# Patent

## Title
Methods of increasing muscle mass using follistatin-like related gene (FLRG)

## Abstract

The present relates to use of follistatin-like related gene (FLRG) to increase muscle mass in a subject. As such, 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. In addition transgenic non-human mammals expressing FLRG and having increased muscle mass as compared to a corresponding mammal having a myostatin-null mutation or a decreased level of myostatin are provided.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a 35 USC §371 National Stage application of International Application No. PCT/US2008/056098 filed Mar. 6, 2008, which claims the benefit under 35 USC §119(e) to U.S. application Ser. No. 60/926,897 filed Apr. 30, 2007, and to U.S. application Ser. No. 60/905,479 filed Mar. 7, 2007. The disclosure of each of the prior applications is considered part of and is incorporated by reference in the disclosure of this application.


GRANT INFORMATION
This invention was made with government support under Grant No. R01 HD35887 awarded by the National Institutes of Health. The government has certain rights in the invention.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to myostatin, follistatin, and follistatin-like related gene (FLRG), and more specifically to use of FLRG to increase muscle growth in a subject.
2. Background Information
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.
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 disulfide-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.
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
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 Follistatin-Like Related Gene (FLRG). 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. The increase in muscle tissue growth following administration of FLRG is expected to be greater as compared to the muscle tissue growth of the corresponding subject treated with an inhibitor of myostatin expression or activity, which is indicative of increasing the growth of muscle tissue in the subject as a result of FLRG administration. In one embodiment the muscle tissue growth is at least two-fold greater than the muscle tissue growth of the corresponding subject treated with a specific inhibitor of myostatin expression or activity in the absence of another agent.
The invention further 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 FLRG either prior to, simultaneously with or following the inhibitor of myostatin expression or activity, thereby increasing muscle tissue growth in the subject. In one embodiment, the inhibitor of myostatin expression or activity is a myostatin prodomain comprising amino acid residues from about 20 to 262 of a promyostatin polypeptide as set forth in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20. In another embodiment, the inhibitor of myostatin expression or activity is an antibody, or functional fragment thereof, which binds to myostatin. The antibody can be a monoclonal antibody or a polyclonal antibody, and can be humanized or chimeric. In another embodiment, the inhibitor of myostatin expression or activity is an antisense or interfering RNA nucleic acid. In another embodiment, the inhibitor of myostatin expression or activity is a polynucleotide encoding a dominant negative myostatin polypeptide or a polynucleotide encoding a truncated myostatin polypeptide. In another embodiment, the muscle tissue growth is at least two-fold greater than the muscle tissue growth of the corresponding subject treated with a specific inhibitor of myostatin expression or activity in the absence of another agent.
The invention further 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 an FLRG polynucleotide or polypeptide and 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. The increase in muscle tissue growth following administration of FLRG is greater as compared to the muscle tissue growth in the subject treated with an inhibitor of myostatin expression or activity, which is indicative of increasing the growth of muscle tissue in the subject as a result of FLRG administration, thereby ameliorating the severity of the pathologic condition. 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.
The invention further 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 FLRG either prior to, simultaneous with or following the inhibitor of myostatin expression or activity. In one embodiment, the inhibitor of myostatin expression or activity is a myostatin prodomain comprising amino acid residues from about 20 to 262 of a promyostatin polypeptide as set forth in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20. In another embodiment, the inhibitor of myostatin expression or activity is an antibody, or functional fragment thereof, which binds to myostatin. The antibody can be a monoclonal antibody or a polyclonal antibody, and can be humanized or chimeric. In another embodiment, the inhibitor of myostatin expression or activity is an antisense or interfering RNA nucleic acid. In another embodiment, the inhibitor of myostatin expression or activity is a polynucleotide encoding a dominant negative myostatin polypeptide or a polynucleotide encoding a truncated myostatin polypeptide. In another 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.
In various embodiments, the subject is a mammal such as ovine, procine, bovine, murine or human. As such, the invention further provides a transgenic non-human mammal whose genome contains a nucleic acid sequence comprising FLRG 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 FLRG 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. In another embodiment, the muscle tissue growth of the transgenic non-human mammal is at least two-fold greater than the muscle tissue growth of the corresponding mammal whose genome lacks FLRG and contains a myostatin-null mutation or a decreased level of myostatin as compared with normal basal level. In another embodiment, the regulatory element includes a myosin light chain promoter and an enhancer. In another embodiment, the regulatory element includes a myosin light chain promoter and myosin light chain 1/3 enhancer. In another embodiment, the genome of the corresponding mammal contains a truncated Activin Type II receptor gene that encodes a truncated dominant negative Activin Type II receptor lacking kinase activity. The Activin Type II receptor can be an Activin RIIA or an Activin RIIB.
The invention further provides a transgenic non-human mammal whose genome contains a myostatin-null mutation or a decreased level of myostatin as compared with normal basal levels, a nucleic acid sequence comprising FLRG, 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 FLRG 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 alone. In another embodiment, the muscle tissue growth of the transgenic non-human mammal is at least two-fold greater than the muscle tissue growth of the corresponding mammal whose genome lacks FLRG and contains a myostatin-null mutation or a decreased level of myostatin as compared with normal basal level. In another embodiment, the regulatory element includes a myosin light chain promoter and an enhancer. In another embodiment, the regulatory element includes a myosin light chain promoter and myosin light chain 1/3 enhancer. In another embodiment, the genome of the corresponding mammal contains a truncated Activin Type II receptor gene that encodes a truncated dominant negative Activin Type II receptor lacking kinase activity. The Activin Type II receptor can be an Activin RITA or an Activin RIIB.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the amino acid sequences of murine promyostatin (SEQ ID NO: 4); rat promyostatin (SEQ ID NO: 6); human promyostatin (SEQ ID NO: 2); baboon promyostatin (SEQ ID NO: 10); bovine promyostatin (SEQ ID NO: 12); porcine promyostatin (SEQ ID NO: 14); ovine promyostatin (SEQ ID NO: 16); chicken promyostatin (SEQ ID NO: 8), turkey promyostatin (SEQ ID NO: 18); and zebrafish promyostatin (SEQ ID NO: 20). Amino acids are numbered relative to the human promyostatin (SEQ ID NO: 2). Dashed lines indicate gaps introduced to maximize homology. Identical residues among sequences are shaded.
FIG. 2 shows the amino acid sequences of murine promyostatin (SEQ ID NO: 4) and zebrafish promyostatin (SEQ ID NO: 20), and portions of the amino acid sequences of salmon allele 1 promyostatin (SEQ ID NO: 27; “salmon 1”) and salmon allele 2 promyostatin (SEQ ID NO: 29; “salmon 2”). Amino acid position relative to human promyostatin is indicated to left of each row (compare FIG. 1; first amino acid of salmon1 corresponds to human promyostatin 218; first amino acid of salmon2 corresponds to human promyostatin 239). Dashed lines indicate gaps introduced to maximize homology. Relative amino acid positions, including gaps, is indicated along top of each row. Identical residues among sequences are shaded.
FIG. 3 is a pictorial diagram showing the results from Northern analysis of FLRG transgenic mice. Total RNA was prepared from various tissues from 10-week old female mice, electrophoresed, blotted, and probed with a fragment derived from SV 40 corresponding to the processing/polyadenylation sequences present in the transgenic construct. The blots were re-hybridized with a probe for the S26 ribosomal protein to control for loading.
FIGS. 4A and 4B are graphical diagrams showing that muscle weight increases in Mstn mutant and F66 transgenic mice. Numbers represent percent increases relative to wild type mice and were calculated from the data shown in Table 4. Muscles analyzed were: pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius (green). FIG. 4B shows the distribution of fiber diameters. Gray bars represent muscles from wild type mice, and red bars represent muscles from Mstn−/−, F66, and F66/Mstn−/− mice.
FIGS. 5A and 5B are pictorial diagrams showing a comparison of wild type (left) and F66/Mstn−/− (right) mice. FIG. 5B shows the muscles of wild type (top panels) and F66/Mstn−/− mice (bottom panels).



