a chimeric polypeptide can be particularly useful in performing methods of the invention, as disclosed herein. For example, in one embodiment, a method of the invention includes modulating a ligand that cooperates with myostatin to suppress muscle growth and/or signal transduction in a cell. Thus, where one peptide component of a chimeric polypeptide encodes a cell compartment localization domain and a second peptide component encodes a dominant negative Smad polypeptide, the functional chimeric polypeptide can be translocated to the cell compartment designated by the cell compartment localization domain and can have the dominant negative activity of the Smad polypeptide, thereby modulating myostatin signal transduction in the cell. [0058] In another aspect, the transgenic organism, for example, a transgenic mouse, can be crossbred with other mice, for example, with ob/ob, db/db, or agouti lethal yellow mutant mice, to determine optimal levels of expression of ACVR2 and/or a myostatin inhibitor useful for treating or preventing a disorder such as obesity, type II diabetes, or the like. Further, the invention provides transgenic non-human organisms that express high levels of ACVR2. Such organisms exhibit dramatic increases in muscle mass, similar to that seen in myostatin knock-out mice (see for example, U.S. Pat. No. 5,994,618, herein incorporated by reference). As discussed herein, such animal models are important to identify agents for enhancing muscle growth for therapeutic purposes and agricultural applications. Methods of producing transgenic non-human animals are known in the art (see for Example U.S. Pat. Nos. 6,140,552; 5,998,698; 6,218,596, all of which are herein incorporated by reference).

[0059] As used herein, the term "animal" refers to any bird, fish or mammal, except a human, and includes any stage of development, including embryonic and fetal stages. Farm animals such as pigs, goats, sheep, cows, horses, rabbits and the like; rodents such as mice; and domestic pets such as cats and dogs are included within the meaning of the term "animal." In addition, the term "organism" is used herein to include animals as described above, as well as other eukaryotes, including, for example, other vertebrates such as reptiles and amphibians, as well as invertebrates as described above.

[0060] The following examples are provided to further illustrate the advantages and features of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLE 1

Effect of inhibin βA and inhibin βB inactivating mutations on muscle mass

[0061] In order to determine the identity of other TGF-β family members that cooperate with myostatin to suppress muscle growth, it was initially investigated whether the key ligand or ligands are one or more of the activin isoforms. The activins, which are dimers of inhibin- β subunits, were attractive as candidates because they had been shown to have in vitro activities on muscle cells (Link and Nishi, 1997; He et al., 2005; Souza et al, 2008; Trendelenburg et al, 2009). Moreover, a recent study showed that activin A is capable of inducing atrophy when overexpressed in muscle (Gilson et al, 2009).

[0062] In mice, four genes encoding inhibin-β subunits have been identified, InhβA, InhβB, InhβC, and InhβE (for review, see Chang et al, 2002). Inhibin-β A and inhibin-βB can form either homodimers or heterodimers with each other to generate activin A, activin B, or activin AB. Inhibin-β A and inhibin-βB can also heterodimerize with inhibin-α to form inhibin A and inhibin B, respectively, which generally have counteracting activities to the activins. Much less is known about the functions of inhibin-βC and inhibin-βE, which are more closely related to each other in terms of their amino acid sequences and expression patterns and are also closely linked on the chromosome. Mice carrying targeted mutations in each of these genes have been generated and characterized previously (Schrewe et al, 1994; Vassalli et al, 1994; Matzuk et al, 1995a; Lau et al, 2000), and for InhβA and InhβB, the existing mutant mouse lines were analyzed. For InhβC and InhβE, however, a double mutant mouse line was analyzed, where the line was generated independently in which the exon encoding the C-terminal domain of InhβC and the entire coding sequence of InhβE were deleted in the same mutant allele. All of these Inhβ mutant alleles were backcrossed at least 6 times onto a C57BL/6 genetic background prior to analysis.

[0063] For the InhβB and InhβC/βE mutations, the effect of complete loss of function was analyzed, since the homozygous mutants are viable as adults. In the case of InhβA, however, homozygous loss has been shown to lead to embryonic lethality (Matzuk et al, 1995a), so the effect of heterozygous loss of InhβA was only able to be analyzed. As shown in Table 1 and Figure 2, the most significant effect observed was, in fact, in mice heterozygous for the InhβA loss-of-function mutation, which exhibited about a 10% increase in weights of all four muscles that were examined. The effects seen in Inhβ A^^' mice were highly statistically significant, with p values ranging from 2XlO^"4 to 2xlO^"6 depending on the specific muscle. Mutations in each of the other genes had little or no effect, except in the case of InhβB homozygous mutants, in which two muscles (pectoralis and triceps) also showed statistically significant increases, although the magnitude of the effects was lower than that seen in Inhβ <A^+/~ mice. These data provide the first loss-of-function genetic evidence that activin A may be one of the key ligands that functions with myostatin to limit muscle mass. Table 1 - Muscle weights (mg) of mutant mice.