DETAILED DESCRIPTION OF THE INVENTION
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.
Promyostatin, which previously has been referred to as growth differentiation factor-8 (GDF-8), includes an amino terminal prodomain and a C-terminal mature myostatin peptide (see U.S. Pat. No. 5,827,733, incorporated herein by reference). Myostatin activity is effected by the mature myostatin peptide following its cleavage from promyostatin. Thus, promyostatin is a precursor polypeptide that is proteolytically cleaved to produce active myostatin. As provided in U.S. Pat. No. 6,891,082, incorporated herein by reference in its entirety, the myostatin prodomain can inhibit myostatin activity.
Promyostatin is a member of the transforming growth factor-β (TGF-β) superfamily, which contains multifunctional polypeptides that control proliferation, differentiation, and other functions in various cell types. The TGF-β superfamily, which encompasses a group of structurally-related proteins that affect a wide range of differentiation processes during embryonic development, includes, for example, Mullerian inhibiting substance (MIS), which is required for normal male sex development, Drosophila decapentaplegic (DPP) gene product, which is required for dorsal-ventral axis formation and morphogenesis of the imaginal disks, the Xenopus Vg-1 gene product, which localizes to the vegetal pole of eggs, the activins, which can induce the formation of mesoderm and anterior structures in Xenopus embryos, which can induce de novo cartilage and bone formation. The TGF-β family members can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, hematopoiesis, and epithelial cell differentiation.
Many of the TGF-β family members have regulatory effects (positive or negative) on other peptide growth factors. In particular, certain members of the TGF-β superfamily have expression patterns or possess activities that relate to the function of the nervous system. For example, the inhibins and activins are expressed in the brain, and activin can function as a nerve cell survival molecule. Another family member, growth differentiation factor-1 (GDF-1), is nervous system-specific in its expression pattern, and other family members such as Vgr-1, OP-1, and BMP-4, are also expressed in the nervous system. Because skeletal muscle produces a factor or factors that promote the survival of motor neurons, the expression of myostatin (GDF-8) in muscle suggests that myostatin can be a trophic factor for neurons. As such, methods for modulating the activity of myostatin have been shown to be useful for treating neurodegenerative diseases such as amyotrophic lateral sclerosis or muscular dystrophy, or for maintaining cells or tissues in culture prior to transplantation.
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.
Promyostatin polypeptides from various species share substantial sequence identity, and the amino acid sequences of human, murine, rat and chicken mature myostatin C-terminal sequence are 100% identical (see FIG. 1). Promyostatin polypeptides are exemplified herein (see FIG. 1) by human promyostatin (SEQ ID NO: 2); murine promyostatin (SEQ ID NO: 4); rat promyostatin (SEQ ID NO: 6); baboon promyostatin (SEQ ID NO: 10); bovine promyostatin (SEQ ID NO: 12); porcine promyostatin (SEQ ID NO: 14); ovine promyostatin (SEQ ID NO: 16); chicken promyostatin (SEQ ID NO: 8), turkey promyostatin (SEQ ID NO: 18); and zebrafish promyostatin (SEQ ID NO: 20). Promyostatin polypeptides also are exemplified herein by a polypeptide comprising the portions of salmon allele 1 (SEQ ID NO: 27; “salmon1”) and of salmon allele 2 (SEQ ID NO: 29; “salmon2”; see FIG. 2). Nucleic acid molecules encoding these promyostatin polypeptides are disclosed herein as SEQ ID NOs: 1, 3, 5, 9, 11, 13, 15, 7, 17, 19, 26 and 28, respectively (see, also, McPherron and Lee, Proc. Natl. Acad. Sci., USA 94:12457, 1997, which is incorporated herein by reference). A pro-GDF-11 polypeptide is exemplified herein by human pro-GDF-11 (SEQ ID NO: 25), which is encoded by SEQ ID NO: 24.
Previous studies have identified several proteins that are normally found in a complex with myostatin in the blood. These include the myostatin propeptide, follistatin-like related gene (FLRG), and GDF-associated serum protein-1 (GASP-1), each of which has been demonstrated to be capable of inhibiting myostatin activity in vitro. In addition, follstatin, has also been shown to be a potent myostatin inhibitor, and transgenic mice overexpressing follstatin in muscle have been shown to have dramatic increases in muscle mass. The data presented herein demonstrates that FLRG, like follstatin, can promote muscle growth when expressed as a transgene in skeletal muscle and that both of these molecules appear to act by blocking not only myostatin but also other ligands with similar activity to myostatin. As such, the present invention demonstrates that the effect of follistatin and FLRG, a protein related to follistatin, does not result solely from the inhibition of myostatin activity.
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 FLRG. 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 FLRG administration.
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.
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 FLRG 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.
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.
As used herein, the term “myostatin (GDF-8) 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, peptoids such as vinylogous peptoids, a small organic molecule, and a chemical agent that mimics the action of the GDF prodomain.
The proteins of the TGF-β family are synthesized as large precursor proteins, which subsequently undergo proteolytic cleavage at a cluster of basic residues approximately 110 to 140 amino acids from the C-terminus, resulting in the formation of a prodomain peptide and a C-terminal mature peptide. The C-terminal mature peptides of the members of this family of proteins are 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 20% to 50%. In each case, the active species appears to be a disulfide-linked dimer of C-terminal peptide fragments.
As used herein, reference to a “pro-GDF,” for example, promyostatin or pro-GDF-11, refers to the full length polypeptide, including the amino terminal prodomain and the carboxy terminal biologically active GDF peptide. In addition, the prodomain includes a signal peptide (leader sequence), which comprises about the first 15 to 30 amino acids at the amino terminus of the prodomain. The signal peptide can be cleaved from the full length pro-GDF polypeptide, which can be further cleaved at an Arg-Xaa-Xaa-Arg (SEQ ID NO: 21) proteolytic cleavage site.
Reference herein to amino acid residues is made with respect to the full length pro-GDF polypeptides. It should also be recognized that reference is made herein to particular peptides beginning or ending at “about” a particular amino acid residue. The term “about” is used in this context because it is recognized that a particular protease can cleave a pro-GDF polypeptide at or immediately adjacent to a proteolytic cleavage recognition site, or one or a few amino acids from the recognition site. As such, reference, for example, to a myostatin prodomain having a sequence of about amino acid residues 1 to 263 of SEQ ID NO: 4 would include an amino terminal peptide portion of promyostatin that includes the signal peptide and has a carboxy terminus ending at amino acid residue 257 to amino acid residue 269, preferably at amino acid residue 260 to amino acid residue 266.
Similarly, the signal peptide can be cleaved at any position from about amino acid residue 15 to 30 of a pro-GDF polypeptide, for example, at residue 15, 20, 25 or 30, without affecting the function, for example, of a remaining prodomain. Thus, for convenience, reference is made generally herein to a peptide portion of a pro-GDF polypeptide, from which the signal peptide has been cleaved, as beginning at about amino acid residue 20. However, it will be recognized that cleavage of the signal peptide can be at any amino acid position within about the first 15 to 30 amino terminal amino acids of a pro-GDF polypeptide. As such, reference, for example, to a myostatin prodomain having a sequence of about amino acid residues 20 to 263 of SEQ ID NO: 4 would include a peptide portion of promyostatin that lacks about the first 15 to 30 amino acids of promyostatin, comprising the signal peptide, and that has a carboxy terminus ending at amino acid residue 257 to amino acid residue 269, preferably at amino acid residue 260 to amino acid residue 266.
In general, reference is made herein to a pro-GDF polypeptide or a GDF prodomain as beginning at about amino acid 1. In view of the above disclosure, however, it will be recognized that such pro-GDF polypeptides or GDF prodomains that lack the signal peptide also are encompassed within the present invention. Further in this respect, it should be recognized that the presence or absence of a signal peptide in a peptide of the invention can influence, for example, the compartments of a cell through which a peptide, for example, a myostatin prodomain will traverse and to which the peptide ultimately will localize, including whether the peptide will be secreted from the cell. Thus, the present invention further includes a substantially purified signal peptide portion of a pro-GDF polypeptide. As disclosed herein, such a signal peptide can be used to target an agent, particularly a peptide agent, to the same cellular compartments as the naturally occurring GDF from which the signal peptide is derived.
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.
As such, a functional peptide portion of a promyostatin polypeptide is characterized, in part, in that it has an activity of myostatin. Thus, peptides exhibiting a biological activity of myostatin are included in the invention, as are epitopic peptides, which provide an epitope substantially unique to myostatin. As used herein, the term “functional peptide portion,” when used in reference to a promyostatin polypeptide, means a contiguous amino acid sequence of a C-terminal myostatin polypeptide that can affect muscle growth or fat content of a subject, or that can specifically interact with a reagent that is known to specifically interact with myostatin; or of a myostatin prodomain, which can inhibit the activity of a C-terminal myostatin polypeptide. An activin type II receptor (Act RII) such as Act RIIA or Act RIIB (see, for example, U.S. Pat. No. 5,885,794, which is incorporated herein by reference), or an anti-myostatin antibody, which specifically binds myostatin, but not other members of the TGF-β family, are examples of reagents that specifically interact with a myostatin peptide.
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.
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.
The sequence of a peptide inhibitor of the invention also can be modified in comparison to the corresponding sequence in a promyostatin polypeptide 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. Examples of positions of a promyostatin polypeptide that can be modified are evident from examination of FIG. 1, which shows various amino acid differences in the myostatin prodomain and mature myostatin peptide that do not substantially affect promyostatin or myostatin activity.
A method 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 FLRG. FLRG may be administered prior to, simultaneously with or following the agent, such that the resulting increase in muscle growth is due to FLRG. 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 FLRG 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 FLRG 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.
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.
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 a myostatin prodomain with myostatin. A specific interaction can be characterized by a dissociation constant of at least about 1×10−6 M, generally at least about 1×10−7 M, usually at least about 1×10−8 M, and particularly at least about 1×10−9 M or 1×10−10 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. In addition, a specific interaction such as the extracellular interaction of a myostatin prodomain and myostatin generally is stable under conditions such as those used for aquaculture of a commercially valuable marine organism. Methods for determining whether two molecules interact specifically are well known and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.
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. As disclosed herein, myostatin specifically interacts with an activin type II receptor. The identification of this interaction provides targets for identifying antagonists and agonists useful for agricultural and human therapeutic purposes, for example, for treating in various pathological conditions such as obesity, type II diabetes, and cachexia. The identification of this specific interaction also provides a means to identify other myostatin receptors, as well as the specific receptors of other growth differentiation factors. Accordingly, the present invention provides GDF receptors, which specifically interact with a GDF or combination of GDFs, for example, with myostatin, GDF-11, or both.
A GDF receptor of the invention is exemplified herein by a myostatin receptor, particularly an activin type II receptor, which specifically interacts with myostatin. For convenience of discussion, the receptors of the invention are referred to herein generally as a “GDF receptor” and are exemplified by a myostatin receptor, which is a receptor that specifically interacts at least with myostatin. As such, while reference is made generally to a specific interaction of myostatin with a myostatin receptor, it will be recognized that the present disclosure more broadly encompasses any GDF receptor, including a receptors that specifically interact at least with FLRG and/or follistatin.
As such, present invention includes use of a substantially purified polynucleotide encoding all or a peptide portion of a GDF receptor. Although a GDF receptor is exemplified herein by an activin type II receptor, polynucleotides encoding activin type II receptors previously have been described (U.S. Pat. No. 5,885,794, incorporated herein by reference). A polynucleotide useful in the methods of the invention can also encode a polypeptide having a myostatin receptor activity, for example, myostatin binding activity, or can encode a mutant myostatin receptor, for example, a mutant myostatin receptor having a mutation in a kinase domain, such that the mutant acts as a dominant negative myostatin receptor (see above). Thus, a polynucleotide useful in the methods of the invention can be a naturally occurring, synthetic, or intentionally manipulated polynucleotide. For example, portions of the mRNA sequence can be altered due to alternate RNA splicing patterns or the use of alternate promoters for RNA transcription. As another example, the polynucleotide can be subjected to site directed mutagenesis. The polynucleotide also can be antisense nucleotide sequence. GDF receptor polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included within the invention, provided the amino acid sequence of the GDF receptor polypeptide encoded by the polynucleotide is functionally unchanged. Also included are nucleotide sequences that encode myostatin receptor polypeptides
Oligonucleotide portions of a polynucleotide encoding a GDF receptor of the invention also are encompassed within the present invention. Such oligonucleotides generally are at least about 15 bases in length, which is sufficient to permit the oligonucleotide to selectively hybridize to a polynucleotide encoding the receptor, and can be at least about 18 nucleotides or 21 nucleotides or more in length. As used herein, the term “selective hybridization” or “selectively hybridize” refers to hybridization under moderately stringent or highly stringent physiological conditions, which can distinguish related nucleotide sequences from unrelated nucleotide sequences.
In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (for example, relative GC:AT content), and nucleic acid type, i.e., whether the oligonucleotide or the target nucleic acid sequence is DNA or RNA, can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. Methods for selecting appropriate stringency conditions can be determined empirically or estimated using various formulas, and are well known in the art (see, for example, Sambrook et al., supra, 1989).
An example of progressively higher stringency conditions is as follows: 2×SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and 0.1×SSC at about 68° C. (high stringency conditions). Washing can be carried out using only one of these conditions, for example, high stringency conditions, or each of the conditions can be used, for example, for 10 to 15 minutes each, in the order listed above, repeating any or all of the steps listed.
A GDF receptor-encoding polynucleotide of the invention can be obtained by any of several methods. For example, the polynucleotide can be isolated using hybridization or computer based techniques, as are well known in the art. These methods include, but are not limited to, 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences; 2) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features; 3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the DNA sequence of interest; 4) computer searches of sequence databases for similar sequences (see above); 5) differential screening of a subtracted DNA library; and 6) two hybrid assays using, for example, a mature GDF peptide in one of the hybrids.
Polynucleotides encoding mutant GDF receptors and mutant GDF receptor polypeptides are also encompassed within the invention. An alteration in a polynucleotide encoding a GDF receptor can be an intragenic mutation such as point mutation, nonsense (STOP) mutation, missense mutation, splice site mutation or frameshift, or can be a heterozygous or homozygous deletion, and can be a naturally occurring mutation or can be engineered using recombinant DNA methods, for example. Such alterations can be detected using standard methods known to those of skill in the art, including, but not limited to, nucleotide sequence analysis, Southern blot analysis, a PCR based analysis such as multiplex PCR or sequence tagged sites (STS) analysis, or in situ hybridization analysis. GDF receptor polypeptides can be analyzed by standard SDS-PAGE, immunoprecipitation analysis, western blot analysis, or the like. Mutant GDF receptors are exemplified by truncated GDF receptors, including a soluble extracellular domain, which can have the ability to specifically bind its cognate GDF, but lacks a kinase domain; an intracellular GDF receptor kinase domain, which can exhibit constitutive kinase activity; as well as by GDF receptors that contain a point mutation, which disrupts the kinase activity of the receptor or the ligand binding ability of the receptor; and the like. Such GDF receptor mutants are useful for modulating GDF signal transduction and, therefore, for practicing various methods of the invention.
A polynucleotide encoding a GDF receptor can be expressed in vitro by introducing the polynucleotide into a suitable host cell. “Host cells” can be any cells in which the particular vector can be propagated, and, where appropriate, in which a polynucleotide contained in the vector can be expressed. The term “host cells” includes any progeny of an original host cell. It is understood that all progeny of the host cell may not be identical to the parental cell due, for example, to mutations that occur during replication. However, such progeny are included when the term “host cell” is used. Methods of obtaining a host cell that transiently or stably contains an introduced polynucleotide of the invention are well known in the art.
A GDF receptor polynucleotide of the invention can be inserted into a vector, which can be a cloning vector or a recombinant expression vector. The term “recombinant expression vector” refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of a polynucleotide, particularly, with respect to the present invention, a polynucleotide encoding all or a peptide portion of a GDF receptor. Such expression vectors contain a promoter sequence, which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector generally contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, but are not limited to, the T7-based expression vector for expression in bacteria, the pMSXND expression vector for expression in mammalian cells and baculovirus-derived vectors for expression in insect cells. The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter, which can be a T7 promoter, metallothionein I promoter, polyhedrin promoter, or other promoter as desired, particularly tissue specific promoters or inducible promoters.
A polynucleotide sequence encoding a GDF receptor can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing polynucleotides having eukaryotic or viral sequences in prokaryotes are well known in the art, as are biologically functional viral and plasmid DNA vectors capable of expression and replication in a host. Methods for constructing an expression vector containing a polynucleotide of the invention are well known, as are factors to be considered in selecting transcriptional or translational control signals, including, for example, whether the polynucleotide is to be expressed preferentially in a particular cell type or under particular conditions (see, for example, Sambrook et al., supra, 1989).
A variety of host cell/expression vector systems can be utilized to express a GDF receptor coding sequence, including, but not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors; yeast cells transformed with recombinant yeast expression vectors; plant cell systems infected with recombinant virus expression vectors such as a cauliflower mosaic virus or tobacco mosaic virus, or transformed with recombinant plasmid expression vector such as a Ti plasmid; insect cells infected with recombinant virus expression vectors such as a baculovirus; animal cell systems infected with recombinant virus expression vectors such as a retrovirus, adenovirus or vaccinia virus vector; and transformed animal cell systems genetically engineered for stable expression. Where the expressed GDF receptor is post-translationally modified, for example, by glycosylation, it can be particularly advantageous to select a host cell/expression vector system that can effect the desired modification, for example, a mammalian host cell/expression vector system.
Depending on the host cell/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, and the like can be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage .lambda., plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells, for example, a human or mouse metallothionein promoter, or from mammalian viruses, for example, a retrovirus long terminal repeat, an adenovirus late promoter or a vaccinia virus 7.5K promoter, can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide for transcription of the inserted GDF receptors coding sequence.
In yeast cells, a number of vectors containing constitutive or inducible promoters can be used (see Ausubel et al., supra, 1987, see chapter 13; Grant et al., Meth. Enzymol. 153:516-544, 1987; Glover, DNA Cloning Vol. II (IRL Press, 1986), see chapter 3; Bitter, Meth. Enzymol. 152:673-684, 1987; see, also, The Molecular Biology of the Yeast Saccharomyces (Eds., Strathern et al., Cold Spring Harbor Laboratory Press, 1982), Vols. I and II). A constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL can be used (Rothstein, DNA Cloning Vol. II (supra, 1986), chapter 3). Alternatively, vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome.
Eukaryotic systems, particularly mammalian expression systems, allow for proper post-translational modifications of expressed mammalian proteins. Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and advantageously, plasma membrane insertion of the gene product can be used as host cells for the expression of a GDF receptor polypeptide, or functional peptide portion thereof.
Mammalian cell systems which utilize recombinant viruses or viral elements to direct expression can be engineered. For example, when using adenovirus expression vectors, the GDF receptors coding sequence can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. Alternatively, the vaccinia virus 7.5K promoter can be used (Mackett et al., Proc. Natl. Acad. Sci., USA 79:7415-7419, 1982; Mackett et al., J. Virol. 49:857-864, 1984; Panicali et al., Proc. Natl. Acad. Sci., USA 79:4927-4931, 1982). Particularly useful are bovine papilloma virus vectors, which can replicate as extrachromosomal elements (Sarver et al., Mol. Cell. Biol. 1:486, 1981). Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host cell chromosome, thereby yielding a high level of expression. These vectors can be used for stable expression by including a selectable marker in the plasmid, such as, for example, the neo gene. Alternatively, the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the GDF receptors gene in host cells (Cone and Mulligan, Proc. Natl. Acad. Sci., USA 81:6349-6353, 1984). High level expression can also be achieved using inducible promoters, including, but not limited to, the metallothionein IIA promoter and heat shock promoters.
For long term, high yield production of recombinant proteins, stable expression is preferred. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the GDF receptors cDNA controlled by appropriate expression control elements such as promoter, enhancer, sequences, transcription terminators, and polyadenylation sites, and a selectable marker. The selectable marker in the recombinant plasmid can confer resistance to the selection, and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which, in turn can be cloned and expanded into cell lines. For example, following the introduction of foreign DNA, engineered cells can be allowed to grow for 1 to 2 days in an enriched media, and then are switched to a selective media. A number of selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci., USA 48:2026, 1982), and adenine phosphoribosyltransferase (Lowy, et al., Cell 22:817, 1980) genes can be employed in tk-, hgprt- or aprt-cells respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., Proc. Natl. Acad. Sci. USA 77:3567, 1980; O'Hare et al., Proc. Natl. Acad. Sci., USA 78: 1527, 1981); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci., USA 78:2072, 1981); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147, 1984) genes. Additional selectable genes, including trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman and Mulligan, Proc. Natl. Acad. Sci., USA 85:8047, 1988); and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, Curr. Comm. Mol. Biol. (Cold Spring Harbor Laboratory Press, 1987), also have been described.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors can be used. Eukaryotic cells can also be cotransformed with DNA sequences encoding the GDF receptors of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein. (Gluzman, Eukaryotic Viral Vectors (Cold Spring Harbor Laboratory Press, 1982)).
The invention also provides stable recombinant cell lines, the cells of which express GDF receptor polypeptides and contain DNA that encodes GDF receptors. Suitable cell types include, but are not limited to, NIH 3T3 cells (murine), C2C12 cells, L6 cells, and P19 cells. C2C12 and L6 myoblasts differentiate spontaneously in culture and form myotubes depending on the particular growth conditions (Yaffe and Saxel, Nature 270:725-727, 1977; Yaffe, Proc. Natl. Acad. Sci., USA 61:477-483, 1968). P19 is an embryonal carcinoma cell line. Such cells are described, for example, in the Cell Line Catalog of the American Type Culture Collection (ATCC). These cells can be stably transformed using well known methods (see, for example, Ausubel et al., supra, 1995, see sections 9.5.1-9.5.6).
A GDF receptor can be expressed from a recombinant polynucleotide of the invention using inducible or constitutive regulatory elements, as described herein. The desired protein encoding sequence and an operably linked promoter can be introduced into a recipient cell either as a non-replicating DNA (or RNA) molecule, which can either be a linear molecule or a covalently closed circular molecule. Expression of the desired molecule can occur due to transient expression of the introduced sequence, or the polynucleotide can be stably maintained in the cell, for example, by integration into a host cell chromosome, thus allowing a more permanent expression. Accordingly, the cells can be stably or transiently transformed (transfected) cells.
An example of a vector that can be employed is one which is capable of integrating the desired gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker can complement an auxotrophy in the host such as leu2, or ura3, which are common yeast auxotrophic markers; can confer a biocide resistance, for example, to an antibiotic or to heavy metal ions such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or can be introduced into the same cell by cotransfection.
The introduced sequence can be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a variety of vectors can be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include the ease with which recipient cells that contain the vector can be recognized and selected from those cells that do not contain the vector; the number of copies of the vector desired in a particular host cell; and whether it is desirable to be able to “shuttle” the vector between host cells of different species.
For a mammalian host, several vector systems are available for expression. One class of vectors utilizes DNA elements that provide autonomously replicating extra-chromosomal plasmids derived from animal viruses, for example, a bovine papilloma virus, polyoma virus, adenovirus, or SV40 virus. A second class of vectors includes vaccinia virus expression vectors. A third class of vectors relies upon the integration of the desired gene sequences into the host chromosome. Cells that have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers genes (as described above), which allow selection of host cells that contain the expression vector. The selectable marker gene can be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransfection. Additional elements can be included to provide for optimal synthesis of an encoded mRNA or peptide, including, for example, splice signals, transcription promoters or enhancers, and transcription or translation termination signals. cDNA expression vectors incorporating appropriate regulatory elements are well known in the art (see, for example, Okayama, Mol. Cell. Biol. 3:280, 1983).
Once the vector or DNA sequence containing the construct has been prepared for expression, the DNA construct can be introduced into an appropriate host. Various methods can be used for introducing the polynucleotide into a cell, including, for example, methods of transfection or transformation such as protoplast fusion, calcium phosphate precipitation, and electroporation or other conventional techniques, for example, infection where the vector is a viral vector
An agent that has been identified as having the ability to specifically interact with a myostatin receptor such as the Act RIIB receptor, can be used to screen for additional myostatin receptors or other GDF receptors. Such a method can include incubating components such as the agent (or myostatin or other GDF) and a cell expressing a GDF receptor, which can be a truncated membrane bound receptor or a soluble receptor, under conditions sufficient to allow the agent (or GDF) to interact specifically with the receptor; measuring the agent (or GDF) bound to the receptor; and isolating the receptor. A method of molecular modeling as described above also can be useful as a screening method for identifying a GDF receptor, or a functional peptide portion thereof.
An agent that alters a specific interaction of myostatin with its receptor can act, for example, by binding to myostatin such that it cannot interact specifically with its cellular receptor, by competing with myostatin for binding to its receptor, or by otherwise by-passing the requirement that myostatin specifically interact with its receptor in order to induce myostatin signal transduction. A truncated myostatin receptor such as a soluble extracellular domain of a myostatin receptor is an example of an agent that can bind myostatin, thereby sequestering myostatin and reducing or inhibiting its ability to interact specifically with a cell surface myostatin receptor. A myostatin prodomain or functional peptide portion thereof is another example of an agent that can bind myostatin, thereby reducing or inhibiting the ability of the myostatin to interact specifically with a cell surface myostatin receptor. Such myostatin antagonists are useful in practicing a method of the invention, particularly for reducing or inhibiting myostatin signal transduction in a cell.
Follistatin and FLRG are other examples of agents that can bind to myostatin, thereby reducing or inhibiting the ability of myostatin to interact specifically with its receptor. Follistatin can bind to and inhibit the activity of various TGF-β family members, including myostatin (GDF-8; U.S. Pat. No. 6,004,937, incorporated herein by reference) and GDF-11 (Gamer et al., Devel. Biol. 208:222-232, 1999) and, therefore, can be used for performing a method as disclosed. Although the use of follistatin for modulating the effects of myostatin previously has been described (U.S. Pat. No. 6,004,937), and that follistatin reduces or inhibits the ability of myostatin to interact specifically with a myostatin receptor such as Act RIIB (U.S. Pat. No. 6,891,082, incorporated herein by reference), it was not known, prior to the present disclosure, that FLRG interacts with other ligands so as to result in a greater increase in muscle tissue growth as compared to muscle tissue growth resulting from inhibition of myostatin expression or activity alone.
In another embodiment, the agent is a substantially purified proteolytic fragment of a growth differentiation factor (GDF) polypeptide (a pro-GDF polypeptide) or a functional peptide portion thereof. Proteolytic fragments of a pro-GDF polypeptide are exemplified herein by proteolytic fragments of a promyostatin polypeptide and proteolytic fragments of a pro-GDF-11 polypeptide. As disclosed herein, a peptide portion of a pro-GDF polypeptide that is equivalent to a proteolytic fragment of a pro-GDF can be produced by a chemical method or a recombinant DNA method. In view of the present disclosure, proteolytic fragments of other GDF polypeptides readily can be made and used.
In general, peptides corresponding to proteolytic fragments of a pro-GDF polypeptide are exemplified by a carboxy terminal (C-terminal) mature GDF fragment, which can interact specifically with a GDF receptor and affect GDF signal transduction, and an amino terminal prodomain fragment, which can include a signal peptide and, as disclosed herein, can interact specifically with a pro-GDF polypeptide or mature GDF peptide and affect its ability to effect GDF signal transduction. For example, proteolytic fragments of a promyostatin polypeptide include a C-terminal mature myostatin peptide, which can interact specifically with a myostatin receptor and induce myostatin signal transduction; and an amino terminal prodomain fragment, which can interact specifically with myostatin, thereby reducing or inhibiting the ability of myostatin to induce myostatin signal transduction.
A proteolytic fragment of a pro-GDF polypeptide, or a functional peptide portion thereof, is characterized, in part, by having or affecting an activity associated with the stimulation or inhibition of GDF signal transduction. For example, a promyostatin polypeptide or functional peptide portion thereof can have myostatin receptor binding activity, myostatin signal transduction stimulatory or inhibitory activity, myostatin binding activity, promyostatin binding activity, or a combination thereof. Thus, the term “functional peptide portion,” when used herein in reference to a pro-GDF polypeptide, means a peptide portion of the pro-GDF polypeptide that can interact specifically with its receptor and stimulate or inhibit GDF signal transduction; that can interact specifically with a mature GDF or a pro-GDF; or that exhibits cellular localization activity, i.e., the activity of a signal peptide. It should be recognized that a functional peptide portion of full length mature myostatin peptide, for example, need not have the same activity of the mature myostatin, including the ability to stimulate myostatin signal transduction, since functional peptide portions of the mature peptide can have, for example, an ability to specifically interact with a myostatin receptor without also having the ability to activate the signal transduction pathway. Methods for identifying such a functional peptide portion of a pro-GDF polypeptide, which can be useful as a myostatin antagonist, are disclosed herein or otherwise known in the art. Thus, in one embodiment, a functional peptide portion of a promyostatin polypeptide can interact specifically with a myostatin receptor, and can act as an agonist to stimulate myostatin signal transduction or as an antagonist to reduce or inhibit myostatin signal transduction.
In another embodiment, a functional peptide portion of a promyostatin polypeptide interacts specifically with a promyostatin polypeptide or with a mature myostatin peptide, thereby blocking myostatin signal transduction. Such a functional peptide portion of promyostatin can act, for example, by preventing cleavage of a promyostatin polypeptide to mature myostatin; by forming a complex with a mature myostatin peptide; or by some other mechanism. Where a peptide-myostatin complex is formed, the complex can block myostatin signal transduction, for example, by reducing or inhibiting the ability of the myostatin to interact specifically with its receptor, or by binding to the receptor in the form that lacks the ability to induce myostatin signal transduction.
In another embodiment, an agent useful in the methods of the invention is GDF a peptide variant. Exemplary variants include, but are not limited to, analogs, homologs, muteins and mimetics of a GDF peptide or GDF receptor that retain the ability to specifically bind to their respective GDF receptor or GDFs peptide, respectively. Peptides of the GDF receptors refer to portions of the amino acid sequence of GDF receptors that have these abilities. The variants can be generated directly from GDF peptides or receptors themselves by chemical modification, by proteolytic enzyme digestion, or by combinations thereof. Additionally, genetic engineering techniques, as well as methods of synthesizing polypeptides directly from amino acid residues, can be employed.
Peptides can be synthesized by such commonly used methods as t-BOC or FMOC protection of alpha-amino groups. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C terminus of the peptide (Coligan, et al., Current Protocols in Immunology (Wiley Interscience, 1991), Unit 9, which is incorporated herein by reference). Peptides useful in the methods of the invention can also be synthesized by the well known solid phase peptide synthesis methods (Merrifield, J. Am. Chem. Soc., 85:2149, 1962; Stewart and Young, Solid Phase Peptides Synthesis (Freeman, San Francisco, 1969), see pages 27-62, each of which is incorporated herein by reference), using a copoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer. On completion of chemical synthesis, the peptides can be deprotected and cleaved from the polymer by treatment with liquid HF-10% anisole for about 1⁄4-1 hours at 0° C. After evaporation of the reagents, the peptides are extracted from the polymer with 1% acetic acid solution which is then lyophilized to yield the crude material. This can normally be purified by such techniques as gel filtration on Sephadex G-15 using 5% acetic acid as a solvent. Lyophilization of appropriate fractions of the column will yield the homogeneous peptide or peptide derivatives, which can then be characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and quantitated by the solid phase Edman degradation.
Non-peptide compounds that mimic the binding and function of GDF peptides or GDF receptors (“mimetics”) can be produced by the approach outlined by Saragovi et al. (Science 253: 792-95, 1991, which is incorporated herein by reference). Mimetics are molecules which mimic elements of protein secondary structure (Johnson et al., “Peptide Turn Mimetics,” in Biotechnology and Pharmacy (Pezzuto et al., Eds.; Chapman and Hall, New York 1993), which is incorporated herein by reference). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions. For the purposes of the present invention, an appropriate mimetic can be considered to be the equivalent of a GDF peptide or GDF receptor.
In another embodiment, an agent useful in the methods of the invention is a GDF receptor-binding agent that blocks the specific binding of a GDF to its receptor. Such agents are also useful, for example, as research and diagnostic tools in the study of muscle wasting disorder as described above and as effective therapeutics, and can be identified using the methods as disclosed herein, for example, a molecular modeling method. In addition, pharmaceutical compositions comprising GDF receptor-binding agents can represent effective therapeutics. In the context of the invention, the phrase “GDF receptor-binding agent” denotes a naturally occurring ligand of a GDF receptor, for example, myostatin; a synthetic ligand of GDF receptors, or an appropriate derivative of the natural or synthetic ligands. The determination and isolation of ligands is well known in the art (Lerner, Trends Neurosci. 17:142-146, 1994, which is incorporated herein by reference).
In another embodiment, the present invention includes GDF receptor-binding agents that interfere with binding between a GDF receptor and a GDF. Such binding agents can interfere by competitive inhibition, by non-competitive inhibition or by uncompetitive inhibition. Interference with normal binding between GDF receptors and one or more GDF can result in a useful pharmacological effect.
Thus, an agent useful in a method of the invention can modulate the level or activity of an intracellular polypeptide involved in a myostatin signal transduction pathway. As disclosed herein, regulation of muscle growth by myostatin can involve components of a signal transduction pathway that is activated by activin type II receptors (see Examples 7 and 9; see, also, Example 14). Myostatin specifically interacts with activin type IIB receptors (Act RIIB) expressed in COS cells in culture (Example 7). The low affinity of binding indicates that myostatin binding to Act RIIB in vivo may involve other factors, similar to TGF-β, which has significantly higher affinity for the type II receptor when the type I receptor also is present (Attisano et al., Cell 75:671-680, 1993), or to other systems that require other molecules for presenting the ligand to the signaling receptor (Massague, supra, 1998; Wang et al., Cell 67:795-805, 1991).
The specific interaction of myostatin with Act RIIB indicates that myostatin signal transduction can involve components of the Smad signal transduction pathway. Thus, the Smad signal transduction pathway provides a target for modulating the effect of myostatin on a cell, and agents that affect the Smad pathway can be useful for modulating myostatin signal transduction in a cell.
Agents useful for modulating the level or activity of intracellular polypeptide components of a GDF signal transduction include agonists, which can increase signal transduction activity, and antagonists, which can reduce or inhibit signal transduction activity. With respect to myostatin, for example, agents that can increase myostatin signal transduction activity are exemplified by phosphatase inhibitors, which can reduce or inhibit dephosphorylation of Smad polypeptides, thereby prolonging the signal transducing activity of the Smad. Dominant negative Smad 6 or Smad 7 polypeptides, which can negate the inhibitory effect of Smad 6 and Smad 7 on myostatin signal transduction, are additional examples of agents that can increase myostatin signal transduction activity by increasing Smad signal transduction.
As used herein, the term “myostatin signal transduction” refers to the series of events, generally a series of protein-protein interactions, that occurs in a cell due to the specific interaction of myostatin with a myostatin receptor expressed on the surface of the cell. As such, myostatin signal transduction can be detected, for example, by detecting a specific interaction of myostatin with its receptor on a cell, by detecting phosphorylation of one or more polypeptides involved in a myostatin signal transduction pathway in the cell, by detecting expression of one or more genes that are specifically induced due to myostatin signal transduction, or by detecting a phenotypic change that occurs in response to myostatin signal transduction (see Examples). As disclosed herein, an agent useful in a method of the invention can act as an agonist to stimulate myostatin signal transduction or as an antagonist to reduce or inhibit myostatin signal transduction.
A myostatin signal transduction pathway is exemplified herein by the Smad pathway, which is initiated upon myostatin specifically interacting with the extracellular domain of an activin type II receptor and propagated through interactions of intracellular polypeptides, including Smad proteins, in the cell. In general, myostatin signal transduction is associated with phosphorylation or dephosphorylation of specific intracellular polypeptides such as Smad polypeptides. Thus, myostatin signal transduction in a cell can be detected by detecting an increased level of phosphorylation of one or more Smad polypeptides in the presence of myostatin as compared to the level of phosphorylation of the polypeptides in the absence of myostatin. A method of the invention provides a means to increase or decrease myostatin signal transduction and, therefore, the level of phosphorylation of an Smad polypeptide involved in a myostatin signal transduction pathway will be increased above a normal level or decreased below a level expected in the presence of myostatin, respectively.
Antagonist agents that can reduce or inhibit myostatin signal transduction activity are exemplified by dominant negative Smad polypeptides such as dominant negative Smad 2, Smad 3 or Smad 4, in which the C-terminal phosphorylation sites have been mutated. The inhibitory Smad polypeptides such as Smad 6 and Smad 7, which inhibit the activation of Smad 2 and Smad 3; and a c-ski polypeptide, which binds Smad polypeptides and inhibits signal transduction, are additional examples of antagonists useful for reducing or inhibiting myostatin signal transduction by decreasing Smad signal transduction.
Where the agent that acts intracellularly is a peptide, it can be contacted with the cell directly, or a polynucleotide encoding the peptide (or polypeptide) can be introduced into the cell and the peptide can be expressed in the cell. 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 the peptide agent, which can be expressed in the cell. The expressed peptide agent can be a mutant GDF receptor or peptide portion thereof. Example of a mutant GDF receptor include a kinase-deficient form of a myostatin receptor such as a dominant negative Act RIIA or Act RIIB, which can, but need not, have the ability to specifically bind a ligand (e.g., myostatin); and a truncated myostatin or other GDF receptor, such as a soluble form of a myostatin receptor, which binds myostatin, thereby sequestering it from interacting specifically with a cellular myostatin receptor; a dominant-negative form of a Smad polypeptide such as a dominant negative Smad 3, in which the C-terminal phosphorylation sites have been mutated (Liu et al., Proc. Natl. Acad. Sci., USA 94:10669-10674, 1997); a Smad 7 polypeptide, which inhibits the activation of Smad 2 and Smad 3 (Heldin et al., Nature 390:465-471, 1997); or a c-ski polypeptide, which can bind a Smad polypeptide and inhibit signal transduction by the Smad (Suprave et al., Genes Devel. 4:1462-1472, 1990).
Expression of a c-ski peptide agent in a cell can be particularly useful for modulating myostatin signal transduction. Mice lacking c-ski show a severe reduction in skeletal muscle mass (Berk et al., Genes Devel. 11:2029-2039, 1997), whereas transgenic mice that overexpress c-ski in muscle show dramatic muscle hypertrophy (Suprave et al., supra, 1990). c-ski interacts with and blocks the activity of certain Smad proteins, including Smad 2, Smad 3 and Smad 4, which mediate signaling of TGF-β and activin type II receptors (Luo et al., Genes Devel. 13:2196-1106, 1999; Stroschein et al., Science 286:771-774, 1999; Sun et al., Mol. Cell. 4:499-509, 1999a; Sun et al., Proc. Natl. Acad. Sci., USA 96:112442-12447, 1999b; Akiyoshi et al., J. Biol. Chem. 274:35269, 1999). Thus, in view of the present disclosure that myostatin activity can be mediated through Act RIIB binding, it will be recognized that the activity of myostatin, or of any GDF that utilizes an Smad pathway, can be modulated by increasing or decreasing c-ski expression in a target cell.
In another embodiment, an agent useful in a method of the invention can be a polynucleotide, which can be contacted with or introduced into a cell as described above. Generally, but not necessarily, the polynucleotide is introduced into the cell, where it effects its function either directly, or following transcription or translation or both. For example, the polynucleotide can encode a peptide agent, which is expressed in the cell and modulates myostatin activity. Such an expressed peptide can be, for example, a mutant promyostatin polypeptide, which cannot be cleaved into active myostatin; or can be a mutant myostatin receptor, for example, a truncated myostatin receptor extracellular domain; a myostatin receptor extracellular domain operatively associated with a membrane anchoring domain; or a mutant myostatin receptor lacking protein kinase activity. Methods for introducing a polynucleotide into a cell are exemplified below or otherwise known in the art.
A polynucleotide agent useful in a method of the invention also can be, or can encode, an antisense molecule, a ribozyme, interfering RNA, or a triplexing agent. For example, the polynucleotide can be (or can encode) an antisense nucleotide sequence such as an antisense c-ski nucleotide sequence, which can act as an agonist to increase myostatin signal transduction in a cell; or an antisense Smad nucleotide sequence, which can act either as an agonist to increase myostatin signal transduction or as antagonist to reduce or inhibit myostatin signal transduction, depending on the particular Smad antisense nucleotide sequence. Such polynucleotides can be contacted directly with a target cell and, upon uptake by the cell, can effect their antisense, ribozyme or triplexing activity; or can be encoded by a polynucleotide that is introduced into a cell, whereupon the polynucleotide is expressed to produce, for example, an antisense or interfering RNA molecule or ribozyme, which effects its activity.
An antisense polynucleotide, ribozyme or triplexing agent is complementary to a target sequence, which can be a DNA or RNA sequence, for example, messenger RNA, and can be a coding sequence, a nucleotide sequence comprising an intron-exon junction, a regulatory sequence such as a Shine-Delgarno sequence, or the like. The degree of complementarity is such that the polynucleotide, for example, an antisense polynucleotide, can interact specifically with the target sequence in a cell. Depending on the total length of the antisense or other polynucleotide, one or a few mismatches with respect to the target sequence can be tolerated without losing the specificity of the polynucleotide for its target sequence. Thus, few if any mismatches would be tolerated in an antisense molecule consisting, for example, of 20 nucleotides, whereas several mismatches will not affect the hybridization efficiency of an antisense molecule that is complementary, for example, to the full length of a target mRNA encoding a cellular polypeptide. The number of mismatches that can be tolerated can be estimated, for example, using well known formulas for determining hybridization kinetics (see Sambrook et al., supra, 1989) or can be determined empirically using methods as disclosed herein or otherwise known in the art, particularly by determining that the presence of the antisense polynucleotide, ribozyme, or triplexing agent in a cell decreases the level of the target sequence or the expression of a polypeptide encoded by the target sequence in the cell.
A polynucleotide useful as an antisense molecule, a ribozyme or a triplexing agent can inhibit translation or cleave the nucleic acid molecule, thereby modulating myostatin signal transduction in a cell. An antisense molecule, for example, can bind to an mRNA to form a double stranded molecule that cannot be translated in a cell. Antisense oligonucleotides of at least about 15 to 25 nucleotides are preferred since they are easily synthesized and can hybridize specifically with a target sequence, although longer antisense molecules can be expressed from a polynucleotide introduced into the target cell. Specific nucleotide sequences useful as antisense molecules can be identified using well known methods, for example, gene walking methods (see, for example, Seimiya et al., J. Biol. Chem. 272:4631-4636 (1997), which is incorporated herein by reference). Where the antisense molecule is contacted directly with a target cell, it can be operatively associated with a chemically reactive group such as iron-linked EDTA, which cleaves a target RNA at the site of hybridization. A triplexing agent, in comparison, can stall transcription (Maher et al., Antisense Res. Devel. 1:227 (1991); Helene, Anticancer Drug Design 6:569 (1991)). Thus, a triplexing agent can be designed to recognize, for example, a sequence of a Smad gene regulatory element, thereby reducing or inhibiting the expression of a Smad polypeptide in the cell, thereby modulating myostatin signal transduction in a target cell.
In another embodiment, an agent useful in a method of the invention can be proteolytic fragments of a pro-GDF polypeptide. Proteolytic fragments of a pro-GDF polypeptide can be produced by cleavage of the polypeptide at a proteolytic cleavage site having a consensus amino acid sequence Arg-Xaa-Xaa-Arg (SEQ ID NO: 21). Such proteolytic recognition sites are exemplified by the Arg-Ser-Arg-Arg (SEQ ID NO: 22) sequence shown as amino acid residues 263 to 266 in SEQ ID NO: 1 (promyostatin) or amino acid residues 295 to 298 of SEQ ID NO: 25 (human pro-GDF-11; see, also, relative positions 267 to 270 of FIG. 2), and by the Arg-Ile-Arg-Arg (SEQ ID NO: 23) sequence shown as amino acid residues 263 to 266 in SEQ ID NO: 20.
In addition to the proteolytic cleavage site for the signal peptide, promyostatin polypeptides, for example, contain two additional potential proteolytic processing sites (Lys-Arg and Arg-Arg). Cleavage of a promyostatin polypeptide at or near the latter proteolytic processing site, which is contained within the consensus Arg-Xaa-Xaa-Arg (SEQ ID NO: 21) proteolytic cleavage recognition site (see, for example, amino acid residues 263 to 266 of SEQ ID NO: 2), generates a biologically active C-terminal mature human myostatin fragment. The exemplified full length mature myostatin peptides contain about 103 to about 109 amino acids and have a predicted molecular weight of approximately 12,400 daltons (Da). In addition, myostatin can form dimers, which have an expected molecular weight of about 23 to 30 kiloDaltons (kDa). The dimers can be myostatin homodimers or can be heterodimers, for example, with GDF-11 or another GDF or TGF-β family member.
A proteolytic fragment of the invention is exemplified by a GDF prodomain, for example, a myostatin prodomain, which includes about amino acid residues 20 to 262 of a promyostatin polypeptide, or a functional peptide portion thereof, or a GDF-11 prodomain, which includes about amino acid residues 20 to 295 of a pro-GDF-11 polypeptide, or a functional peptide portion thereof, each of which can further contain the signal peptide comprising about amino acids 1 to 20 of the respective pro-GDF polypeptide. Myostatin prodomains are further exemplified by about amino acid residues 20 to 263 as set forth in SEQ ID NO: 4 and SEQ ID NO: 6; as well as by about amino acid residues about 20 to 262 as set forth in SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 20, which can be produced by proteolytic cleavage of a corresponding promyostatin polypeptide, can be chemically synthesized, or can be expressed from a recombinant polynucleotide encoding the proteolytic fragment. A functional peptide portion of a myostatin prodomain is exemplified by a peptide portion of a myostatin prodomain that can interact specifically with myostatin or with promyostatin. A GDF-11 prodomain is exemplified by about amino acid residues 20 to 295 of SEQ ID NO: 25, which can further include the signal peptide comprising about amino acid residues 1 to 20 of SEQ ID NO: 25, and a functional peptide portion of a GDF-11 prodomain is exemplified by a peptide portion of a GDF-11 prodomain that can specifically interact with mature GDF-11 or a pro-GDF-11 polypeptide. Preferably, the functional peptide portion of a GDF prodomain inhibits the ability of the corresponding GDF or a related GDF to stimulate signal transduction, for example, by reducing or inhibiting the ability of the GDF to interact specifically with its receptor, or by binding to the receptor as an inactive complex. In one embodiment, the present invention includes a functional fragment of a pro-GDF polypeptide, particularly a functional fragment of a GDF prodomain, operably linked to a GDF signal peptide, preferably a myostatin signal peptide or a GDF-11 signal peptide comprising about the first 15 to 30 amino terminal amino acids of promyostatin or pro-GDF-11, respectively.
As disclosed herein, a myostatin prodomain or GDF-11 prodomain can interact with mature myostatin, thereby reducing or inhibiting the ability of the mature GDF to interact specifically with its receptor (see Examples 7 and 8). Thus, a functional peptide portion of a myostatin prodomain, for example, can be obtained by examining peptide portions of a myostatin prodomain using methods as provided herein, and identifying functional peptide portions of the prodomain that can interact specifically with myostatin or with promyostatin and can reduce or inhibit the ability of myostatin to interact specifically with a myostatin receptor or to stimulate myostatin signal transduction.
A functional peptide portion of a myostatin prodomain that can specifically interact with myostatin, or a functional peptide portion of another GDF prodomain, also can be identified using any of various assays known to be useful for identifying specific protein-protein interactions. Such assays include, for example, methods of gel electrophoresis, affinity chromatography, the two hybrid system of Fields and Song (Nature 340:245-246, 1989; see, also, U.S. Pat. No. 5,283,173; Fearon et al., Proc. Natl. Acad. Sci., USA 89:7958-7962, 1992; Chien et al., Proc. Natl. Acad. Sci. USA 88:9578-9582, 1991; Young, Biol. Reprod. 58:302-311 (1998), each of which is incorporated herein by reference), the reverse two hybrid assay (Leanna and Hannink, Nucl. Acids Res. 24:3341-3347, 1996, which is incorporated herein by reference), the repressed transactivator system (U.S. Pat. No. 5,885,779, which is incorporated herein by reference), the phage display system (Lowman, Ann. Rev. Biophys. Biomol. Struct. 26:401-424, 1997, which is incorporated herein by reference), GST/HIS pull down assays, mutant operators (WO 98/01879, which is incorporated herein by reference), the protein recruitment system (U.S. Pat. No. 5,776,689, which is incorporated herein by reference), and the like (see, for example, Mathis, Clin. Chem. 41:139-147, 1995 Lam, Anticancer Drug Res. 12:145-167, 1997; Phizicky et al., Microbiol. Rev. 59:94-123, 1995; each of which is incorporated herein by reference).
A functional peptide portion of a GDF prodomain also can be identified using methods of molecular modeling. For example, an amino acid sequence of a mature myostatin peptide can be entered into a computer system having appropriate modeling software, and a three dimensional representation of the myostatin (“virtual myostatin”) can be produced. A promyostatin amino acid sequence also can be entered into the computer system, such that the modeling software can simulate portions of the promyostatin sequence, for example, portions of the prodomain, and can identify those peptide portions of the prodomain that can interact specifically with the virtual myostatin. A base line for a specific interaction can be predefined by modeling the virtual myostatin and a full length promyostatin prodomain, and identifying the amino acid residues in the virtual myostatin that are “contacted” by the prodomain, since such an interaction is known to inhibit the activity of the myostatin.
It should be recognized that such methods, including two hybrid assays and molecular modeling methods, also can be used to identify other specifically interacting molecules encompassed within the present invention. Thus, methods such as the two hybrid assay can be used to identify a GDF receptor such as a myostatin receptor using, for example, a myostatin peptide or a peptide portion thereof that specifically interacts with an Act RIIA or Act RIIB receptor as one binding component of the assay, and identifying a GDF receptor, which specifically interacts with the myostatin peptide. Similarly, methods of molecular modeling can be used to identify an agent that interacts specifically with a mature GDF peptide such as mature myostatin, or with a GDF receptor and, therefore, can be useful as an agonist or an antagonist of signal transduction mediated by the GDF or the GDF receptor. Such an agent can be, for example, a functional peptide portion of a myostatin prodomain or GDF-11 prodomain, or a chemical agent that mimics the action of the GDF prodomain.
Modeling systems useful for the purposes disclosed herein can be based on structural information obtained, for example, by crystallographic analysis or nuclear magnetic resonance analysis, or on primary sequence information (see, for example, Dunbrack et al., “Meeting review: the Second meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP2) (Asilomar, Calif., Dec. 13-16, 1996). Fold Des. 2 (2): R27-42, (1997); Fischer and Eisenberg, Protein Sci. 5:947-55, 1996; (see, also, U.S. Pat. No. 5,436,850); Havel, Prog. Biophys. Mol. Biol. 56:43-78, 1991; Lichtarge et al., J. Mol. Biol. 274:325-37, 1997; Matsumoto et al., J. Biol. Chem. 270:19524-31, 1995; Sali et al., J. Biol. Chem. 268:9023-34, 1993; Sali, Molec. Med. Today 1:270-7, 1995a; Sali, Curr. Opin. Biotechnol. 6:437-51, 1995b; Sali et al., Proteins 23: 318-26, 1995c; Sali, Nature Struct. Biol. 5:1029-1032, 1998; U.S. Pat. Nos. 5,933,819; 5,265,030, each of which is incorporated herein by reference).
The crystal structure coordinates of a promyostatin polypeptide or a GDF receptor can be used to design compounds that bind to the protein and alter its physical or physiological properties in a variety of ways. The structure coordinates of the protein can also be used to computationally screen small molecule data bases for agents that bind to the polypeptide to develop modulating or binding agents, which can act as agonists or antagonists of GDF signal transduction. Such agents can be identified by computer fitting kinetic data using standard equations (see, for example, Segel, “Enzyme Kinetics” (J. Wiley & Sons 1975), which is incorporated herein by reference).
Methods of using crystal structure data to design inhibitors or binding agents are known in the art. For example, GDF receptor coordinates can be superimposed onto other available coordinates of similar receptors, including receptors having a bound inhibitor, to provide an approximation of the way the inhibitor interacts with the receptor. Computer programs employed in the practice of rational drug design also can be used to identify compounds that reproduce interaction characteristics similar to those found, for example, between a mature myostatin and a co-crystallized myostatin prodomain. Detailed knowledge of the nature of the specific interactions allows for the modification of compounds to alter or improve solubility, pharmacokinetics, and the like, without affecting binding activity.
Where it is desired to identify a chemical entity that interacts specifically with myostatin or with a GDF receptor, any of several methods to screen chemical entities or fragments for their ability to interact specifically with the molecule can be used. This process may begin by visual inspection, for example, of myostatin and a myostatin prodomain on the computer screen. Selected peptide portions of the prodomain, or chemical entities that can act as mimics, then can be positioned in a variety of orientations, or docked, within an individual binding site of the myostatin. Docking can be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARMM and AMBER.
Specialized computer programs can be particularly useful for selecting peptide portions of a prodomain, or chemical entities useful, for example, as a GDF receptor agonist or antagonist. Such programs include, for example, GRID (Goodford, J. Med. Chem., 28:849-857, 1985; available from Oxford University, Oxford, UK); MCSS (Miranker and Karplus, Proteins: Structure. Function and Genetics 11:29-34, 1991, available from Molecular Simulations, Burlington Mass.); AUTODOCK (Goodsell and Olsen, Proteins: Structure. Function, and Genetics 8:195-202, 1990, available from Scripps Research Institute, La Jolla Calif.); DOCK (Kuntz, et al., J. Mol. Biol. 161:269-288, 1982, available from University of California, San Francisco Calif.), each of which is incorporated herein by reference.
Suitable peptides or agents that have been selected can be assembled into a single compound or binding agent. Assembly can be performed by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen, followed by manual model building using software such as Quanta or Sybyl. Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include, for example, CAVEAT (Bartlett et al, Special Pub., Royal Chem. Soc. 78:182-196, 1989, available from the University of California, Berkeley Calif.); 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro Calif.; for review, see Martin, J. Med. Chem. 35:2145-2154, 1992); HOOK (available from Molecular Simulations, Burlington, Mass.), each of which is incorporated herein by reference.
In addition to the method of building or identifying such specifically interacting agents in a step-wise fashion, one fragment or chemical entity at a time as described above, the agents can be designed as a whole or de novo using either an empty active site or, optionally, including some portions of a known agent that specifically interacts, for example, a full length myostatin prodomain, which interacts specifically with myostatin. Such methods include, for example, LUDI (Bohm, J. Comp. Aid. Molec. Design 6:61-78, 1992, available from Biosym Technologies, San Diego Calif.); LEGEND (Nishibata and Itai, Tetrahedron 47:8985, 1991, available from Molecular Simulations, Burlington Mass.); LeapFrog (available from Tripos Associates, St. Louis Mo.), and those described by Cohen et al. (J. Med. Chem. 33:883-894, 1990) and by Navia and Murcko, Curr. Opin. Struct. Biol. 2:202-210, 1992, each of which is incorporated herein by reference). Additional molecular modeling methods and computer software examples are provided in U.S. Pat. No. 6,891,082, incorporated herein by reference in its entirety.
Thus, specific inhibitors of myostatin expression or activity can be identified by the methods disclosed herein. It will therefore be appreciated that one can identify a peptide portion of a promyostatin prodomain that can interact specifically with myostatin and reduce or inhibit the ability of myostatin to interact with its receptor or otherwise affect the ability of the myostatin to effect signal transduction. Similarly, one can identify small organic molecules that mimic the action of a GDF prodomain, thereby reducing or inhibiting myostatin signal transduction. The methods also can be used to identify agents that interact specifically with a GDF receptor, for example, an Act RITA, Act RIIB or other GDF receptor, such agents being useful as GDF receptor agonists or antagonists, which can modulate GDF signal transduction in a cell. In addition, the methods provide a means to identify previously unknown pro-GDF polypeptides or GDF receptors, for example, by identifying conserved structural features of the particular polypeptides.