¹ p < 0.001 vs. wild type; p < 0.05 vs. wild type; ^c p< 0.001 vs. Mstn ^~; p < 0.001 vs. Mstri^'; ^e p < 0.01 vs. Mstri'^~; ^f p < 0.05 vs. Mstri'^'; ^r statistically significant decreases relative to controls; ^A statistically significant increases relative to controls; bold = controls.

EXAMPLE 2

Effect of the soluble ACVR2 receptor in enhancing muscle growth

[0064] In previous studies, it has been shown that a soluble form of the activin type HB receptor (ACVR2B/Fc) is a potent inhibitor of myostatin signaling and is capable of inducing significant muscle growth when administered to wild type adult mice (Lee et ah, 2005). We investigated the possibility that the other activin type II receptor, ACVR2, might also be capable of inducing muscle growth in vivo. We generated a Chinese hamster ovary (CHO) cell line producing high levels of a fusion protein consisting of the extracellular, ligand- binding domain of the ACVR2 receptor fused to an Fc domain and then purified the fusion protein (ACVR2/Fc) from the conditioned medium of these CHO cells using a protein A Sepharose affinity column. We tested this fusion protein for in vivo activity by administering it by intraperitoneal injection weekly for 4 weeks beginning at 6 weeks of age. At 10 weeks of age, we sacrificed the animals and measured weights of the pectoralis, triceps, quadriceps, gastrocnemius muscles. As shown in Table 2 and Figure 2, mice injected with the ACVR2/Fc fusion protein at doses of either 10 mg/kg or 25 mg/kg exhibited significant increases in muscle mass compared to mice receiving vehicle (PBS). Hence, this soluble receptor represents a novel biologic that is capable of increasing muscle growth in vivo. Although the targets for this receptor in vivo are not yet known, the fact that ACVR2 has a lower affinity for myostatin than ACVR2B (Lee and McPherron, 2001; Rebbapragada et ah, 2003) raises the possibility that ACVR2 may be targeting other ligands besides myostatin. If so, likely candidates would be the activins based on the results presented in Table 1 and Figure 2 showing that activin A and perhaps activin B play analogous roles to myostatin in suppressing muscle growth.

Table 2 - Muscle weights (mg) of mutant mice.

a p < 0.05 vs. PBS; ^D p < 0.01 vs. PBS; ^c p< 0.001 vs. PBS

[0065] References — all references cited are incorporated herein by reference in their entireties.

[0066] Akiyoshi, et ah, (1999) "c-Ski acts as a transcriptional co-repressor in transforming growth factor-β signaling through interaction with Smads." JBioi Chem 274: 35269-35277.

[0067] Amthor, et al., 2004. Follistatin complexes Myostatin and antagonises Myostatin- mediated inhibition of myogenesis. Dev Biol 270:19-30.

[0068] Attisano, et ah, (1992) "Novel activin receptors: Distinct genes and alternative mRNA splicing generate a repertoire of serine/threonine kinase receptors." Cell 68: 97-108. [0069] Berk, et al, (1997). "Mice Jacking the ski proto-oncogene have defects in neurulation, craniofacial patterning, and skeletal muscle development." Genes Dev 11 : 2029- 2039.

[0070] Bogdanovich, et al , (2002). "Functional improvement of dystrophic muscle by myostatin blockade." Nature 420: 418-421.

[0071] Bogdanovich, et al, (2005) "Myostatin propeptide-mediated amelioration of dystrophic pathophysiology." FASEB J. 19: 543-549.

[0072] Chang, et al, 2002. Genetic analysis of the mammalian transforming growth factor-β superfamily. Endocrine Rev 23:787-823.

[0073] Clop, et al., (2006). "A mutation creating a potential illegitimate micro RNA target site in the myostatin gene affects muscularity in sheep." Nature Genet. 38: 813-818.

[0074] de Winter, et al, (1996). "Follistatins neutralize activin bioactivity by inhibition of activin binding to its type II receptors." MoI Cell Endocrin 116: 105-114.

[0075] Fainsod, et al, (1997). "The dorsalizing and neural inducing gene follistatin is an antagonist of BMP -4. "Meek Dev. 63: 39-50.

[0076] Gilson, et al. 2009., Follistatin induces muscle hypertrophy through satellite cell proliferation and inhibition of both myostatin and activin. Am J Physiol Endocrinol Metab 297:E157-E164.

[0077] Grobet, et al, (1997). "A deletion in the bovine myostatin gene causes the double- muscled phenotype in cattle." Nature Genet 17: 71-74.

[0078] Grobet, et al, (1998). "Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle." Mamm Genome 9: 210-213.

[0079] Haidet, et al., 2008. Long-term enhancement of skeletal muscle mass and strength by single gene administration of myostatin inhibitors. Proc Natl Acad Sci USA 105:4318- 4322. [0080] Hayette, et al. , (1998). "FLRG (follistatin-related gene), a new target of chromosomal rearrangement in malignant blood disorders." Oncogene 16: 2949-2954.

[0081] He, et al., (2005) "Activin A inhibits formation of skeletal muscle during chick development." Anat Embryol 209: 401-407.

[0082] Hill, et al., (2002). "The myostatin propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum." JBioi Chem 111: 40735-40741.

[0083] Hill, et al., (2003). "Regulation of myostatin in vivo by growth and differentiation factor-associated serum protein- 1 : a novel protein with protease inhibitor and follistatin domains." MoI Endocrinol 17: 1144-1154.

[0084] Iemura, et al., (1998). "Direct binding of follistatin to a complex of bone- morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early Xenopus embryo." Proc Natl Acad Sci USA 95: 9337-9342.

[0085] Kambadur, et al., (1997). "Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle." Genome Res 7: 910-915.

[0086] Kondas, et al., (2008). "Both WFIKKNl and WFIKKN2 have high affinity for growth and differentiation factors 8 and 11." JBioi Chem 283: 23677-23684.

[0087] Langley, et al., (2002). "Myostatin inhibits myoblast differentiation by down- regulating MyoD expression." JBioi Chem 277: 49831-49840.

[0088] Lau, et ah, 2000. Activin betaC and betaE genes are not essential for mouse liver growth, differentiation, and regeneration. MoI Cell Biol 20:6127-6137.

[0089] Lee, S.-J. (2004). "Regulation of muscle mass by myostatin. " Ann Rev Cell Dev Biol 20: 61-86.

[0090] Lee, S.-J. (2007). "Quadrupling muscle mass in mice by targeting TGF-β signaling pathways: PLoS ONE 2: e789.

[0091] Lee, S.-J. (2008). "Genetic analysis of the role of proteolysis in the activation of latent myostatin." PLoS ONE 2,: el 628. [0092] Lee, et ah, (2001). "Regulation of myostatin activity and muscle growth." Proc NatlAcadSci USA 98: 9306-9311.

[0093] Lee, et al., (2005). "Regulation of muscle growth by multiple ligands signaling through activin type II receptors." Proc. Natl. Acad. Sci, USA 102: 18117-18122.

[0094] Link, et al., (1997) "Opposing effects of activin A and follistatin on developing skeletal muscle cells." Exp Cell Res 233: 350-362.

[0095] Luo, et ah, (1999). "The Ski oncoprotein interacts with the Smad proteins to repress TGFβ signaling." Genes Dev 13:2196-2206.

[0096] Massague. J. (1998). "TGF-β signal transduction." Annu. Rev. Biochm. 67: 753- 791.

[0097] Mathews, et al., (1991) "Expression cloning of an activin receptor, a predicted transmembrane serine kinase." Cell 65: 973-982.

[0098] Matzuk, et al., (1995a). "Functional analysis of activins during mammalian development." Nature 374: 354-356.

[0099] Matzuk, et al., (1995b). "Multiple defects and perinatal death in mice deficient in follistatin." Nature 374: 360-363.

[0100] McPherron, et ah, 2009. Redundancy of myostatin and growth/differentiation factor 11. BMC Dev Biol 9:24-32.

[0101] McPherron, et ah, (1997). "Regulation of skeletal muscle mass in mice by a new TGF-β superfamily member." Nature 387: 83-90.

[0102] McPherron, et al., (1997). "Double muscling in cattle due to mutations in the myostatin gene." Proc Natl Acad Sci USA 94: 12457-12461.

[0103] Mosher, et al., (2007). "A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs." PLOS Genetics 3(5): 779-786.

[0104] Moustakas, et ah, 2009. The regulation of TGFβ signal transduction. Development 136:3699-3714. [0105] Nakamura, et al, (1990). "Activin-binding protein from rat ovary is follistatin." Science 247: 836-838.