Similar to other members of the TGF-β superfamily, active GDF peptides are expressed as precursor polypeptides, which are cleaved to a mature, biologically active form. Accordingly, in still another embodiment, the proteolytic fragment of a pro-GDF polypeptide is a mature GDF peptide, or a functional peptide portion of a mature GDF peptide, where, as discussed above, the functional peptide portion can have the activity of a GDF agonist or antagonist. The proteolytic fragment can be a mature C-terminal myostatin peptide, which includes about amino acid residues 268 to 374 of a promyostatin polypeptide (see FIG. 1; see, also, FIG. 2), or a mature C-terminal GDF-11 peptide, which includes about amino acid residues 299 to 407 of a pro-GDF-11 polypeptide. Full length mature myostatin peptides are exemplified by amino acid residues about 268 to 375 as set forth in SEQ ID NO: 4 and SEQ ID NO: 6; by amino acid residues about 267 to 374 as set forth in SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 20, and by amino acid residues about 49 to 157 of SEQ ID NO: 27 and amino acid residues about 28 to 136 of SEQ ID NO: 29. A full length mature GDF-11 peptide is exemplified by amino acid residues about 299 to 407 of SEQ ID NO: 25. Functional peptide portions of the mature GDF peptides are exemplified by peptide portions of mature myostatin or mature GDF-11 that have an agonist or antagonist activity with respect to the activity of a mature GDF peptide. Preferably, the mature GDF peptide activity is an ability to interact specifically with its receptor.
As disclosed herein, a mature myostatin peptide (referred to herein generally as “myostatin”) can induce myostatin signal transduction activity by interacting specifically with a myostatin receptor expressed on the surface of a cell (see Example 7). Thus, a functional peptide portion of myostatin can be obtained by examining peptide portions of a mature myostatin peptide using a method as described herein (Example 7) or otherwise known in the art, and identifying functional peptide portions of myostatin that specifically interact with a myostatin receptor, for example, an activin type IIA receptor (Act RIIA) or Act RIIB receptor expressed on a cell (Act RIIA, Cell 65:973-982, 1991; Act RIIB Cell 68:97-108, 1992, both herein incorporated by reference in their entirety).
A myostatin prodomain can reduce or inhibit myostatin signal transduction activity. In one embodiment, the myostatin prodomain can interact specifically with myostatin, thereby reducing or inhibiting the ability of the myostatin peptide to interact specifically with its receptor. As disclosed herein, a precursor promyostatin also lacks the ability to interact specifically with a myostatin receptor, and, therefore, mutations in promyostatin that reduce or inhibit the ability of promyostatin to be cleaved into mature myostatin provide a means to reduce or inhibit myostatin signal transduction. Accordingly, in another embodiment, the present invention includes a mutant pro-GDF polypeptide, which contains one or more amino acid mutations that disrupt proteolytic cleavage of the mutant pro-GDF to an active mature GDF peptide.
A mutant pro-GDF polypeptide of the invention can have a mutation that affects cleavage at a proteolytic cleavage site such as the consensus proteolytic cleavage recognition site Arg-Xaa-Xaa-Arg (SEQ ID NO: 21), which is present in pro-GDF polypeptides. Thus, the mutation can be a mutation of an Arg residue of SEQ ID NO: 21, such that a mutant promyostatin, for example, cannot be cleaved into a myostatin prodomain and a mature myostatin peptide. However, the mutation also can be at a site other than the proteolytic cleavage site, and can alter the ability of the protease to bind to the pro-GDF polypeptide so as to effect proteolysis at the cleavage site. A mutant pro-GDF polypeptide of the invention, for example, a mutant promyostatin can have a dominant negative activity with respect to myostatin and, therefore, can be useful for reducing or inhibiting myostatin signal transduction in a cell.
In addition, an agent useful in a method of the invention can be a mutant myostatin receptor, which, for example, lacks myostatin signal transduction activity in response to myostatin binding, or has constitutive myostatin signal transduction activity. For example, a mutant myostatin receptor can have a point mutation, a deletion, or the like in its kinase domain such that the receptor lacks kinase activity. Such a dominant negative mutant myostatin receptor lacks the ability to transmit myostatin signal transduction despite the fact that it can specifically bind myostatin.
In another embodiment, the present invention includes use of a substantially purified polynucleotide, which encodes a peptide portion of a promyostatin polypeptide or a mutant promyostatin, or a peptide portion of a pro-GDF-11 polypeptide or mutant pro-GDF-11, as described above. As discussed in greater detail below, the invention also includes polynucleotides useful as agents for modulating the affect of myostatin on a cell, and further provides a polynucleotide encoding a GDF receptor, or functional peptide portion thereof. Examples of such polynucleotides are provided in the following disclosure. As such, it should be recognized that the following disclosure is relevant to the various embodiments of the invention as disclosed herein.
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.
In general, the nucleotides comprising a polynucleotide are naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2′-deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose. However, a polynucleotide also can contain nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides. Such nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220-5234 (1994); Jellinek et al., Biochemistry 34:11363-11372 (1995); Pagratis et al., Nature Biotechnol. 15:68-73 (1997), each of which is incorporated herein by reference).
The covalent bond linking the nucleotides of a polynucleotide generally is a phosphodiester bond. However, the covalent bond also can be any of numerous other bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides (see, for example, Tam et al., Nucl. Acids Res. 22:977-986 (1994); Ecker and Crooke, BioTechnology 13:351360 (1995), each of which is incorporated herein by reference). The incorporation of non-naturally occurring nucleotide analogs or bonds linking the nucleotides or analogs can be particularly useful where the polynucleotide is to be exposed to an environment that can contain a nucleolytic activity, including, for example, a tissue culture medium or upon administration to a living subject, since the modified polynucleotides can be less susceptible to degradation.
A polynucleotide comprising naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template. In comparison, a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally will be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template (Jellinek et al., supra, 1995).
Where a polynucleotide encodes a peptide, for example, a peptide portion of promyostatin or a peptide agent, the coding sequence generally is contained in a vector and is operatively linked to appropriate regulatory elements, including, if desired, a tissue specific promoter or enhancer. The encoded peptide can be further operatively linked, for example, to peptide tag such as a His-6 tag or the like, which can facilitate identification of expression of the agent in the target cell. A polyhistidine tag peptide such as His-6 can be detected using a divalent cation such as nickel ion, cobalt ion, or the like. Additional peptide tags include, for example, a FLAG epitope, which can be detected using an anti-FLAG antibody (see, for example, Hopp et al., BioTechnology 6:1204 (1988); U.S. Pat. No. 5,011,912, each of which is incorporated herein by reference); a c-myc epitope, which can be detected using an antibody specific for the epitope; biotin, which can be detected using streptavidin or avidin; and glutathione S-transferase, which can be detected using glutathione. Such tags can provide the additional advantage that they can facilitate isolation of the operatively linked peptide or peptide agent, for example, where it is desired to obtain a substantially purified peptide corresponding to a proteolytic fragment of a myostatin polypeptide.
As used herein, the term “operatively linked” or “operatively associated” means that two or more molecules are positioned with respect to each other such that they act as a single unit and effect 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 operatively 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 effect 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 operatively 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.
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 myostatin 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.
Cell compartmentalization domains are well known and include, for example, a plasma membrane localization domain, a nuclear localization signal, a mitochondrial membrane localization signal, an endoplasmic reticulum localization signal, or the like (see, for example, Hancock et al., EMBO J. 10:4033-4039, 1991; Buss et al., Mol. Cell. Biol. 8:3960-3963, 1988; U.S. Pat. No. 5,776,689 each of which is incorporated herein by reference). Such a domain can be useful to target an agent to a particular compartment in the cell, or to target the agent for secretion from a cell. For example, the kinase domain of a myostatin receptor such as Act RIIB generally is associated with the inner surface of the plasma membrane. Thus, a chimeric polypeptide comprising a dominant negative myostatin receptor kinase domain, for example, a dominant negative Act RIIB receptor, which lacks kinase activity, can further comprise a plasma membrane localization domain, thereby localizing the dominant negative Act RIIB kinase domain to the inner cell membrane.
As disclosed herein, a pro-GDF signal peptide has cellular localization activity. As used herein, the term “cellular localization activity” refers to the ability of a signal peptide to direct translocation of a peptide operably linked thereto to one or more specific intracellular compartments or to direct secretion of the molecule from the cell. As such, a pro-GDF signal peptide can be particularly useful for directing translocation of a peptide or other agent operably linked to the signal peptide to the same intracellular compartments as a naturally expressed GDF having substantially the same signal peptide. Furthermore, the signal peptide, for example, a promyostatin signal peptide comprising about the first 15 to 30 amino acids of promyostatin, can direct secretion of an operably linked agent from the cell through the same pathway as the naturally occurring pro-GDF having comprising the signal peptide. Thus, particularly useful agents for performing a method of the invention include a GDF prodomain or functional peptide portion thereof that is operably linked to a GDF signal peptide, preferably a promyostatin signal peptide.
A polynucleotide of the invention, including a polynucleotide agent useful in performing a method of the invention, can be contacted directly with a target cell. For example, oligonucleotides useful as antisense molecules, ribozymes, or triplexing agents can be directly contacted with a target cell, whereupon they enter the cell and effect their function. A polynucleotide agent also can interact specifically with a polypeptide, for example, a myostatin receptor (or myostatin), thereby altering the ability of myostatin to interact specifically with the receptor. Such polynucleotides, as well as methods of making and identifying such polynucleotides, are disclosed herein or otherwise well known in the art (see, for example, O.quadrature. Connell et al., Proc. Natl. Acad. Sci., USA 93:5883-5887, 1996; Tuerk and Gold, Science 249:505-510, 1990; Gold et al., Ann. Rev. Biochem. 64:763-797, 1995; each of which is incorporated herein by reference).
A polynucleotide of the invention, which can encode a peptide portion of a pro-GDF polypeptide such as promyostatin, or can encode a mutant promyostatin polypeptide, or can encode a GDF receptor or functional peptide portion thereof, or can be a polynucleotide agent useful in performing a method of the invention, can be contained in a vector, which can facilitate manipulation of the polynucleotide, including introduction of the polynucleotide into a target cell. The vector can be a cloning vector, which is useful for maintaining the polynucleotide, or can be an expression vector, which contains, in addition to the polynucleotide, regulatory elements useful for expressing the polynucleotide and, where the polynucleotide encodes a peptide, for expressing the encoded peptide in a particular cell. An expression vector can contain the expression elements necessary to achieve, for example, sustained transcription of the encoding polynucleotide, or the regulatory elements can be operatively linked to the polynucleotide prior to its being cloned into the vector.
An expression vector (or the polynucleotide) generally contains or encodes a promoter sequence, which can provide constitutive or, if desired, inducible or tissue specific or developmental stage specific expression of the encoding polynucleotide, a poly-A recognition sequence, and a ribosome recognition site or internal ribosome entry site, or other regulatory elements such as an enhancer, which can be tissue specific. The vector also can contain elements required for replication in a prokaryotic or eukaryotic host system or both, as desired. Such vectors, which include plasmid vectors and viral vectors such as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virus and adeno-associated virus vectors, are well known and can be purchased from a commercial source (Promega, Madison Wis.; Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Md.) or can be constructed by one skilled in the art (see, for example, Meth. Enzymol., Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Canc. Gene Ther. 1:51-64, 1994; Flotte, J. Bioenerg. Biomemb. 25:37-42, 1993; Kirshenbaum et al., J. Clin. Invest. 92:381-387, 1993; each of which is incorporated herein by reference).
A tetracycline (tet) inducible promoter can be particularly useful for driving expression of a polynucleotide of the invention, for example, a polynucleotide encoding a truncated or dominant negative form of myostatin, in which the proteolytic processing site has been mutated, or encoding a myostatin prodomain, which can form a complex with a mature myostatin peptide, or encoding a truncated or dominant negative form of a GDF receptor. Upon administration of tetracycline, or a tetracycline analog, to a subject containing a polynucleotide operatively linked to a tet inducible promoter, expression of the encoded peptide is induced, whereby the peptide can effect its activity, for example, whereby a peptide agent can reduce or inhibit myostatin signal transduction. Such a method can be used, for example, to induce muscle hypertrophy in a subject.
The polynucleotide also can be operatively linked to tissue specific regulatory element, for example, a muscle cell specific regulatory element, such that expression of an encoded peptide is restricted to the muscle cells in an individual, or to muscle cells in a mixed population of cells in culture, for example, an organ culture. Muscle cell specific regulatory elements including, for example, the muscle creatine kinase promoter (Sternberg et al., Mol. Cell. Biol. 8:2896-2909, 1988, which is incorporated herein by reference) and the myosin light chain enhancer/promoter (Donoghue et al., Proc. Natl. Acad. Sci., USA 88:5847-5851, 1991, which is incorporated herein by reference) are well known in the art.
Viral expression vectors can be particularly useful for introducing a polynucleotide into a cell, particularly a cell in a subject. Viral vectors provide the advantage that they can infect host cells with relatively high efficiency and can infect specific cell types. For example, a polynucleotide encoding a myostatin prodomain or functional peptide portion thereof can be cloned into a baculovirus vector, which then can be used to infect an insect host cell, thereby providing a means to produce large amounts of the encoded prodomain. The viral vector also can be derived from a virus that infects cells of an organism of interest, for example, vertebrate host cells such as mammalian, avian or piscine host cells. Viral vectors can be particularly useful for introducing a polynucleotide useful in performing a method of the invention into a target cell. Viral vectors have been developed for use in particular host systems, particularly mammalian systems and include, for example, retroviral vectors, other lentivirus vectors such as those based on the human immunodeficiency virus (HIV), adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, vaccinia virus vectors, and the like (see Miller and Rosman, BioTechniques 7:980-990, 1992; Anderson et al., Nature 392:25-30 Suppl., 1998; Verma and Somia, Nature 389:239-242, 1997; Wilson, New Engl. J. Med. 334:1185-1187 (1996), each of which is incorporated herein by reference).
When retroviruses, for example, are used for gene transfer, replication competent retroviruses theoretically can develop due to recombination of retroviral vector and viral gene sequences in the packaging cell line utilized to produce the retroviral vector. Packaging cell lines in which the production of replication competent virus by recombination has been reduced or eliminated can be used to minimize the likelihood that a replication competent retrovirus will be produced. All retroviral vector supernatants used to infect cells are screened for replication competent virus by standard assays such as PCR and reverse transcriptase assays. Retroviral vectors allow for integration of a heterologous gene into a host cell genome, which allows for the gene to be passed to daughter cells following cell division.
A polynucleotide, which can be contained in a vector, can be introduced into a cell by any of a variety of methods known in the art (Sambrook et al., Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1987, and supplements through 1995), each of which is incorporated herein by reference). Such methods include, for example, transfection, lipofection, microinjection, electroporation and, with viral vectors, infection; and can include the use of liposomes, microemulsions or the like, which can facilitate introduction of the polynucleotide into the cell and can protect the polynucleotide from degradation prior to its introduction into the cell. The selection of a particular method will depend, for example, on the cell into which the polynucleotide is to be introduced, as well as whether the cell is isolated in culture, or is in a tissue or organ in culture or in situ.
Introduction of a polynucleotide into a cell by infection with a viral vector is particularly advantageous in that it can efficiently introduce the nucleic acid molecule into a cell ex vivo or in vivo (see, for example, U.S. Pat. No. 5,399,346, which is incorporated herein by reference). Moreover, viruses are very specialized and can be selected as vectors based on an ability to infect and propagate in one or a few specific cell types. Thus, their natural specificity can be used to target the nucleic acid molecule contained in the vector to specific cell types. As such, a vector based on an HIV can be used to infect T cells, a vector based on an adenovirus can be used, for example, to infect respiratory epithelial cells, a vector based on a herpesvirus can be used to infect neuronal cells, and the like. Other vectors, such as adeno-associated viruses can have greater host cell range and, therefore, can be used to infect various cell types, although viral or non-viral vectors also can be modified with specific receptors or ligands to alter target specificity through receptor mediated events.
In another embodiment, an agent useful in a method of the invention can interact with a cellular myostatin receptor, thereby competing with myostatin for the receptor. Such an agent can be, for example, an antibody that specifically binds a cell surface myostatin receptor, including all or a portion of the myostatin binding domain, thereby preventing myostatin from interacting specifically with the receptor. Such an anti-myostatin receptor antibody can be selected for its ability to specifically bind the receptor without activating myostatin signal transduction and, therefore, can be useful as a myostatin antagonist for reducing or inhibiting myostatin signal transduction; or can be selected for its ability to specifically bind the receptor and activate myostatin signal transduction, thus acting as a myostatin agonist. The antibody can be raised using a myostatin receptor, or the extracellular domain of the receptor, as an immunogen, or can be an anti-idiotype antibody, which is raised against an anti-myostatin antibody and mimics myostatin.
Thus, the present invention also includes antibodies that specifically bind a peptide portion of a promyostatin polypeptide or a mutant promyostatin polypeptide. Particularly useful antibodies of the invention include antibodies that specifically bind a myostatin prodomain, or a functional peptide portion thereof, and antibodies that bind a promyostatin polypeptide and reduce or inhibit proteolytic cleavage of the promyostatin to a mature myostatin peptide. In addition, an antibody of the invention can be an antibody that specifically binds a GDF receptor, or functional peptide portion thereof, as described below. Methods of preparing and isolating an antibody of the invention are described in greater detail below, the disclosure of which is incorporated herein by reference.
As used herein, the term “antibody” is used in its broadest sense to include naturally occurring and non-naturally occurring polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies. An antibody useful in a method of the invention, or an antigen binding fragment thereof, is characterized, for example, by having specific binding activity for an epitope of a GDF receptor, for example, a myostatin receptor. In addition, as discussed above, an antibody of the invention can be an antibody that specifically binds a peptide portion of a promyostatin polypeptide, particularly a myostatin prodomain or functional peptide portion thereof. It will be recognized that the following methods, which exemplify the preparation and characterization of GDF receptor antibodies, further are applicable to the preparation and characterization of additional antibodies of the invention, including antibodies that specifically bind a myostatin prodomain, antibodies that specifically bind a promyostatin polypeptide and reduce or inhibit proteolytic cleavage of the promyostatin to myostatin, and the like.
The term “binds specifically” or “specific binding activity,” when used in reference to an antibody means that an interaction of the antibody and a particular epitope has a dissociation constant of at least about 1×10−6, generally at least about 1×10−7, usually at least about 1×10−8, and particularly at least about 1×10−9 or 1×10−10 or less. As such, Fab, F(ab′)2, Fd and Fv fragments of an antibody that retain specific binding activity for an epitope of a GDF receptor, are included within the definition of an antibody. For purposes of the present invention, an antibody that reacts specifically with an epitope of a myostatin receptor, for example, is considered to not substantially react with a TGF-β receptor if the antibody has at least a two-fold greater binding affinity, generally at least a five-fold greater binding affinity, and particularly at least a ten-fold greater binding affinity for the myostatin receptor as compared to the TGF-β receptor.
Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains (see Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference). These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246, 1993; Ward et al., Nature 341:544-546, 1989; Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1988); Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995); each of which is incorporated herein by reference).
Antibodies that bind specifically with a GDF peptide or GDF receptor can be raised using the receptor as an immunogen and removing antibodies that cross-react, for example, with a TGF-β type I or type II receptor, with an activin receptor such as Act RIB, Act RIIA or Act RIIB, or a BMP receptors such as BMP RII, BMP RIA and BMP RIB (see Massague, supra, 1998). An antibody of the invention conveniently can be raised using a peptide portion of a myostatin receptor that is not present in a TGF-β, activin, or BMP receptor. Similarly, an antibody that specifically binds a myostatin prodomain can be raised using the prodomain, or a functional peptide portion thereof as the immunogen. Where such a peptide is non-immunogenic, it can be made immunogenic by coupling the hapten to a carrier molecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), or by expressing the peptide portion as a fusion protein. Various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art (see, for example, by Harlow and Lane, supra, 1988).
Methods for raising polyclonal antibodies, for example, in a rabbit, goat, mouse or other mammal, are well known in the art (see, for example, Green et al., “Production of Polyclonal Antisera,” in Immunochemical Protocols (Manson, ed., Humana Press 1992), pages 1-5; Coligan et al., “Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters,” in Curr. Protocols Immunol. (1992), section 2.4.1; each or which is incorporated herein by reference). In addition, monoclonal antibodies can be obtained using methods that are well known and routine in the art (Harlow and Lane, supra, 1988). For example, spleen cells from a mouse immunized with a myostatin receptor, or an epitopic fragment thereof, can be fused to an appropriate myeloma cell line such as SP/02 myeloma cells to produce hybridoma cells. Cloned hybridoma cell lines can be screened using labeled antigen to identify clones that secrete monoclonal antibodies having the appropriate specificity, and hybridomas expressing antibodies having a desirable specificity and affinity can be isolated and utilized as a continuous source of the antibodies. The antibodies can be further screened for the inability to bind specifically with the myostatin receptor. Such antibodies are useful, for example, for preparing standardized kits for clinical use. A recombinant phage that expresses, for example, a single chain anti-myostatin receptor antibody also provides an antibody that can used for preparing standardized kits.
Methods of preparing monoclonal antibodies are also well known (see, for example, Kohler and Milstein, Nature 256:495, 1975, which is incorporated herein by reference; see, also, Coligan et al., supra, 1992, see sections 2.5.1-2.6.7; Harlow and Lane, supra, 1988). Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well established techniques, including, for example, affinity chromatography with Protein-A SEPHAROSE, size exclusion chromatography, and ion exchange chromatography (Coligan et al., supra, 1992, see sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; see, also, Barnes et al., “Purification of Immunoglobulin G (IgG),” in Meth. Molec. Biol. 10:79-104 (Humana Press 1992), which is incorporated herein by reference). Methods of in vitro and in vivo multiplication of monoclonal antibodies is well known to those skilled in the art. Multiplication in vitro can be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture. Multiplication in vivo can be carried out by injecting cell clones into mammals histocompatible with the parent cells, for example, syngeneic mice, to cause growth of antibody producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.
Therapeutic applications for antibodies disclosed herein are also part of the present invention. For example, antibodies of the present invention can also be derived from subhuman primate antibody. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication WO 91/11465 (1991); and Losman et al., Int. J. Cancer 46:310, 1990, each of which is incorporated herein by reference.
A therapeutically useful anti-GDF receptor antibody also can be derived from a “humanized” monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are known (see, for example, Orlandi et al., Proc. Natl. Acad. Sci., USA 86:3833, 1989, which is hereby incorporated in its entirety by reference). Techniques for producing humanized monoclonal antibodies also are known (see, for example, Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci., USA 89:4285, 1992; Sandhu, Crit. Rev. Biotechnol. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993; each of which is incorporated herein by reference).
Antibodies of the invention also can be derived from human antibody fragments isolated from a combinatorial immunoglobulin library (see, for example, Barbas et al., METHODS: A Companion to Methods in Immunology 2:119, 1991; Winter et al., Ann. Rev. Immunol. 12:433, 1994; each of which is incorporated herein by reference). Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, Calif.).
An antibody of the invention also can be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been “engineered” to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994; and Taylor et al., Int. Immunol. 6:579, 1994; each of which is incorporated herein by reference.
Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and an Fc fragment directly (see, for example, Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, each of which is incorporated by reference, and references contained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230. 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Meth. Enzymol., 1:422 (Academic Press 1967), each of which is incorporated herein by reference; see, also, Coligan et al., supra, 1992, see sections 2.8.1-2.8.10 and 2.10.1-2.10.4).
Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light/heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques can also be used, provided the fragments specifically bind to the antigen that is recognized by the intact antibody. For example, Fv fragments comprise an association of VH and VL chains. This association can be noncovalent (Inbar et al., Proc. Natl. Acad. Sci., USA 69:2659, 1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde (Sandhu, supra, 1992). Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow et al., Methods: A Companion to Methods in Enzymology 2:97, 1991; Bird et al., Science 242:423-426, 1988; Ladner et al., U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology 11:1271-1277, 1993; each of which is incorporated herein by reference; see, also Sandhu, supra, 1992.
Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides (“minimal recognition units”) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991, which is incorporated herein by reference).
Thus, in one embodiment, an agent useful in a method of the invention is a monoclonal antibody that binds specifically to a GDF receptor, particularly to a myostatin receptor, can increase the development of skeletal muscles. In another embodiment a GDF receptor monoclonal antibody, polypeptide, or polynucleotide is administered to a patient suffering from a pathologic condition such as a muscle wasting disease, a neuromuscular disorder, muscle atrophy, aging, or the like. The GDF receptor antibody, particularly an anti-myostatin receptor antibody, can also be administered to a patient suffering from a pathologic condition such as a muscular dystrophy, spinal cord injury, traumatic injury, congestive obstructive pulmonary disease (COPD), AIDS or cachexia. In one embodiment, the anti-myostatin receptor antibody is administered to a patient with muscle wasting disease or disorder by intravenous, intramuscular or subcutaneous injection; preferably, a monoclonal antibody is administered within a dose range between about 0.1 .mu.g/kg to about 100 mg/kg; more preferably between about 1 .mu.g/kg to 75 mg/kg; most preferably from about 10 mg/kg to 50 mg/kg. The antibody can be administered, for example, by bolus injunction or by slow infusion. Slow infusion over a period of 30 minutes to 2 hours is preferred. The anti-myostatin receptor antibody, or other anti-GDF receptor antibody, can be formulated in a formulation suitable for administration to a patient. Such formulations are known in the art.
The dosage regimen will be determined by the attending physician considering various factors which modify the action of the myostatin receptor protein, 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. The dosage can vary with the type of matrix used in the reconstitution and the types of agent, such as anti-myostatin receptor antibodies, to be used in the composition. Generally, systemic or injectable administration, such as intravenous, intramuscular or subcutaneous injection. Administration generally is initiated at a dose which is minimally effective, and the dose is increased over a preselected time course until a positive effect is observed. Subsequently, incremental increases in dosage are made limiting such incremental increases 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, which can aid in increasing muscle mass, to the final composition, can also affect the dosage. In the embodiment where an anti-myostatin receptor antibody is administered, the antibody is generally administered within a dose range of about 0.1 .mu.g/kg to about 100 mg/kg.; more preferably between about 10 mg/kg to 50 mg/kg.
In another embodiment, an agent useful in a method of the invention can be an agent that reduces or inhibits proteolytic cleavage of a pro-GDF polypeptide to an active mature GDF peptide, thereby reducing or inhibiting GDF signal transduction. Such an agent can be a protease inhibitor, particularly one that inhibits the activity of a protease that recognizes and cleaves an Arg-Xaa-Xaa-Arg (SEQ ID NO: 21) proteolytic recognition site. Where the pro-GDF is promyostatin, an anti-myostatin antibody that reduces or inhibits the specific binding of a protease to the Arg-Xaa-Xaa-Arg (SEQ ID NO: 21) proteolytic cleavage site in myostatin also can be used reduce or inhibit proteolysis of promyostatin, thereby reducing the amount of mature myostatin produced. Such an antibody can bind the proteolytic cleavage site, or can bind some other site on the pro-GDF polypeptide such that binding of and cleavage by the protease is reduced or inhibited.
In another embodiment, an agent useful in a method of the invention can be a composition that binds to a GDF receptor or otherwise interferes with the specific binding of a GDF to its receptor. Such compositions may be identified by incubating components comprising the composition and a GDF receptor under conditions sufficient to allow the components to interact specifically, and measuring the binding of the composition to GDF receptors. Compositions that bind to GDF receptors include peptides, peptidomimetics, polypeptides, chemical compounds and biologic agents as described above. Incubating includes exposing the reactants to conditions that allow contact between the test composition and GDF receptors, and provide conditions suitable for a specific interaction as would occur in vivo. Contacting can be in solution or in solid phase. The test ligand/composition can optionally be a combinatorial library for screening a plurality of compositions, as described above. Compositions so identified can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (Saiki et al., Bio/Technology 3:1008-1012, 1985, which is incorporated herein by reference), allele-specific oligonucleotide (ASO) probe analysis (Conner et al., Proc. Natl. Acad. Sci., USA 80:278, 1983, which is incorporated herein by reference), oligonucleotide ligation assays (OLAs) (Landegren et al., Science 241:1077 1988, which is incorporated herein by reference), and the like (see Landegren et al., Science 242:229-237, 1988, which is incorporated herein by reference).
To determine if a composition can functionally complex with the receptor protein, induction of an exogenous gene can be monitored by monitoring changes in the protein level of a protein encoded for by the exogenous gene, or any other method as disclosed herein. When a composition is identified that can induce transcription of the exogenous gene, it is concluded that this composition can specifically bind to the receptor protein coded for by the nucleic acid encoding the initial sample test composition.
Expression of the exogenous gene can be monitored by a functional assay or assay for a protein product, for example. The exogenous gene is therefore a gene that provides an assayable/measurable expression product in order to allow detection of expression of the exogenous gene. Such exogenous genes include, but are not limited to, reporter genes such as chloramphenicol acetyltransferase gene, an alkaline phosphatase gene, β-galactosidase, a luciferase gene, a green fluorescent protein gene, guanine xanthine phosphoribosyltransferase, alkaline phosphatase, and antibiotic resistance genes such as neomycin phosphotransferase.
Expression of the exogenous gene is indicative of a specific interaction of a composition and a GDF receptor; thus, the binding or blocking composition can be identified and isolated. The compositions of the present invention can be extracted and purified from the culture media or a cell by using known protein purification techniques commonly employed, including, for example, extraction, precipitation, ion exchange chromatography, affinity chromatography, gel filtration and the like. Compositions can be isolated by affinity chromatography using the modified receptor protein extracellular domain bound to a column matrix or by heparin chromatography.
Myostatin is essential for proper regulation of skeletal muscle mass. As compared to wild type mice, myostatin knock-out mice, which lack myostatin, have two to three times the amount of muscle due to a combination of hyperplasia and hypertrophy. As disclosed herein, myostatin knock-out mice also have a dramatic reduction in fat accumulation due, at least in part, to an increased anabolic state of skeletal muscle tissue throughout the body. Conversely, overexpression of myostatin in nude mice induced a wasting syndrome that resembles the cachectic state observed in human patients suffering from chronic diseases such as cancer or AIDS. As further disclosed herein, myostatin activity can be mediated through a signal transduction having the characteristics of the Smad signal transduction pathway. Accordingly, the invention includes modulating an effect of myostatin on a cell by contacting the cell with an agent that affects myostatin signal transduction in the cell.
As used herein, the term “modulate,” when used in reference to an effect of myostatin on a cell, means that myostatin 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 myostatin or myostatin signal transduction activity, which can be the level of activity of the signal transduction pathway in the absence of myostatin, or the level of activity in a normal cell in the presence of myostatin. For example, the myostatin signal transduction pathway exhibits a particular activity in a muscle cell contacted with myostatin, and, upon further contacting the muscle cell with a myostatin prodomain, myostatin signal transduction activity can be reduced or inhibited. As such, a myostatin prodomain is an agent useful for reducing or inhibiting myostatin signal transduction. Similarly, a prodomain of another GDF family member such as a GDF-11 prodomain, or of another TGF-β family member such as an activin prodomain, MIS prodomain, or the like, can be useful for reducing myostatin signal transduction. The terms “reduce or inhibit” are used together herein because it is recognized that, in some cases, the level of myostatin 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.
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.
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.
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 myostatin 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.
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).
Thus, in one embodiment, the methods of ameliorating 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 include contacting a muscle cell of a subject in need thereof with an FLRG polynucleotide or polypeptide and 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. As demonstrated in the examples below, the increase in muscle tissue growth following administration of FLRG is greater as compared to the muscle tissue growth in the subject treated with an inhibitor of myostatin expression or activity.
In another embodiment, the methods of ameliorating 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 include contacting a muscle cell of a subject in need thereof with an inhibitor of myostatin expression or activity in combination with an FLRG polynucleotide or polypeptide and 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 alone. As demonstrated in the examples below, the increase in muscle tissue growth following administration of FLRG is greater as compared to the muscle tissue growth in the subject treated with an inhibitor of myostatin expression or activity.
As used herein, the term a “corresponding subject” refers to an individual that is age-matched and/or of the same sex as the individual being administered the composition of the invention. Similarly, a “corresponding normal cell” or “corresponding normal sample” refers to cells, or a sample from a subject, that is from the same organ and of the same type as the cells being examined. In one aspect, the corresponding normal cells comprise a sample of cells obtained from a healthy individual that does not have skin cancer. Such corresponding normal cells can, but need not be, from an individual that is age-matched and/or of the same sex as the individual providing the cells being examined.
The presence of the agent or agents in the target cell can be identified directly, for example, by operatively linking a detectable label to the agent, by using an antibody specific for the agent or agents, particularly a peptide agent, or by detecting a downstream effect due to the agent, for example, decreased phosphorylation of an Smad polypeptide in the cell. An agent can be labeled so as to be detectable using methods well known in the art (Hermanson, “Bioconjugate Techniques” (Academic Press 1996), which is incorporated herein by reference; see, also, Harlow and Lane, supra, 1988). For example, a peptide or polynucleotide agent can be labeled with various detectable moieties including a radiolabel, an enzyme such as alkaline phosphatase, biotin, a fluorochrome, and the like. Where the agent is contained in a kit, the reagents for labeling the agent also can be included in the kit, or the reagents can be purchased separately from a commercial source.
An agent useful in a method of the invention can be administered to the site of the pathologic condition, or can be administered by any method that provides the target cells with the polynucleotide or peptide. 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.
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.
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, Fla. 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). In 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).
The route of administration of a pharmaceutical composition containing FLRG and/or an agent that alters myostatin signal transduction will depend, in part, on the chemical structure of the molecule. Polypeptides and polynucleotides, for example, are not particularly useful when administered orally because they can be degraded in the digestive tract. However, methods for chemically modifying polypeptides, for example, to render them less susceptible to degradation by endogenous proteases or more absorbable through the alimentary tract are well known (see, for example, Blondelle et al., supra, 1995; Ecker and Crook, supra, 1995). In addition, a peptide agent can be prepared using D-amino acids, or can contain one or more domains based on peptidomimetics, which are organic molecules that mimic the structure of peptide domain; or based on a peptoid such as a vinylogous peptoid.
A pharmaceutical composition as disclosed herein can be administered to an individual by various routes including, for example, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intrarectally, intracisternally or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively. Furthermore, the pharmaceutical composition can be administered by injection, intubation, orally or topically, the latter of which can be passive, for example, by direct application of an ointment, or active, for example, using a nasal spray or inhalant, in which case one component of the composition is an appropriate propellant. A pharmaceutical composition also can be administered to the site of a pathologic condition, for example, intravenously or intra-arterially into a blood vessel supplying a tumor.
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.
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).
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 various myostatin inhibitor constructs disclosed herein, and the transgenic non-human organism can be examined directly to determine the effect produced by expressing various levels of FLRG 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 comprising FLRG and a regulatory element comprising a muscle-specific promoter operably linked and integrated into the genome of the mammal. As shown below, the result of expression of the nucleic acid sequence is elevated levels of FLRG 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.
In another embodiment, a transgenic non-human organism of the invention is a mammal whose genome contains a myostatin-null mutation or a decreased level of myostatin as compared with normal basal levels, a nucleic acid sequence comprising FLRG, and a regulatory element comprising a muscle-specific promoter operably linked and integrated into the genome of the mammal. The result of expression of the nucleic acid sequence is elevated levels of FLRG 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 alone.
In addition, 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 FLRG and/or a myostatin inhibitor useful for treating or preventing a disorder such as obesity, type II diabetes, or the like. As such, the present invention provides transgenic non-human organisms, particularly transgenic organisms containing a polynucleotide encoding a myostatin prodomain, which can include the myostatin signal peptide, a pro-peptide (see Examples) or a polynucleotide encoding a mutant promyostatin polypeptide. Further, the invention provides transgenic non-human organisms that express high levels of FLRG or that express a dominant negative Act IIB receptor (see Examples). 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).
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.
As such, a myostatin polynucleotide of the invention is derived from any organism, including mouse, rat, cow, pig, human, chicken, ovine, turkey, finfish and other aquatic organisms and other species. Such polynucleotides can be used to make transgenic animals as described herein. Examples of aquatic animals for which transgenics can be made (and from which myostatin polynucleotide can be derived) include those belonging to the class Piscine such as salmon, trout, char, ayu, carp, crucian carp, goldfish, roach, whitebait, eel, conger eel, sardine, zebrafish, flying fish, sea bass, sea bream, parrot bass, snapper, mackerel, horse mackerel, tuna, bonito, yellowtail, rockfish, fluke, sole, flounder, blowfish, filefish; those belonging to the class Cephalopoda such as squid, cuttlefish, octopus; those belonging to the class Pelecypoda such as clam (e.g., hardshell, Manila, Quahog, Surf, Soft-shell); cockles, mussels, periwinkles; scallops (e.g., sea, bay, calloo); conch, snails, sea cucumbers; ark shell; oyster (e.g., C. virginica, Gulf, New Zealand, Pacific); those belonging to the class Gastropoda such as turban shell, abalone (e.g. green, pink, red); and those belonging to the class Crustacea such as lobster, including but not limited to Spiny, Rock, and American; prawn; shrimp, including but not limited to M. rosenbergii, P. styllrolls, P. indicus, P. jeponious, P. monodon, P. vannemel, M. ensis, S. melantho, N. norvegious, cold water shrimp; crab, including but not limited to Blue, rook, stone, king, queen, snow, brown, dungeness, Jonah, Mangrove, soft-shelled, squilla, krill, langostinos; crayfish/crawfish, including but not limited to Blue, Marron, Red Claw, Red Swamp, Soft-shelled, white; Annelida; Chordates, including but not limited to reptiles, such as alligators and turtles; and Amphibians, including frogs; and Echinoderms, including but not limited to sea urchins.
As used herein, the term “transgenic,” when used in reference to an animal or an organism, means that cells of the animal or organism have been genetically manipulated to contain an exogenous polynucleotide sequence that is stably maintained with the cells. The manipulation can be, for example, microinjection of a polynucleotide or infection with a recombinant virus containing the polynucleotide. Thus, the term “transgenic” is used herein to refer to animals (organisms) in which one or more cells receive a recombinant polynucleotide, which can be integrated into a chromosome in the cell, or can be maintained as an extrachromosomally replicating polynucleotide, such as might be engineered into a yeast artificial chromosome. The term “transgenic animal” also includes a “germ cell line” transgenic animal. A germ cell line transgenic animal is a transgenic animal in which the genetic information has been taken up and incorporated into a germ line cell, therefore conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that information, the offspring also are considered to be transgenic animals. The invention further encompasses transgenic organisms.
A transgenic organism can be any organism whose genome has been altered by in vitro manipulation of an early stage embryo or a fertilized egg, or by any transgenic technology to induce a specific gene knock-out. The term “gene knock-out” refers to the targeted disruption of a gene in a cell or in vivo that results in complete loss of function. A target gene in a transgenic animal can be rendered nonfunctional by an insertion targeted to the gene to be rendered nonfunctional, for example, by homologous recombination, or by any other method for disrupting the function of a gene in a cell. Accordingly, the present invention provides transgenic non-human organisms, as well as food products produced by these organisms. Such food products have increased nutritional value because of the increase in muscle tissue. The transgenic non-human animals can be any species as disclosed herein, including vertebrate organisms such as cattle, pigs, sheep, chicken, turkey and fish, and invertebrate species such as shrimp, lobster, crabs, squid, oysters and abalone.
In one embodiment, the transgene to be used in the practice of the subject invention can be a DNA sequence comprising a modified GDF receptors coding sequence. Preferably, the modified GDF receptor gene is one that is disrupted by homologous targeting in embryonic stem cells. For example, the entire mature C-terminal region of the GDF receptors gene can be deleted (see Example 13). Optionally, the disruption (or deletion) can be accompanied by insertion of or replacement with another polynucleotide, for example, a nonfunctional GDF receptor sequence. A “knock-out” phenotype also can be conferred by introducing or expressing an antisense GDF receptor polynucleotide in a cell in the organism, or by expressing an antibody or a dominant negative GDF receptor in the cells. Where appropriate, polynucleotides that encode proteins having GDF receptor activity, but that differ in nucleotide sequence from a naturally occurring GDF gene sequence due to the degeneracy of the genetic code, can be used herein, as can truncated forms, allelic variants and interspecies homologs.
Various methods are known for producing a transgenic animal. In one method, an embryo at the pronuclear stage (a “one cell embryo”) is harvested from a female and the transgene is microinjected into the embryo, in which case the transgene will be chromosomally integrated into the germ cells and somatic cells of the resulting mature animal. In another method, embryonic stem cells are isolated and the transgene is incorporated into the stem cells by electroporation, plasmid transfection or microinjection; the stem cells are then reintroduced into the embryo, where they colonize and contribute to the germ line. Methods for microinjection of polynucleotides into mammalian species are described, for example, in U.S. Pat. No. 4,873,191, which is incorporated herein by reference. In yet another method, embryonic cells are infected with a retrovirus containing the transgene, whereby the germ cells of the embryo have the transgene chromosomally integrated therein.
When the animals to be made transgenic are avian, microinjection into the pronucleus of the fertilized egg is problematic because avian fertilized ova generally go through cell division for the first twenty hours in the oviduct and, therefore, the pronucleus is inaccessible. Thus, the retrovirus infection method is preferred for making transgenic avian species (see U.S. Pat. No. 5,162,215, which is incorporated herein by reference). If microinjection is to be used with avian species, however, the embryo can be obtained from a sacrificed hen approximately 2.5 hours after the laying of the previous laid egg, the transgene is microinjected into the cytoplasm of the germinal disc and the embryo is cultured in a host shell until maturity (Love et al., Biotechnology 12, 1994). When the animals to be made transgenic are bovine or porcine, microinjection can be hampered by the opacity of the ova, thereby making the nuclei difficult to identify by traditional differential interference-contrast microscopy. To overcome this problem, the ova first can be centrifuged to segregate the pronuclei for better visualization.
Non-human transgenic animals of the invention can be bovine, porcine, ovine, piscine, avian or other animals. The transgene can be introduced into embryonal target cells at various developmental stages, and different methods are selected depending on the stage of development of the embryonal target cell. The zygote is the best target for microinjection. The use of zygotes as a target for gene transfer has a major advantage in that the injected DNA can incorporate into the host gene before the first cleavage (Brinster et al., Proc. Natl. Acad. Sci., USA 82:4438-4442, 1985). As a consequence, all cells of the transgenic non-human animal carry the incorporated transgene, thus contributing to efficient transmission of the transgene to offspring of the founder, since 50% of the germ cells will harbor the transgene.
A transgenic animal can be produced by crossbreeding two chimeric animals, each of which includes exogenous genetic material within cells used in reproduction. Twenty-five percent of the resulting offspring will be transgenic animals that are homozygous for the exogenous genetic material, 50% of the resulting animals will be heterozygous, and the remaining 25% will lack the exogenous genetic material and have a wild type phenotype.
In the microinjection method, the transgene is digested and purified free from any vector DNA, for example, by gel electrophoresis. The transgene can include an operatively associated promoter, which interacts with cellular proteins involved in transcription, and provides for constitutive expression, tissue specific expression, developmental stage specific expression, or the like. Such promoters include those from cytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes virus, as well as those from the genes encoding metallothionein, skeletal actin, Phosphenolpyruvate carboxylase (PEPCK), phosphoglycerate (PGK), dihydrofolate reductase (DHFR), and thymidine kinase (TK). Promoters from viral long terminal repeats (LTRs) such as Rous sarcoma virus LTR also can be employed. When the animals to be made transgenic are avian, preferred promoters include those for the chicken .quadrature.-globin gene, chicken lysozyme gene, and avian leukosis virus. Constructs useful in plasmid transfection of embryonic stem cells will employ additional regulatory elements, including, for example, enhancer elements to stimulate transcription, splice acceptors, termination and polyadenylation signals, ribosome binding sites to permit translation, and the like.
In the retroviral infection method, the developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, Proc. Natl. Acad. Sci, USA 73:1260-1264, 1976). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan et al., Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, 1986). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al., Proc. Natl. Acad. Sci., USA 82:6927-6931, 1985; Van der Putten et al., Proc. Natl. Acad. Sci, USA 82:6148-6152, 1985). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus producing cells (Van der Putten et al., supra, 1985; Stewart et al., EMBO J. 6:383-388, 1987). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al., Nature 298:623-628, 1982). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic nonhuman animal. Further, the founder can contain various retroviral insertions of the transgene at different positions in the genome, which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by intrauterine retroviral infection of the mid-gestation embryo (Jahner et al., supra, 1982).
Embryonal stem cell (ES) also can be targeted for introduction of the transgene. ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. Nature 292:154-156, 1981; Bradley et al., Nature 309:255-258, 1984; Gossler et al., Proc. Natl. Acad. Sci., USA 83:9065-9069, 1986; Robertson et al., Nature 322:445-448, 1986). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a nonhuman animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (see Jaenisch, Science 240:1468-1474, 1988).
As disclosed herein, myostatin can exert its activity, at least in part, through the Smad signal transduction pathway, and myostatin expression can be associated with various pathological conditions. As such, the present invention provides new targets for the treatment of various pathological conditions associated with myostatin, including metabolic conditions such as obesity and type II diabetes. Accordingly, the present invention provides methods for ameliorating the severity of a pathological condition in a subject, wherein the pathologic condition is characterized at least in part by an abnormal amount, development or metabolic activity of muscle or adipose tissue, by modulating myostatin signal transduction in a muscle cell or adipose tissue cell in the subject.
Myostatin functions as a negative regulator of muscle growth (McPherron et al., supra, 1997). Myostatin knock-out mice weighed approximately 25% to 30% more than wild type littermates, and this increase in body weight in the mice examined resulted entirely from a dramatic increase in skeletal muscle tissue weight. In mice lacking myostatin, the skeletal muscles weighed about 2 to 3 times as much as the corresponding muscles of wild type littermates. This increased muscle weight in the homozygous knock-out mice resulted from a combination of hyperplasia and hypertrophy.
As disclosed herein, heterozygous myostatin knock-out mice also have increased skeletal muscle mass, although to a lesser extent than that observed in homozygous mutant mice, thus demonstrating that myostatin acts in a dose-dependent manner in vivo (see Example 1). Furthermore, overexpression of myostatin in animals had the opposite effect with respect to muscle growth. For example, nude mice carrying myostatin-expressing tumors developed a wasting syndrome characterized by a dramatic loss of muscle and fat weight (see Example 8). This syndrome in the nude mice resembled the cachectic state that occurs in patients with chronic diseases such as cancer or AIDS.
The serum levels of myostatin immunoreactive material have been correlated with the status of patients with respect to muscle wasting (Gonzalez-Kadavid et al., Proc. Natl. Acad. Med., USA 95:14938-14943, 1998, which is incorporated herein by reference). Thus, patients with AIDS, who also showed signs of cachexia as measured by loss of total body weight, had slightly increased serum levels of myostatin immunoreactive material compared to either normal males without AIDS or to AIDS patients that did not have weight loss. However, the interpretation of these results was complicated because the myostatin immunoreactive material detected in the serum samples did not have the mobility on SDS gels that was expected for authentic processed myostatin.
As disclosed herein, myostatin not only affects muscle mass, but also affects the overall metabolism of an organism. For example, myostatin is expressed in adipose tissue, and myostatin deficient mice have a dramatic reduction in fat accumulation as the animals age (see Examples II and III). Although no mechanism for myostatin action is proposed herein, the effect of myostatin can be direct effect of myostatin on adipose tissue, or can be an indirect effect caused by the lack of myostatin activity on skeletal muscle tissue. Regardless of the mechanism, the overall anabolic effect on muscle tissue that results in response to decreased myostatin activity can alter the overall metabolism of the organism and affect the storage of energy in the form of fat, as demonstrated by the introduction of a myostatin mutation into an obese mouse strain (agouti lethal yellow (Ay) mice), which suppressed fat accumulation by five-fold (see Example 5). Abnormal glucose metabolism also was partially suppressed in agouti mice containing the myostatin mutation. These results demonstrate that methods of inhibition of myostatin can be used to treat or prevent metabolic diseases such as obesity and type II diabetes.
In addition, it was previously shown that the myostatin binding protein, follstatin, can induce dramatic increases in muscle mass when overexpressed as a transgene in mice. In order to determine whether this effect of follstatin results solely from inhibition of myostatin activity, the effect of this transgene in myostatin-null (Mstn−/−) mice was analyzed. As demonstrated in the examples below, Mstn−/− mice carrying a follistatin transgene had about four times the muscle mass of wild type mice, demonstrating the existence of other regulators of muscle mass with similar activity to myostatin.
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
Myostatin Acts in a Dose Dependent Manner
This example demonstrates that the activity of myostatin in inhibiting muscle growth is dependent on the level of myostatin expression in vivo.
Myostatin is a negative regulator of skeletal muscle mass (McPherron et al., supra, 1997; McPherron and Lee, supra, 1997). Myostatin knock-out mice that were homozygous for a deletion of the myostatin gene had a 25-30% increase in total body mass. An examination of the homozygous knock-out mice revealed that the increased muscle mass was due to about a 100-200% increase in skeletal muscle mass throughout the body.
Mice that were heterozygous for the myostatin mutation also had an increase in total body mass. However, the increase mass of the heterozygotes was less than that of the homozygotes, and was statistically significant in only one age and sex group among the many examined. In order to determine whether heterozygous mice have an intermediate phenotype between that of wild type mice and homozygous myostatin knock-out mice, the analysis of muscle weights was extended to the heterozygous mice. Individual muscles sampled from heterozygous mice weighed approximately 25-50% more than those of wild type mice. These results demonstrate that mice that are heterozygous for deletion of a myostatin gene have a phenotype that is intermediate between that of wild type mice and homozygous myostatin knock-out mice, and demonstrate that myostatin produces a dose-dependent effect in vivo.
These results indicate that the manipulation of myostatin activity can be useful in treating muscle wasting diseases and other metabolic disorders associated with myostatin activity. Furthermore, the dose-dependent effect of myostatin indicates that a therapeutic effect can be obtained without achieving complete inhibition of myostatin activity, thereby allowing for an adjustment of myostatin activity if, for example, a certain level of activity produces undesirable effects in a subject.
EXAMPLE 2
Myostatin Effect Decreases with Age in Knock-out Mice
This example demonstrates that a decreased difference in body weight between wild type mice and homozygous myostatin knock-out mice is associated with a decline in muscle weight of the mutant mice.
Myostatin knock-out mice weighed approximately 25-30% more than wild type mice at five months of age (McPherron et al., supra, 1997). However, this difference in total body weights became significantly smaller or disappeared altogether as the animals aged. In order to determine whether this effect was due to a relative loss of weight in the knock-out mice due, for example, to muscle degeneration, or to a relatively greater weight gain by wild type mice, a detailed analysis of muscle weights was made as a function of age.
At all ages examined from 2 months to 17 months, the pectoralis muscle weighed significantly more in homozygous mutant mice than in wild type littermates. The most dramatic difference was observed at 5 months, at which time the pectoralis weight was approximately 200% greater in the mutant mice. Although the pectoralis weight declined slightly at older ages, the weight of this muscle in mutant mice remained greater than twice that of wild type mice. This same basic trend was observed in all of the other muscles examined, including the triceps brachii, the quadriceps, the gastrocnemius and plantaris, and the tibialis anterior. Similar trends were observed in both male and female mice. These results demonstrate that the decreased difference in total body weights between mutant and wild type mice observed with aging is due to a slight decline in muscle weights in the mutant mice.
EXAMPLE 3
Myostatin Affects Fat Accumulation in a Dose Dependent Manner
This example demonstrates that myostatin knock-out mice fail to accumulate fat, and that the decrease in fat accumulation is associated with the level of myostatin expression in vivo.
Since the decline in muscle weights in myostatin mutants, as demonstrated in Example 2, did not fully account for the observation that the wild type animals eventually weighed about the same as the mutant mice, the amount of fat accumulation in wild type and mutant mice was examined. The inguinal, epididymal and retroperitoneal fat pads in male mice were examined. There was no difference in the weights of any of these fat pads between wild type and mutant mice at two months of age. By 5 to 6 months of age, wild type and heterozygous knock-out mice both exhibited a large range of fat pad weights, and, on average, fat pad weights increased by approximately 3-fold to 5-fold by the time the animals reached 9 to 10 months of age. Due to the large range of fat pad weights observed in these animals, some animals showed a much larger increase (up to 10-fold) than others.
In contrast to the wild type and heterozygous knock-out mice, the fat pad weights of myostatin homozygous mutant mice were in a relatively narrow range and were virtually identical in 2 month old mice and in 9 to 10 month old mice. Thus, the increased fat accumulation that occurred with aging in the wild type mice was not observed in the homozygous myostatin knock-out mice. This difference in fat accumulation, together with the slight decline in muscle weights, as a function of age in the homozygous mutant mice fully accounted for the observation that the wild type animals eventually have the same total body weight as the mutants.
The mean fat pad weights of heterozygous knock-out mice at 9 to 10 months of age was intermediate between that of the wild type mice and the homozygous mutant mice. Although this difference was not statistically significant, due to the wide range of fat pad weights in these and the wild type mice, these results nevertheless indicate that myostatin has a dose-dependent effect on the accumulation of fat, similar to its effect on muscle growth.
EXAMPLE 4
Effect of Myostatin on Metabolism
This example demonstrates that serum insulin and glucose levels, as well as metabolic activity, are affected by the level of myostatin expression.
In order to determine whether the skeletal muscle hypertrophy and the lack of fat accumulation in the myostatin mutant mice is due to an effect on overall metabolism, the metabolic profile of the mutant mice was examined. Serum triglyceride and serum cholesterol levels were significantly lower in myostatin mutant mice as compared to wild type control mice (Table 1). Serum insulin levels also appeared to be lower in the myostatin mutant mice. However, the fed and fasting glucose levels both were indistinguishable among homozygous mutant mice and wild type mice (Table 1), and both groups of mice had a normal response in a glucose tolerance test. The results demonstrate that the homozygous myostatin knock-out mice can maintain normal levels of serum glucose even though their serum insulin level is lower than that of wild type animals.