[0106] Rebbapragada, et al, (2003). "Myostatin signals through a transforming growth factor β-like signaling pathway to block adipogenesis." MoI Cell Biol 23(20): 7230-7242.

[0107] Schrewe, et al., (1994) "Mice homozygous for a mutation of activin BB are viable and fertile." Mech Dev 47: 43-51.

[0108] Schuelke, et al., (2004). "Myostatin mutation associated with gross muscle hypertrophy in a child." N Engl J Med 350: 2682-2688.

[0109] Souza, et al., (2008). "Proteomic identification and functional validation of activins and bone morphogenetic 11 as candidate novel muscle mass regulators." MoI Endocrinol 22: 2689-2702.

[0110] Stroschein, et al, (1999). "Negative feedback regulation of TGF-β signaling by the SnoN oncoprotein." Science 286: 771-774.

[0111] Sun, et al., (1999a). "Interaction of the Ski oncoprotein with Smad3 regulates TGF-β signaling." MoI Cell 4: 499-509.

[0112] Sun, et ah, (1999b). "SnoN and Ski protooncoproteins are rapidly degraded in response to transforming growth factor β signaling." Proc Natl Acad Sci USA 96: 12442- 12447.

[0113] Sutrave, et al., (1990). "ski can cause selective growth of skeletal muscle in transgenic mice." Genes Dev. 4: 1462-1472.

[0114] Thies, et al, (2001). "GDF-8 propeptide binds to GDF-8 and antagonizes biological activity by inhibiting GDF-8 receptor binding." Growth Factors 18: 251-259.

[0115] Trendelenburg, et al, 2009. Myostatin reduces Akt/TORCl/p70S6K signaling, inhibiting myoblast differentiation and myotube size. Am J Physiol Cell Physiol 296:C1258- C1270. [0116] Tsuchida, et al, (2000). "Identification and characterization of a novel follistatin- like protein as a binding protein for the TGF-β family." JBioi Chern 275: 40788-40796.

[0117] Vassalli, et al, (1994). "Activin/inhibin beta B subunit gene disruption leads to defects in eyelid development and female reproduction." Genes Dev 8: 414-427.

[0118] Wagner, et al, (2008). "A Phase 1111 trial of MYO-029 in adult subjects with muscular dystrophy." Ann Neurol 63: 561-571.

[0119] Whittemore, et al, (2003). "Inhibition of myostatin in adult mice increases skeletal muscle mass and strength." BBRC 300: 965-971.

[0120] Wolfman, et al, (2003). "Activation of latent myostatin by the BMP-1/tolloid family of metaloproteinases. " Prøc iVαt/Λcαd Sci USA 100: 15842-15846.

[0121] Yamashita, et al, (1995). "Osteogenic protein- 1 binds to activin type II receptors and induces certain activin-like effects." J Cell Biol 130: 217-226.

[0122] Yang, et al, (2001). "Expression of myostatin pro domain results in muscular transgenic mice." MoI Repro Dev 60: 351-361.

[0123] Zimmers, et al, (2002). "Induction of cachexia in mice by systemically administered myostatin." Science 296: 1486-1488.

[0124] Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. A composition comprising a therapeutically effective amount of soluble Activin Type II Receptor (ACVR2) polypeptide.

2. The composition of claim 1, wherein the composition is formulated for oral or parenteral administration.

3. The composition of claim 1, wherein the soluble ACVR2 polypeptide is fused to an Fc domain.

4. A method for increasing muscle mass in a mammal comprising administering an effective amount of soluble Activin Type II Receptor (ACVR2) polypeptide.

5. The method of claim 4, wherein the mammal is suffering from a disease or disorder chosen from muscular disorder and neuromuscular disorder.

6. The method of claim 5, wherein the disease or disorder is a muscular disorder.

7. The method of claim 6, wherein the muscular disorder is chosen from at least one of muscular dystrophy, muscle atrophy, and muscle wasting disorder.

8. The method of claim 6, wherein the muscular disorder is muscular dystrophy.

9. The method of claim 8, wherein the muscular dystrophy is Duchenne muscular dystrophy.

10. The method of claim 6, wherein the muscular disorder is a muscle wasting disorder.

11. The method of claim 10, wherein the muscle wasting disorder is cachexia.

12. The method of claim 10, wherein the muscle wasting disorder is anorexia.

13. The method of claim 5, wherein the disease or disorder is a neuromuscular disorder.

14. The method of claim 13, wherein the neuromuscular disorder is amyotrophic lateral sclerosis (ALS).

15. The method of claim 4, wherein the mammal is human.