TABLE 1







Serum Parameters











+/+
−/−















triglycerides (mg/dl)
 131.5 ± 16.5
 66.8 ± 11.4
p = 0.012


cholesterol (mg/dl)
138.3 ± 8.1
 94.5 ± 6.8
p = 0.0034


fed glucose (mg/dl)
114.0 ± 4.8
119.3 ± 5.2
n.s. (p = 0.43)


fasting glucose (mg/dl)
 86.5 ± 3.8
103.3 ± 9.3
n.s. (p = 0.13)





+/+ indicates wild type mice;


−/− indicate homozygous knock-out mice






In order to determine whether differences in metabolic rates could explain the lack of fat accumulation in the mutant mice, the rate of oxygen consumption of wild type and mutant mice was compared using a calorimeter. Mutant mice had a lower basal metabolic rate and a lower overall metabolic rate than wild type control mice. These results indicate that the lack of fat accumulation in the myostatin mutant mice is not due to a higher rate of metabolic activity.
EXAMPLE 5
Myostatin Affects Fat Accumulation in Genetically Obese Mice
This example demonstrates that a lack of myostatin expression suppresses fat accumulation in mice that are a genetic model for obesity.
In order to determine whether the loss of myostatin activity could suppress fat accumulation not only in normal mice but also in obese mice, the effect of the myostatin mutation in agouti lethal yellow (Ay) mice, which represent a genetic model of obesity (Yen et al., FASEB J. 8:479-488, 1994), was examined. Mice that were doubly heterozygous for the lethal yellow and myostatin mutations were generated, and offspring from crosses of these doubly heterozygous mice were examined.
The total body weight of the Ay/a, myostatin −/− double mutant mouse was dramatically reduced (approximately 9 grams) compared to that of the Ay/a, myostatin +/+ mouse. This reduction in total body weight was even more dramatic considering that the Ay/a, myostatin −/− double mutant had about 2 to 3 times more skeletal muscle than did the AY/a, myostatin +/+ mouse. The double mutant had approximately 10 grams more muscle than the Ay/a, myostatin +/+ mouse and, therefore, the total weight reduction in the rest of the tissues was about 19 grams.
The reduction in total body weight resulted from a reduction in overall fat content. As shown in Table 2, the weights of the parametrial and retroperitoneal fat pads were reduced 5-fold to 6-fold in the Ay/a, myostatin −/− double mutant as compared to the Ay/a, myostatin +/+ mouse. These results indicate that the presence of the myostatin mutation dramatically suppresses fat accumulation in obesity.
The presence of the myostatin mutation also dramatically affected glucose metabolism. Agouti lethal mice lacking the myostatin mutation had grossly abnormal glucose tolerance test results, with serum glucose levels often reaching 450 to 600 mg/dl and only slowly recovering to baseline levels over a period of 4 hours. Female agouti lethal mice were affected less than male mice, and some females responded almost normally in this test, as previously described (see Yen et al., supra, 1994). In contrast, although the Ay/a, myostatin −/− mice had slightly abnormal glucose tolerance tests, but none of these animals had the gross abnormalities observed in the Ay/a myostatin +/+ mice.
These results indicate that the myostatin mutation at least partially suppressed the development of abnormal glucose metabolism in the agouti lethal mice. Significantly, mice that were heterozygous for the myostatin mutation had an intermediate response compared to myostatin +/+ and myostatin −/− mice, thus confirming the dose-dependent effect of myostatin.
EXAMPLE 6
Purification of Recombinant Myostatin
This example provides a method for preparing and isolating recombinant myostatin.
In order to elucidate the biological activity of myostatin, large quantities of myostatin protein were purified for bioassays. Stable Chinese hamster ovary (CHO) cell lines producing high levels of myostatin protein were generated by co-amplifying a myostatin expression cassette with a dihydrofolate reductase cassette using a methotrexate selection scheme (McPherron et al., supra, 1997). Myostatin was purified from the conditioned medium of the highest producing line by successive fractionation on hydroxyapatite, lentil lectin SEPHAROSE, DEAE agarose, and heparin SEPHAROSE. Silver stain analysis revealed that the purified protein obtained following these four column chromatography steps (referred to as “heparin eluate”) consisted of two species with molecular masses of approximately 35 kilodaltons (kDa) and 12 kDa.
The purified protein preparation was determined by various criteria to represent a complex of two myostatin prodomain peptides and a disulfide-linked dimer of mature C-terminal myostatin peptides. First, western blot analysis, using antibodies raised against specific portions of the promyostatin sequence, identified the 35 kDa band as the prodomain and the 12 kDa band as the mature C-terminal peptide. Second, under non-reducing conditions, the species reacting with antibodies directed against the mature C-terminal peptide had an electrophoretic mobility consistent with a disulfide linked dimer. Third, the molar ratio of prodomain to mature C-terminal peptide was approximately 1:1. Fourth, the prodomain and mature C-terminal peptide copurified through the four column chromatography steps. Finally, the mature C-terminal peptide bound to the lentil lectin column even though the C-terminal region does not contain consensus N-linked glycosylation signals, indicating that the mature C-terminal peptide bound to the column due to its interaction with the prodomain peptide, which contains a potential N-linked glycosylation site.
These results indicate that myostatin produced by the genetically modified CHO cells is secreted in a proteolytically processed form, and that the resulting prodomain and mature C-terminal region associate non-covalently to form a complex containing two prodomain peptides and a disulfide-linked dimer of C-terminal proteolytic fragments, similar to that described for TGF-β. In the TGF-β complex, the C-terminal dimer exists in an inactive, latent form (Miyazono et al., J. Biol. Chem. 263:6407-6415, 1988), and the active species can be released from this latent complex by treatment with acid, chaotropic agents, reactive oxygen species, or plasmin, or by interactions with other proteins, including thrombospondin and integrin αvβ6 (Lawrence et al., Biochem. Biophys. Res. Comm. 133:1026-1034, 1985; Lyons et al., J. Cell Biol. 106:1659-1665, 1988; Schultz-Cherry and Murphy-Ullrich, J. Cell Biol. 122:923-932, 1993; Barcellos-Hoff and Dix, Mol. Endocrinol. 10:1077-1083, 1996; Munger et al., Cell 96:319-328, 1999). Furthermore, the addition of purified prodomain peptide (also known as latency-associated peptide or LAP) to the TGF-β complex inhibits the biological activity of the purified C-terminal dimer in vitro and in vivo (Gentry and Nash, Biochemistry 29:6851-6857, 1990; Bottinger et al., Proc. Natl. Acad. Sci., USA 93:5877-5882, 1996).
The heparin eluate, which consisted of a complex of prodomain and mature C-terminal peptide, was further purified using an HPLC C4 reversed phase column. The C-terminal dimer eluted from the HPLC column earlier than the prodomain, thus allowing the isolation of the C-terminal dimer free of prodomain. Fractions that contained mostly prodomain also were obtained, although these fractions contained small amounts of the C-terminal dimer. Some of the protein also was present as higher molecular weight complexes. The nature of the higher molecular weight complexes is unknown, but, based on western blot analysis in the presence or absence of reducing agents, these complexes can contain at least one prodomain peptide and one C terminal mature myostatin peptide linked by one or more disulfide bonds. In fact, most of the mature C-terminal peptide present in the HPLC fraction enriched for the propeptide (HPLC fractions 35-37) was present in these high molecular weight complexes. These higher molecular weight complexes likely represent improperly folded proteins that are secreted by the genetically modified CHO cells.
EXAMPLE 7
Myostatin Specifically Interacts with an Activin Receptor
This example demonstrates that myostatin specifically binds an activin type II receptor expressed on cells in culture, and that this specific binding is inhibited by a myostatin prodomain.
The receptors for some TGF-β family members have been identified, and most are single membrane spanning serine/threonine kinases (Massague and Weis-Garcia, Cancer Surveys 27:41-64, 1996). The activin type II receptors (Act RIIA and/or Act RIIB), for example, are known to bind members of the TGF-β superfamily. The phenotype of mice lacking the Act RIIB receptor showed anterior/posterior axial patterning defects and kidney abnormalities that were very similar to those observed in GDF-11 knock-out mice (McPherron et al., Nat. Genet. 22:260-264, 1999; Oh and Li, Genes Devel. 11:1812-1826, 1997). Since the amino acid sequences of GDF-11 and myostatin (GDF-8) are 90% identical in the mature C-terminal region, the ability of myostatin to specifically interact with an activin type II receptor was examined.
Myostatin was labeled by radio-iodination, and binding studies were performed using COS cells transfected with an Act RIIB expression construct. Myostatin interacted specifically with the transfected COS cells. Myostatin binding was competed in a dose dependent manner by excess unlabeled myostatin, and was significantly lower in control COS cells, which were transfected with an empty vector. No significant binding occurred to cells transfected with a BMP RII or TGF-β RII expression construct. Myostatin binding to Act RIIB-transfected cells was saturable, and the binding affinity was approximately 5 nM as determined by Scatchard analysis.
The receptor binding assay also was used to examine the ability of the myostatin prodomain to inhibit the ability of the mature C-terminal dimer to interact specifically with Act RIIB in this system. The addition of purified prodomain peptide blocked the ability of the C-terminal dimer to bind the Act RIIB transfected COS cells in a dose-dependent manner. These results indicate that the myostatin prodomain is a natural inhibitor of myostatin.
EXAMPLE 8
Increased Myostatin Levels Induce Weight Loss
This example demonstrates that elevated levels of myostatin can lead to substantial weight loss in vivo.
In one set of experiments, CHO cells that express myostatin were injected into nude mice. The nude mice that had myostatin expressing CHO cell tumors showed severe wasting over the course of approximately 12 to 16 days following injection of the cells. This wasting syndrome was not observed in nude mice injected with any of a variety of control CHO lines that had undergone a similar selection process but did not express myostatin. Furthermore, the myostatin coding sequence in the construct used to transfect the CHO cells was under the control of a metallothionein promoter, and the wasting syndrome was exacerbated when mice bearing the myostatin expressing tumors were maintained on water containing zinc sulfate. Western blot analysis revealed high levels of myostatin protein in the serum of the nude mice that bore the myostatin-expressing CHO cells. These results indicate that the wasting syndrome was induced in response to the elevated level of myostatin in the nude mice and, as discussed below, this result was confirmed by observing similar effects in mice injected with purified myostatin.
The dramatic weight loss observed in the nude mice bearing myostatin expressing CHO cells was due primarily to a disproportionate loss of both fat and muscle weight. White fat pad weights (intrascapular white, uterine, and retroperitoneal fat) were reduced by greater than 90% compared to mice bearing control CHO cell tumors. Muscle weights were also severely reduced, with individual muscles weighing approximately half as much in myostatin expressing mice as in control mice by day 16. This loss in muscle weight was reflected by a corresponding decrease in fiber sizes and protein content.
Mice bearing the myostatin expressing CHO cell tumors also became severely hypoglycemic. However, the weight loss and hypoglycemia were not due to a difference in food consumption, as all of the mice consumed equivalent amounts of food at each time interval examined during the 16 day course of the study. These results indicate that myostatin overexpression induces a dramatic weight loss, which resembles the cachectic wasting syndrome that occurs in patients suffering from chronic diseases such as cancer or AIDS.
With more chronic administrations using lower doses of myostatin, changes in fat weight were observed. For example; twice daily injections of 1 μg of myostatin protein for 7 days resulted in an approximately 50% decrease in the weights of a number of different white fat pads (intrascapular white, uterine, and retroperitoneal fat pads) with no significant effect on brown fat (intrascapular brown). These results confirm that myostatin can induce weight loss and, in extreme cases, a wasting syndrome in vivo.
EXAMPLE 9
Characterization of Myostatin Binding to an Activin Receptor
This example describes a method for characterizing the relationship of myostatin binding to an activin receptor with the biological effects produced by myostatin in vivo.
Act RITA or Act RIIB knock-out mice can be used to confirm that Act RIIA or Act RIIB is a receptor for myostatin in vivo. A detailed muscle analysis of these mice can determine whether knock-out of an activin receptors is associated with a change in muscle fiber number or size. Since Act RIIA/Act RIIB double homozygous mutant die early during embryogenesis (Song et al., Devel. Biol. 213:157-169, 1999), only the various homozygous/heterozygous combinations can be examined. However, tissue-specific or conditional knock-out mice can be generated such that both genes can be “deleted” only in muscle tissue, thus allowing postnatal examination of the double homozygous knock-out mice.
The effect on adipose tissue can be examined with aging of the mice to determine whether the number of adipocytes or the accumulation of lipid by these adipocytes is altered in the knock-out mice. Adipocyte number and size is determined by preparing cell suspensions from collagenase treated tissue (Rodbell, J. Biol. Chem. 239:375-380, 1964; Hirsch and Gallian, J. Lipid Res. 9:110-119, 1968). Total lipid content in the animals is determined by measuring dry carcass weights and then the residual dry carcass weights after lipid extraction (Folch et al., J. Biol. Chem. 226:497-509, 1957).
A variety of serum parameters also can be examined, including fed and fasting glucose and insulin, triglycerides, cholesterol, and leptin. As disclosed above, serum triglycerides and serum insulin are decreased in the myostatin mutant animals. The ability of the activin receptor knock-out mice to respond to an exogenous glucose load also can be examined using glucose tolerance tests. As disclosed above, the response to a glucose load essentially was identical in wild type and myostatin mutant mice at 5 months of age. This observation can be extended by measuring these parameters in the mice as they age. Serum insulin levels also can be measured at various times during the glucose tolerance tests.
Basal metabolic rates also can be monitored using a calorimeter (Columbus Instruments). As disclosed above, myostatin mutant mice have a lower metabolic rate at 3 months of age than their wild type counterparts. This analysis can be extended to older mice, and the respiratory quotient also can be measured in these animals. The ability to maintain normal thermogenesis can be determined by measuring the basal body temperatures as well as their ability to maintain body temperature when placed at 4° C. Brown fat weights and expression levels of UCP1, UCP2, and UCP3 in brown fat, white fat, muscle, and other tissues also can be examined (Schrauwen et al., 1999).
Food intake relative to weight gain can be monitored, and feed efficiency can be calculated. In addition, the weight gain of the animals placed on high fat diets can be monitored. Wild type mice maintained on a high fat diet accumulate fat rapidly, whereas the results disclosed herein indicate the activin receptor mutant animals will remain relatively lean.
The results of these studies can provide a more complete profile of the effect of myostatin mice, particularly with respect to their overall metabolic state, thereby providing insight as to whether the ability of the myostatin knock-out mice to suppress the accumulation of fat is an anabolic effect of the myostatin mutation in muscle that leads to a shift in energy utilization such that little energy is available for storage in the form of fat. For example, the decreased fat accumulation can be due to an increased rate of thermogenesis. These results also will provide a baseline for comparison of the effect of the myostatin activity in the context of different genetic models of obesity and type II diabetes.
EXAMPLE 10
Characterization of Myostatin Effects in Genetic Models of Obesity and Type II Diabetes
This example describes methods for determining the effect of myostatin in treating obesity or type II diabetes.
The dramatic reduction in overall fat accumulation in myostatin mutant mice as compared to wild type mice indicates that myostatin activity can be manipulated to treat or prevent obesity or type II diabetes. The effect of the myostatin mutation can be examined in the context of several well characterized mouse models of these metabolic diseases, including, for example, “obese” mice (ob/ob), “diabetic” mice (db/db), and agouti lethal yellow (Ay) mutant strains. Each of these strains is abnormal for virtually every parameter and test described above (see, for example, Yen et al., supra, 1994, Friedman and Halaas, Nature 395:763-770, 1998). The ability of the myostatin mutation to slow or suppress the development of these abnormalities in mice carrying these other mutations can be examined by constructing double mutants, then subjecting the double mutant animals, along with appropriate control littermates carrying only the ob/ob, db/db, or agouti lethal yellow mutations, to the various tests.
As disclosed above, the myostatin mutation in Ay mice was associated with about a 5-fold suppression of fat accumulation in the myostatin mutant Ay mice, and with a partial suppression of the development of abnormal glucose metabolism as assessed by glucose tolerance tests. These results can be extended to include additional animals at various ages, and similar studies can be performed with the ob/ob and db/db mutants. As both the these mutations are recessive, mice that are doubly homozygous for the myostatin mutation and either the ob or db mutation can be generated. In order to examine the effects of partial loss of myostatin function in these genetic model systems, mice that are homozygous for the ob or db mutation and heterozygous for the myostatin mutation also are examined. Mice that are doubly heterozygous for the myostatin and ob mutations have been generated, and the offspring from matings of these doubly heterozygous mice can be examined, particularly with respect to fat accumulation and glucose metabolism. Partial suppression of either or both of these abnormalities in the obese mutants can indicate that myostatin is a target for the treatment of obesity and type II diabetes.
EXAMPLE 11
Characterization of Transgenic Mice Expressing Dominant Negative Polypeptides that can Affect Myostatin Activity
This example describes methods for characterizing the effect of myostatin postnatally by expressing dominant negative polypeptides that can block myostatin expression or myostatin signal transduction.
Myostatin Inhibitors. The modulation of myostatin activity postnatally can be used to determine the effect of myostatin on muscle fiber number (hyperplasia) and muscle fiber size (hypertrophy). Conditional myostatin knock-out mice, in which the myostatin gene is deleted at defined times during the life of the animal, can be used for these studies. The tet regulator in combination with the cre recombinase provides a system for generating such mice. In this system, the expression of cre is induced by administration of doxycycline.
Transgenic mice expressing an inhibitor of myostatin from an inducible promoter also can be generated such that myostatin activity can be reduced or inhibited at defined times during the life of the animal. The tetracycline regulators are useful for generating such transgenic mice, in which the myostatin expression is induced by doxycycline.
A modification of the tet system, which utilizes co-expression of a hybrid reverse tet-transactivator (fusion protein of the activation domain of VP16 with the mutant reverse tet repressor) and a hybrid tet-transrepressor (fusion protein of the KRAB repressor domain of mammalian Kox1 with the native tet repressor), can be particularly useful for producing the transgenic mice (Rossi et al., Nat. Genet. 20:389-393, 1998; Forster et al., Nucl. Acids Res. 27:708-710, 1999). In this system, the hybrid reverse tet-transactivator binds tet operator sequences, and activates transcription only in the presence of tetracycline; the hybrid tet-transrepressor binds tet operator sequences and represses transcription only in the absence of tetracycline. By co-expressing these two fusion proteins, the basal activity of the target promoter is silenced by the tet-transrepressor in the absence of tetracycline, and is activated by the reverse tet-transactivator upon administration of tetracycline.
Two types of transgenic lines can be generated. In the first type, the transgene encodes a myostatin inhibitor polypeptide under the control of a muscle specific promoter, for example, the muscle creatine kinase promoter (Sternberg et al., supra, 1988) or the myosin light chain enhancer/promoter (Donoghue et al., supra, 1991). Individual transgenic lines are screened for specific expression of the tet regulators in skeletal muscle, and several independent lines for each of the two promoters are selected and examined to confirm that any effects observed are not due, for example, to integration site-specific effects. A construct containing the two tet regulators under the control of the myosin light chain promoter/enhancer has been constructed, and can be used for pronuclear injections. In the second type of line. the transgene contains a myostatin inhibitor polypeptide under the control of a minimal CMV promoter that further contains tet operator sequences.
The myostatin inhibitor can be a dominant negative form of myostatin or a myostatin prodomain, which, as disclosed herein, can inhibit myostatin activity. Dominant negative forms of TGF-β family members have been described (see, for example, Lopez et al., Mol. Cell. Biol. 12:1674-1679, 1992; Wittbrodt and Rosa, Genes Devel. 8:1448-1462, 1994), and contain, for example, a mutant proteolytic cleavage site, thereby preventing the protein from being processed into the biologically active species. When co-expressed in a cell with the endogenous wild type gene, the mutant protein forms non-functional heterodimers with the wild type protein, thus acting as a dominant negative. A mutant myostatin polypeptide containing a mutation in the promyostatin cleavage site has been constructed, and can be examined for a dominant negative effect by co-expressing the mutant with wild type myostatin in varying ratios in 293 cells. Conditioned medium from 293 cells transiently transfected with the constructs can be examined by western blot analysis and the ability of the mutant to block the formation of mature C-terminal dimers can be examined.
An expression construct encoding only the myostatin prodomain also can be utilized. As disclosed above, the prodomain forms a tight complex with the mature C-terminal dimer and blocks the ability of the mature C-terminal myostatin dimer to bind Act RIIB in cells expressing the receptor in culture. By analogy to TGF-β, the myostatin prodomain also can maintain the mature C-terminal dimer in an inactive latent complex in vivo.
These transgenic animals can be bred with those expressing the tet regulators to generate doubly transgenic lines containing both the tet regulators and the inhibitor target construct. These doubly transgenic lines can be screened for those in which all of the different components are expressed appropriately. Northern blot analysis using RNA obtained from various muscles and control tissues from representative mice in each line, before and after administration of doxycycline in the drinking water, can be used to identify such transgenic lines. Transgenic lines will be selected that do not express the transgene in any tissue in the absence of doxycycline, and that express the transgene only in muscle in the presence of doxycycline.
Doxycycline is administered to the selected transgenic animals and the effect on muscle mass is examined. Doxycycline can be administered to pregnant mothers to induce the expression of the inhibitor during embryogenesis. The effect of blocking myostatin activity during development of the transgenic animals can be compared to the effects observed in myostatin knock-out mice. Since the promoters for driving expression of the tet regulators can be induced at a later time during development than the time when myostatin is first expressed, the effect on muscle mass in the transgenic mice can be compared to the effect that occurs in the myostatin knock-out mice.
The effect of inhibiting myostatin activity postnatally can be examined by administering doxycycline to the doubly transgenic mice at various times after birth. Doxycycline treatment can begin, for example, at 3 weeks of age, and the animals can be analyzed at 5 months of age, which is the age at which the difference in muscle weights were at a maximum in the myostatin knock-out mice versus wild type mice. The animals are examined for the effects of the inhibitor on muscle mass. Muscles also can be examined histologically to determine effects on fiber number and fiber size. In addition, a fiber type analysis of various muscles in the transgenic mice can be performed to determine whether there is a selective effect on type I or type II fibers.
Doxycycline can be administered in different doses and at different times to characterize the effect of the myostatin inhibitors. Doubly transgenic mice also can be maintained chronically on doxycycline, then examined for effects on fat pad weights and other relevant metabolic parameters as described above. The results of these studies can confirm that modulating myostatin activity postnatally can increase muscle mass or decrease fat accumulation, thus indicating that targeting myostatin can be useful for the treatment of a variety of muscle wasting and metabolic diseases clinically.
Myostatin. Transgenic mice containing a myostatin transgene also can be examined and the effects produced upon expression of myostatin can be compared with those observed in the nude mice containing the myostatin-expressing CHO cells. Similarly as described above, myostatin can be placed under control of conditional (tet) and tissue specific regulatory elements, and expression of myostatin in the transgenic mice can be examined to determine whether a wasting syndrome occurs similar to that observed in the nude mice. The myostatin transgene can include, for example, processing signals derived from SV40, such that transgene can be distinguished from the endogenous myostatin gene.
Serum samples can be isolated from the myostatin transgenic mice at various times following the administration of doxycycline and the level of myostatin transgene product in the serum can be determined. Total body weights of the animals are monitored over time to determine whether the animals show significant weight loss. In addition, individual muscles and fat pads are isolated and weighed, and the number, size and type of muscle fibers are determined in selected muscle samples.
The level of myostatin transgene expression can be varied by varying the dose of doxycycline administered to the animals. Transgene expression can be monitored using, for example, northern blot analysis of transgene RNA levels in muscle, or myostatin protein levels in serum. The identification of specific levels of myostatin transgene expression allows a correlation of the extent of wasting induced by myostatin. The transgenic lines also can be crossed with the myostatin knock-out mice to generate mice in which the only source of myostatin is expression from the transgene. Expression of myostatin at various times during development can be examined and the effect of myostatin on fiber number, fiber size, and fiber type can be determined. The availability of mice in which the expression of myostatin can be precisely and rapidly controlled provides a powerful tool for further characterizing the myostatin signal transduction pathway and for examining the effects of various agents that potentially can be useful for modulating myostatin signal transduction.
Effectors of myostatin signal transduction. Transgenic mice containing either dominant negative forms of a myostatin signal transduction pathway, which can include components of a TGF-β signal transduction pathway, that is expressed specifically in skeletal muscle can be generated. As disclosed herein, the Smad proteins, which mediate signal transduction through a pathway induced by activin type II receptors, can be involved in myostatin signal transduction.
Act RIIB can bind GDF-11, which is highly related to myostatin (McPherron et al., supra, 1997; Gamer et al., supra, 1999; Nakashima et al., Mech. Devel. 80:185-189, 1999), and expression of c-ski, which can bind an inhibit Smad 2, Smad 3, and Smad 4, dramatically affects muscle growth (Suprave et al., supra, 1990; Berk et al., supra, 1997; see, also, Luo et al., supra, 1999; Stroschein et al., supra, 1999; Sun et al., supra, 1999a and b; Akiyoshi et al., supra, 1999). As disclosed herein, myostatin interacts specifically with Act RIIB and, therefore, can exert its biological effect, at least in part, by binding to activin type II receptors in vivo and activating the Smad signaling pathway.
The role of the Smad signaling pathway in regulating muscle growth can be examined using transgenic mouse lines that are blocked, or capable of being blocked, at specific points in the Act RIIB/Smad signal transduction pathway. The muscle creatine kinase promoter or myosin light chain enhancer/promoter can be used to drive expression of various inhibitors of the Smad signal transduction pathway.
An inhibitor useful in this system can include, for example, follistatin; a dominant negative Act RIIB receptor; a dominant negative Smad polypeptide such as Smad 3; c-ski; or an inhibitory Smad polypeptide such as Smad 7. Follistatin can bind and inhibit the activity of certain TGF-β family members, including GDF-11 (Gamer et al., supra, 1999). Dominant negative forms of an activin type II receptor can be obtained by expressing a truncated GDF receptor, for example, by expressing the extracellular domain, particularly a soluble form of an Act RIIB extracellular domain, or by expressing a truncated Act RIIB receptor that lacks the kinase domain or contains a mutation such that the mutant receptor lacks kinase activity. Smad 7 functions as an inhibitory Smad that can block the signaling pathway induced by activin, TGF-β, and BMP. A dominant negative form of Smad 3, for example, can be constructed by mutating the Smad 3 C-terminal phosphorylation sites, thereby blocking Smad 3 function (Liu et al., supra, 1997). c-ski overexpression has been correlated to muscle hypertrophy in transgenic mice (Suprave et al., supra, 1990).
Transgenic mice can be prepared and each founder line examined for proper, muscle-specific expression of the transgene. The selected mice are examined for total body weights, individual muscle weights, and muscle fiber sizes, numbers and types. Those lines demonstrating a clear effect on muscle mass can be examined further for fat accumulation and other relevant metabolic parameters as described above. The use of these different agents to target specific steps in the activin receptor/Smad signal transduction pathway is particularly informative because the signaling pathways for the different agents overlap at different steps. For example, follistatin binds to and inhibits activin and GDF-11 activity, but not TGF-β, whereas a dominant negative Smad 3 can block signaling through both activin and TGF-β receptors. Smad 7 can have an even more pleotropic because it blocks signaling through BMP receptors as well. The studies can allow the identification of specific targets for modulating myostatin activity, thus providing various strategies for developing drugs or other agents that modulate myostatin signal transduction and, therefore, myostatin activity.
In particular, the transgenic lines described herein can be used to determine the effect of blocking myostatin function or the Smad signaling pathway postnatally on the development of obesity or type II diabetes. For example, the inhibitory transgenes can e crossed into the ob/ob, db/db, and Ay mutant mice. In the absence of doxycycline, an inhibitor transgene is not expressed and, therefore, the animals are indistinguishable from each of the parental mutant mice. In the presence of doxycycline, the inhibitor is expressed and can block myostatin activity. The effect of blocking myostatin activity on development of the metabolic abnormalities in these mutant animals can be examined.
Expression of the inhibitor can be induced at an early age, for example, at 3 weeks of age, to maximize the effect. In addition, myostatin activity can be blocked prior to the time that the metabolic abnormalities become so severe as to be irreversible. Animals can be maintained on doxycycline and assessed at various ages using the tests described above, including those relating to fat accumulation and glucose metabolism. Any delay in the age at which one or more test results becomes abnormal in the ob/ob, db/db, and Ay mutant animals can be identified. Similar studies can be performed using older animals, which have developed some of the signs of obesity or type II diabetes, and the effect of blocking myostatin activity on various parameters, including fat weight and glucose metabolism, can be determined. The results of these studies can further identify specific targets that can be manipulated in an effort to prevent or treat obesity or type II diabetes.
EXAMPLE 12
Characterization of Myostatin Effect on the Induction of Cachexia
This example describes methods for determining the role of myostatin signal transduction in the development and progression of cachexia.
The activin receptor and Smad pathway can constitute at least part of the signal transduction pathway involved in mediating myostatin activity in normal individuals and, therefore, can be involved in mediating the effects that occur in an individual due to excess levels of myostatin. As disclosed herein, cachexia, for example, can be mediated, at least in part, by abnormally high levels of myostatin. As such, methods for manipulating signal transduction through the Smad pathway can provide a new strategy for developing drugs for the treatment of muscle wasting in general and cachexia in particular.
The role of the Smad signaling pathway in cachexia can be examined by examining the susceptibility of the various transgenic lines described above to cachexia, which can be induced, for example, by interleukin-6 (IL-6; Black et al., Endocrinology 128:2657-2659, 1991, which is incorporated herein by reference]), tumor necrosis factor-I (TNF-α; Oliff et al., Cell 50:555-563, 1987, which is incorporated herein by reference), or certain tumor cells. In the case of IL-6 and TNF-α, the inhibitor transgenes can be crossed into a nude mouse background, then the animals can be challenged with CHO cells that produce IL-6 or TNF-α, which induce wasting in nude mice when overexpressed in this manner. CHO cells that overproduce IL-6 or TNF-α can be prepared using the methods described above for generating myostatin overproducing cells. For example, TNF-α cDNA can be cloned into the pMSXND expression vector (Lee and Nathans, J. Biol. Chem. 263:3521-3527, 1988), then cells carrying amplified copies of the expression construct can be selected stepwise in increasing concentrations of methotrexate.
Tumor cells such as Lewis lung carcinoma cells (Matthys et al., Eur. J. Cancer 27:182-187, 1991, which is incorporated herein by reference) or colon 26 adenocarcinoma cells (Tanaka et al., J. Cancer Res. 50:2290-2295, 1990, which is incorporated herein by reference), which can induce cachexia in mice, also can be utilized for these studies. These cell lines cause severe wasting when grown as tumors in mice. Thus, the effect of these tumors can be examined in the various transgenic mice described herein. It is recognized that the various tumor cells will only grow in certain genetic backgrounds. For example, the Lewis lung carcinoma cells are routinely grown in C57 BL/6 mice, and the colon 26 carcinoma cells are routinely grown in BALB/c mice. Thus, the transgenes can be backcrossed into these or other genetic backgrounds to allow growth of the tumor cells.
Various parameters, including total body weight, individual muscle weight, muscle fiber size and number, food intake and serum parameters, including glucose levels, can be monitored. In addition, serum myostatin levels and myostatin RNA levels in muscle can be examined to confirm that increased myostatin expression is correlated with cachexia. The results of these studies can confirm that the action of myostatin is downstream of the cachexia-inducing agents in these experimental models. The results also can confirm that the Smad signaling pathway is essential for development of cachexia in these models, and can demonstrate that a therapeutic benefit can be obtained in the treatment of cachexia by modulating the Smad signaling.
EXAMPLE 13
Identification and Characterization of Growth Differentiation Factor-8 (GDF-8) and GDF-11 Receptors
This example describes methods for identifying and characterizing cell surface receptors for GDF-8 (myostatin) and GDF-11.
The purified GDF-8 and GDF-11 proteins will be used primarily to assay for biological activities. In order to identify potential target cells for GDF-8 and GDF-11 action cells expressing their receptors will be searched. For this purpose, the purified protein will be radio-iodinated using the chloramine T method, which has been used successfully to label other members of this superfamily, like TGF-β (Cheifetz et al., supra, 1987), activins (Sugino et al., J. Biol. Chem. 263:15249-15252, 1988), and BMPs (Paralkar et al., Proc. Natl. Acad. Sci., USA 88:3397-3401, 1991), for receptor binding studies. The mature processed forms of GDF-8 and GDF-11 each contain multiple tyrosine residues. Two different approaches will be taken to identify receptors for these proteins.
One approach will determine the number, affinity, and distribution of receptors. Either whole cells grown in culture, frozen sections of embryos or adult tissues, or total membrane fractions prepared from tissues or cultured cells will be incubated with the labeled protein, and the amount or distribution of bound protein will be determined. For experiments involving cell lines or membranes, the amount of binding will be determined by measuring either the amount of radioactivity bound to cells on the dish after several washes or, in the case of membranes, the amount of radioactivity sedimented with the membranes after centrifugation or retained with the membranes on a filter. For experiments involving primary cultures, where the number of cells can be more limited, binding sites will be visualized directly by overlaying with photographic emulsion. For experiments involving frozen sections, sites of ligand binding will be visualized by exposing these sections to high resolution Beta-max hyperfilm; if finer localization is required, the sections will be dipped in photographic emulsion. For all of these experiments, specific binding will be determined by adding excess unlabeled protein as competitor (for example, see Lee and Nathans, supra, 1988).
A second approach will be to characterize the receptor biochemically. Membrane preparations or potential target cells grown in culture will be incubated with labeled ligand, and receptor/ligand complexes will be covalently cross-linked using disuccinimidyl suberate, which has been commonly used to identify receptors for a variety of ligands, including members of the TGF-β superfamily (Massague and Like, J. Biol. Chem. 260:2636-2645, 1985). Cross-linked complexes are separated by electrophoresis on SDS polyacrylamide gels to look for bands labeled in the absence, but not in the presence, of excess unlabeled protein. The molecular weight of the putative receptor will be estimated by subtracting the molecular weight of the ligand. An important question that these experiments will address is whether GDF-8 and GDF-11 signal through type I and type II receptors like many other members of the TGF-β superfamily (Massague and Weis-Garcia, supra, 1996).
Once a method for detecting receptors for these molecules has been achieved, more detailed analysis will be carried out to determine the binding affinities and specificities. A Scatchard analysis will be used to determine the number of binding sites and dissociation constants. By carrying out cross-competition analyses between GDF-8 and GDF-11, it will be possible to determine whether they are capable of binding to the same receptor and their relative affinities. These studies will give an indication as to whether the molecules signal through the same or different receptors. Competition experiments using other TGF-β family members will be performed to determine specificity. Some of these ligands are available commercially, and some others are available from Genetics Institute, Inc.
For these experiments, a variety of embryonic and adult tissues and cell lines will be tested. Based on the specific expression of GDF-8 in skeletal muscle and the phenotype of GDF-8 knock-out mice, initial studies focus on embryonic and adult muscle tissue for membrane preparation and for receptor studies using frozen sections. In addition, myoblasts will be isolated and cultured from embryos at various days of gestation or satellite cells from adult muscle as described (Vivarelli and Cossu, Devel. Biol. 117:319-325, 1986; Cossu et al., Cell Diff. 9:357-368, 1980). The binding studies on these primary cells after various days in culture will be performed and binding sites localized by autoradiography so that the binding sites can be co-localized with various myogenic markers, such as muscle myosin (Vivarelli et al., J. Cell Biol. 107:2191-2197, 1988), and correlate binding with the differentiation state of the cells, such as formation of multinucleated myotubes. In addition to using primary cells, cell lines will be utilized to look for receptors. In particular, the initial focus will be on three cells lines, C2C12, L6, and P19. C2C12 and L6 myoblasts differentiate spontaneously in culture and form myotubes depending on the particular growth conditions (Yaffe and Saxel, supra, 1977; Yaffe, supra, 1968). P19 embryonal carcinoma cells can be induced to differentiate into various cell types, including skeletal muscle cells in the presence of DMSO (Rudnicki and McBurney, Teratocarcinomas and Embryonic Stem Cells: A practical approach (E. J. Robertson, IRL Press, Cambridge 1987). Receptor binding studies will be carried out on these cell lines under various growth conditions and at various stages of differentiation. Although the initial studies will focus on muscle cells, other tissues and cell types will be examined for the presence of GDF-8 and GDF-11 receptors.
Recombinant human GDF-8 (rhGDF-8) homodimer will be used in these binding studies. RhGDF-8 was expressed using CHO cells and purified to approximately 90% purity. The rhGDF-8 had the expected 25 kDa to 27 kDa molecular weight and, upon reduction, was reduced to the 12 kDa monomer. Using 1-125 labeled GDF-8 in a receptor-ligand binding assay, two myoblast cell lines, L6 and G-8, bound GDF-8. The binding was specific since non labeled GDF-8 effectively competed the binding of the labeled ligand. The dissociation constant (Kd) was 370 pM, and L6 myoblasts have a high number (5,000 receptors/cell) of cell surface binding proteins. GDF-11 (BMP-11) is highly homologous (>90%) to GDF-8. Receptor binding studies revealed that GDF-8 and GDF-11 bound to the same binding proteins on L6 myoblasts. It is important to establish whether or not GDF-8 binds to the known TGF-β receptor. TGF-β did not compete the binding of GDF-8, indicating that the GDF-8 receptor is distinct from the TGF-β receptor. The GDF-8 receptor was not expressed on all myoblast cell lines, including four myoblast cell lines, C2C12, G7, MLB13MYC c14 and BC3H1, which do not bind GDF-8.
The gene or genes encoding receptors for GDF-8 and GDF-11 can be obtained. As a first step towards understanding the mechanism by which GDF-8 and GDF-11 exert their biological effects, it is important to clone the genes encoding their receptors. From the experiments above, it will be more clear as to whether GDF-8 and GDF-11 bind to the same receptor or to different receptors. There will also be considerable information regarding the tissue and cell type distribution of these receptors. Using this information, two different approaches will be taken to clone the receptor genes.
The first approach will be to use an expression cloning strategy. In fact, this was the strategy that was originally used by Mathews and Vale (Cell 65:973-982, 1991) and Lin et al. (Cell 68:775-785, 1992) to clone the first activin and TGF-β receptors. Poly A-selected RNA from the tissue or cell type that expresses the highest relative number of high affinity binding sites will be obtained, and used to prepare a cDNA library in the mammalian expression vector pcDNA-1, which contains a CMV promoter and an SV40 origin of replication. The library will be plated, and cells from each plate will be pooled into broth and frozen. Aliquots from each pool will be grown for preparation of DNA. Each individual pool will be transiently transfected into COS cells in chamber slides, and transfected cells will be incubated with iodinated GDF-8 or GDF-11. After washing away the unbound protein, the sites of ligand binding will be visualized by autoradiography. Once a positive pool is identified, the cells from that pool will be replated at lower density, and the process will be repeated. Positive pools will then be plated, and individual colonies will be picked into grids and re-analyzed as described (Wong et al., Science 228:810-815, 1985).
Initially, using pool sizes of 1500 colonies will be screened. In order to be certain to identify a positive clone in a mixture of this complexity, a control experiment using TGF-β and a cloned type II receptor will be performed. The coding sequence for the TGF-β type II receptor will be cloned into the pcDNA-1 vector, and bacteria transformed with this construct will be mixed with bacteria from the library at various ratios, including 1:1500. The DNA prepared from this mixture then will be transfected into COS cells, incubated with iodinated TGF-β, and visualized by autoradiography. If positive signals are observed at a ratio of 1:1500, pools of 1500 clones will be screened. Otherwise, smaller pool sizes corresponding to ratios at which the procedure is sensitive enough to identify a positive signal in control experiments will be used.
A second parallel strategy to attempt to clone the GDF-8 and GDF-11 receptors also will be used, taking advantage of the fact that most receptors for members of the TGF-β superfamily that have been identified belong to the membrane-spanning serine/threonine kinase family (Massague and Weis-Garcia, supra, 1996). Because the cytoplasmic domains of these receptors are related in sequence, degenerate PCR probes will be used to clone members of this receptor family that are expressed in tissues that contain binding sites for GDF-8 and GDF-11. In fact, this is the approach that has been used to identify most of the members of this receptor family. The general strategy will be to design degenerate primers corresponding to conserved regions of the known receptors, to use these primers for PCR on cDNA prepared from the appropriate RNA samples (most likely from skeletal muscle), to subclone the PCR products, and finally to sequence individual subclones. As sequences are identified, they will be used as hybridization probes to eliminate duplicate clones from further analysis. The receptors that are identified then will be tested for their ability to bind purified GDF-8 and GDF-11. Because this screen will yield only small PCR products, full-length cDNA clones will be obtained for each receptor from cDNA libraries prepared from the appropriate tissue, inserted into the pcDNA-1 vector, transfected into COS cells, and the transfected cells will be assayed for their ability to bind iodinated GDF-8 or GDF-11. Ideally, every receptor that is identified in this screen will be tested for the ability to bind these ligands. However, the number of receptors that are identified can be large, and isolating all of the full-length cDNAs and testing them can require considerable effort. Almost certainly some of the receptors that are identified will correspond to known receptors, and for these, either obtaining full-length cDNA clones from other investigators or amplifying the coding sequences by PCR based on the published sequences should be straightforward. For novel sequences, the tissue distribution will be determined by northern blot analysis and the highest priority will be directed to those receptors whose expression pattern most closely resembles the distribution of GDF-8 and/or GDF-11 binding sites as determined above.
In particular, it is known that these receptors fall into two classes, type I and type II, which can be distinguished based on the sequence and which are both required for full activity. Certain ligands cannot bind type I receptors in the absence of type II receptors while others are capable of binding both receptor types (Massague and Weis-Garcia, supra, 1996). The cross-linking experiments outlined above should give some indication as to whether both type I and type II receptors are also involved in signaling GDF-8 and GDF-11. If so, it will be important to clone both of these receptor subtypes in order to fully understand how GDF-8 and GDF-11 transmit their signals. Because it cannot be predicted as to whether the type I receptor is capable of interacting with GDF-8 and GDF-11 in the absence of the type II receptor, type II receptor(s) will be cloned first. Only after at least one type II receptor has been identified for these ligands, will an attempt be made to identify the type I receptors for GDF-8 and GDF-11. The general strategy will be to cotransfect the type II receptor with each of the type I receptors that are identified in the PCR screen, then assay the transfected cells by crosslinking. If the type I receptor is part of the receptor complex for GDF-8 or GDF-11, two cross-linked receptor species should be detected in the transfected cells, one corresponding to the type I receptor and the other corresponding to the type II receptor.
The search for GDF-8 and GDF-11 receptors is further complicated by the fact at least one member of the TGF-β superfamily, namely, GDNF, is capable of signaling through a completely different type of receptor complex involving a GPI-linked component (GDNFR-alpha) and a receptor tyrosine kinase (c-ret; Trupp et al., Nature 381:785-789, 1996; Durbec et al., Nature 381:789-793, 1996; Treanor et al., Nature 382:80-83, 1996; Jing et al., Cell 85:1113-1124, 1996). Although GDNF is the most distantly-related member of the TGF-β superfamily, it is certainly possible that other TGF-β family members can also signal through an analogous receptor system. If GDF-8 and GDF-11 do signal through a similar receptor complex, the expression screening approach should be able to identify at least the GPI-linked component (indeed GDNFR-alpha was identified using an expression screening approach) of this complex. In the case of GDNF, the similar phenotypes of GDNF- and c-ret-deficient mice suggested c-ret as a potential receptor for GDNF.
EXAMPLE 14
Production of Transgenic Mice Expressing Myostatin Pro-Peptide, Follistatin or a Dominant Negative Act Riib
Purification of myostatin. A Chinese hamster ovary (CHO) cell line carrying amplified copies of a myostatin expression construct was transfected with an expression construct for the furin protease PACE in order to improve processing of the precursor protein. Conditioned medium (prepared by Cell Trends, Middletown, Md.) was passed successively over hydroxylapatite (eluted with 200 mM sodium phosphate pH 7.2), lentil lectin Sepharose (eluted with 50 mM Tris pH 7.4, 500 mM NaCl, 500 mM methyl mannose), DEAE agarose (collected material that flowed through the column in 50 mM Tris pH 7.4, 50 mM NaCl), and heparin Sepharose (eluted with 50 mM Tris pH 7.4, 200 mM NaCl). The eluate from the heparin column was then bound to a reverse phase C4 HPLC column and eluted with an acetonitrile gradient in 0.1% trifluoroacetic acid. Antibodies directed against the mature C-terminal protein were described previously (see U.S. Pat. No. 5,827,733, incorporated herein by reference). In order to raise antibodies against the pro peptide, the portion of the human myostatin protein spanning amino acids 122-261 was expressed in bacteria using the RSET vector (Invitrogen, San Diego, Calif.), purified by nickel chelate chromatography, and injected into rabbits. Immunizations were carried out by Spring Valley Labs (Woodbine, Md.).
Receptor binding. Purified myostatin was radioiodinated using the chloramine T method (Frolik, et al. (1984) J Biol Chem 259, 10995-11000). COS-7 cells grown in 6 or 12 well plates were transfected with 1-2 μg pCMV5 or pCMV5/receptor construct using lipofectamine (Gibco, Rockville, Md.). Crosslinking experiments were carried out 2 days post transfection as described (Franzén, et al. (1993) Cell 75, 681-692). For quantitative receptor binding assays, cell monolayers were washed twice with PBS containing 1 mg/ml BSA and incubated with labeled myostatin in the presence or absence of various concentrations of unlabeled myostatin, pro peptide, or follistatin at 4° C. Cells were then washed 3 times with the same buffer, lysed in 0.5N NaOH, and counted in a gamma counter. Specific binding was calculated as the difference in bound myostatin between cells transfected with Act RIIB and cells transfected with vector. This method of calculating specific binding was especially important in assessing the effect of the pro peptide as the addition of the pro peptide also reduced non-specific binding in a concentration-dependent manner.
Transgenic mice. DNAs encoding a truncated form of murine Act RIIB spanning amino acids 1-174, the murine myostatin pro peptide spanning amino acids 1-267, and the human follistatin short form were cloned into the MDAF2 vector containing the myosin light chain promoter and myosin light chain 1/3 enhancer (McPherron, A. C. & Lee, S.-J. (1993) J Biol Chem 268, 3444-3449). Purified transgenes including the myosin light chain regulatory sequences and SV40 processing sites were used for microinjections. All microinjections and embryo transfers were carried out by the Johns Hopkins School of Medicine Transgenic Core Facility. Transgenic founders in a hybrid SJL/C57BL/6 background were mated to wild type C57BL/6 mice, and all studies were carried out using F1 offspring. For analysis of muscle weights, individual muscles were dissected from both sides of nearly all animals, and the average of the left and right muscle weights was used. Analysis of fiber numbers and sizes was carried out as described (McPherron, A. C., Lawler, A. M. & Lee, S.-J. (1997) Nature 387, 83-90). RNA isolation and Northern analysis were carried out as described (McPherron, A. C. & Lee, S.-J. (1993) J Biol Chem 268, 3444-3449).
In order to overproduce myostatin protein, a CHO cell line carrying amplified copies of a myostatin expression construct was produced (McPherron, A. C., Lawler, A. M. & Lee, S.-J. (1997) Nature 387, 83-90). Myostatin was purified from the conditioned medium of this cell line by successive fractionation on hydroxylapatite, lentil lectin Sepharose, DEAE agarose, and heparin Sepharose. Silver stain analysis of the purified protein preparation revealed the presence of two protein species of 29 kd and 12.5 kd. A variety of data suggested that this purified protein consisted of a non-covalent complex of two pro peptide molecules bound to a disulfide-linked C-terminal dimer. First, by Western analysis, the 29 kd and 12.5 kd species were immunoreactive with antibodies raised against bacterially-expressed fragments of myostatin spanning the pro peptide and C-terminal mature region, respectively. Second, in the absence of reducing agents, the C-terminal region had an electrophoretic mobility consistent with that of a dimer. Third, the two species were present in a molar ratio of approximately 1:1. And fourth, the C-terminal dimer was retained on the lectin column and could be eluted with methyl mannose even though this portion of the protein contains no potential N-linked glycosylation sites; the simplest interpretation of these data is that the C-terminal region bound the lectin indirectly by being present in a tight complex with the pro peptide, which does have a glycosylation signal.
Because the C-terminal dimer is known to be the biologically active molecule for other TGF-β family members, the C-terminal dimer of myostatin was purified away from its pro peptide by reverse phase HPLC. The fractions containing the purified C-terminal dimer (32-34) appeared to be homogeneous. However, the fractions most enriched for the pro peptide (35-37) were contaminated with small amounts of C-terminal dimer and with high molecular weight complexes that most likely represented misfolded proteins.
Most members of the TGF-β superfamily have been shown to signal by binding serine/threonine kinase receptors followed by activation of Smad proteins (Heldin, C.-H., Miyazono, K. & ten Dijke, P. (1997) Nature 390, 465-471; Massagué, J., Blain, S. W. & Lo, R. S. (2000) Cell 103, 295-309). The initial event in triggering the signaling pathway is the binding of the ligand to a type II receptor. In order to determine whether myostatin is capable of binding any of the known type II receptors for related ligands, cross-linking studies were carried out with radio-iodinated myostatin C-terminal dimer on COS-7 cells transfected with expression constructs for either TGF-β, BMP, or activin type II receptors. Cross-linked complexes of the predicted size (full length receptor bound to myostatin) were detected for cells expressing either Act RIIA or Act RIIB. Higher levels of binding to Act RIIB than to Act RIIA were observed in both cross-linking and standard receptor binding assays, therefore receptor binding studies were focused on Act RIIB. Binding of myostatin to Act RIIB was specific (binding could be competed by excess unlabeled myostatin) and saturable, and assuming that all of the myostatin protein was bioactive, it was estimated that the dissociation constant by Scatchard analysis to be approximately 10 nM. It is known in the case of TGF-β that the affinity for the type II receptor is significantly higher in the presence of the appropriate type I receptor and that other molecules are involved in presenting the ligand to the receptor.
In order to determine whether activin type II receptors may be involved in myostatin signaling in vivo, the effect of expressing a dominant negative form of Act RIIB in mice was investigated. For this purpose, a construct in which a truncated form of Act RIIB lacking the kinase domain was placed downstream of a skeletal muscle-specific myosin light chain promoter/enhancer was generated. From pronuclear injections of this construct, a total of 7 founder animals positive for the transgene were identified. Analysis of these founder animals at 7 months of age revealed that all seven had significant increases in skeletal muscle mass with individual muscles of these founder animals weighing up to 125% more than those of control non-transgenic animals derived from similar injections (Table 2).
Three lines of evidence suggested that the increases in muscle weights in these founder animals resulted from the expression of the transgene. First, analysis of offspring derived from matings of three founder animals (the other four founder animals did not generate sufficient numbers of offspring for analysis) with wild type C57BL/6 mice showed that the increases in muscle weights correlated with the presence of the transgenes (Table 3). Second, although muscle weights varied among the different transgenic lines, the magnitude of the increase was highly consistent among animals in any given line for all muscles examined and for both males and females (Table 3). For example, all muscles of both male and female mice from the C5 line weighed approximately 30-60% more than those of control animals, whereas all muscles from C11 mice weighed approximately 110-180% more. Third, Northern analysis of RNA samples prepared from transgenic animals showed that the expression of the transgene was restricted to skeletal muscle and that the relative levels of transgene expression correlated with the relative magnitude of the increase in muscle weights (Table 3). For example, animals from the C11 line, which had the greatest increases in muscle weights, also had the highest levels of transgene expression.
These data showed that expression of a dominant negative form of Act RIIB can cause increases in muscle mass similar to those seen in myostatin knockout mice. In myostatin knockout mice, the increase in muscle mass has been shown to result from increases in both fiber number and fiber size. In order to determine whether expression of dominant negative Act RIIB also causes both hyperplasia and hypertrophy, sections of the gastrocnemius and plantaris muscles of animals from the C27 line were analyzed. Compared to control muscles, the muscles of the C27 animals showed a clear increase in overall cross-sectional area. This increase in area resulted partially from an increase in fiber number. At the widest point, the gastrocnemius and plantaris muscles had a total of 10015+1143 fibers in animals from the C27 line (n=3) compared to 7871+364 fibers in control animals (n=3). However, muscle fiber hypertrophy also contributed to the increase in total area. The mean fiber diameter was 51 μm in animals of the C27 line compared to 43 μm in control animals. Hence, the increase in muscle mass appeared to result from an approximately 27% increase in the number of fibers and 19% increase in fiber diameter (assuming the fibers to be roughly cylindrical, this increase in diameter result in an approximately 40% increase in cross sectional area). Except for the increase in fiber number and size, however, the muscles from the transgenic animals looked grossly normal. In particular, there were no obvious signs of degeneration, such as widely varying fiber sizes (the standard deviation of fiber sizes was similar between control and transgenic animals) or extensive fibrosis or fat infiltration.
These approaches were used to explore other possible strategies for inhibiting myostatin. First, the effect of the myostatin pro peptide was investigated. In the case of TGF-β, it is known that the C-terminal dimer is held in an inactive, latent complex with other proteins, including its pro peptide, and that the pro peptide of TGF-β can have an inhibitory effect on TGF-β activity both in vitro and in vivo (Miyazono, et al. (1988) J Biol Chem 263, 6407-6415; Gentry, L. E. & Nash, B. W. (1990) Biochem. 29, 6851-6857; Böttinger, et al. (1996) Proc Natl Acad Sci, USA 93, 5877-5882). The observation that the myostatin C-terminal dimer and pro peptide co-purified raised the possibility that myostatin may normally exist in a similar latent complex and that the myostatin pro peptide may have inhibitory activity. Second, the effect of follistatin, which has been shown to be capable of binding and inhibiting the activity of several TGF-β family members was examined. In particular, follistatin can block the activity of GDF-11, which is highly related to myostatin, and follistatin knockout mice have been shown to have reduced muscle mass at birth, which would be consistent with over-activity of myostatin (Gamer, et al. (1999) Dev Biol 208, 222-232; Matzuk, et al. (1995) Nature 374, 360-363).
The effect of the pro peptide and follistatin in vitro was then studied. Both the myostatin pro peptide and follistatin were capable of blocking the binding of the C-terminal dimer to Act RIIB. The Ki of follistatin was estimated to be approximately 470 pM and that of the pro peptide to be at least 50-fold higher. The calculation of the Ki for the pro peptide, however, assumes that all of the protein in the final preparation represented biologically active pro peptide and therefore is likely to be an overestimate. As discussed above, the pro peptide preparation was contaminated both with small amounts of C-terminal dimer and with misfolded high molecular weight species.
In order to determine whether these molecules are also capable of blocking myostatin activity in vivo, transgenic mice were generated in which the myosin light chain promoter/enhancer was used to drive expression of either the myostatin pro peptide or follistatin. From pronuclear injections of the pro peptide construct, three transgenic mouse lines (two of these, B32A and B32B, represented independently segregating transgene insertion sites in one original founder animal) were identified that showed increased muscling. As shown in Table 3, muscle weights of animals from each line were increased by approximately 20-110% compared to those of non-transgenic control animals. Northern analysis of RNA samples prepared from representative animals of each of these lines showed that the expression levels of the transgene correlated with the magnitude of the increase in muscle weights. Specifically, animals from the B32A line, which had only an approximately 20-40% increase in muscle mass, had the lowest levels of transgene expression, and animals from the B32B and B53 lines, which had an approximately 70-110% increase in muscle mass, had the highest levels of transgene expression. Perhaps significantly, muscle weights in animals that were doubly transgenic for the B32A and B32B insertion sites were similar to those observed in animals transgenic only for the B32B insertion site (Table 3) despite the fact that the doubly transgenic animals appeared to have higher levels of transgene expression. These findings suggest that the effects seen in the B32B line (and B53 line) were the maximal achievable from overexpressing the pro peptide. As in the case of animals expressing the dominant negative form of Act RIIB, animals expressing the pro peptide showed increases in both muscle fiber number and size. Analysis of the gastrocnemius and plantaris muscles from two animals that were doubly transgenic for the B32A and B32B insertion sites showed that fiber numbers were increased by approximately 40% (the two animals had 11940 and 10420 fibers), and fiber diameters were increased by approximately 21% (to 52 μm) compared to control animals.
The most dramatic effects on skeletal muscle were obtained using the follistatin construct. Two founder animals (F3 and F66) showed increased muscling (Table 2). In one of these animals (F3), muscle weights were increased by 194-327% relative to control animals, resulting from a combination of hyperplasia (66% increase in fiber number to 13051 in the gastrocnemius/plantaris) and hypertrophy (28% increase in fiber diameter to 55 μm). Although the muscle weights of myostatin knockout mice in a hybrid SJL/C57BL/6 background have not been analyzed, the increases in muscle mass observed in the F3 founder animal were significantly greater than the increases seen in myostatin null animals in other genetic backgrounds. These results suggest that at least part of the effect of follistatin may result from inhibition of another ligand besides myostatin. Clearly, analysis of additional follistatin transgenic lines will be essential in determining whether other ligands may also be involved in negatively regulating muscle growth.
Following proteolytic processing, the myostatin C-terminal dimer is likely maintained in a latent complex with its pro peptide and perhaps other proteins as well. Myostatin is also negatively regulated by follistatin, which binds the C-terminal dimer and inhibits its ability to bind to receptors. Release of the C-terminal dimer from these inhibitory proteins by unknown mechanisms allows myostatin to bind to activin type II receptors. By analogy with other family members, it is presumed that activation of these receptors then leads to activation of a type I receptor and Smad proteins.
This overall model for myostatin regulation and signaling is consistent not only with the data presented here but also with other genetic data. As discussed earlier, follistatin knockout mice have been shown to have reduced muscle mass at birth, which is what one might expect for uninhibited myostatin activity. A similar muscle phenotype has been reported for mice lacking ski, which has been shown to inhibit the activity of Smad2 and 3, and the opposite phenotype, namely excess skeletal muscle, has been observed in mice overexpressing ski. Based on the present findings, one hypothesis is that these observed phenotypes reflect the over-activity and under-activity, respectively, of myostatin in these mice.
Although all of the in vitro and genetic data are consistent with the overall model that have put forth herein, these data would also be consistent with alternative models involving other receptors and ligands. For example, the mechanism by which the truncated form of Act RIIB enhances muscle growth in the transgenic mice is not known. It is possible that the truncated receptor is not acting to block signaling in the target cell but is rather merely acting as a sink to deplete extracellular concentrations of myostatin. It is also possible that the truncated receptor is blocking signaling of other ligands besides myostatin. In this regard, it has been shown that dominant negative forms of type II activin receptors can block signaling of a variety of different TGF-β related ligands in other species. Similarly, the data provided herein does not show definitively that follistatin is blocking myostatin activity in vivo to promote muscle growth. In this regard, the extraordinary degree of muscling seen in one of the follistatin expressing founder animals suggests that other follistatin-sensitive ligands may be involved in regulating muscle growth.







TABLE 2







Muscle weights (mg)











transgenic animals
pectoralis
triceps
quadriceps
gastroc./plantaris





male controls (7 mo., n = 10)
100.8 ± 5.4 
115.6 ± 5.5
 243.8 ± 12.5
168.1 ± 7.6







dom. neg. Act RIIB (7 mo.)











C5 male founder
148
155
318
252


C11 male founder
227
250
454
338


C33 male founder
158
176
352
244


C42 male founder
196
212
309
269


female controls (7 mo., n = 10)
68.9 ± 2.7
 96.9 ± 3.5
208.3 ± 7.1
140.3 ± 4.3







dom. neg. Act RIIB (7 mo.)











C2 female founder
104
163
352
263


C4 female founder
103
139
303
194


C27 female founder
135
117
181
256


male controls (4 mo., n = 12)
98.3 ± 3.3
110.9 ± 2.9
251.7 ± 8.5
169.3 ± 4.7







follistatin (4 mo.)











F3 male founder
296
494
736
568


F66 male founder
169
263
421
409





All animals (including controls) represent hybrid SJL/C57BL/6 F0 mice born from injected embryos.













TABLE 3







Muscle weights (mg)











transgenic line
pectoralis
triceps
quadriceps
gastroc./plantaris










MALES











controls (n = 50)
104.6 ± 1.5
113.9 ± 1.6
246.2 ± 3.0
167.7 ± 2.1







dom. neg. Act RIIB











C5 (n = 11)
153.7 ± 6.0***
177.5 ± 6.0***
322.7 ± 9.3***
247.1 ± 8.1***


C27 (n = 5)
190.4 ± 7.1***
230.8 ± 13.0***
406.8 ± 11.6***
283.8 ± 6.9***


C11 (n = 2)
278.0 ± 18.4*
244.5 ± 4.9**
515.5 ± 7.8**
366.0 ± 21.2*







pro peptide











B32A (n = 8)
139.9 ± 7.1***
160.6 ± 7.8***
322.5 ± 10.3***
222.6 ± 7.1***


B32B (n = 4)
214.0 ± 19.9**
206.5 ± 6.7***
435.8 ± 15.0***
289.5 ± 8.6***


B32A + B (n = 8)
212.4 ± 8.4***
220.3 ± 6.4***
429.1 ± 11.1***
288.3 ± 8.5***


B53 (n = 8)
215.1 ± 6.4***
229.6 ± 8.1***
413.3 ± 13.2***
293.5 ± 10.5***







FEMALES











controls (n = 50)
 64.7 ± 1.4
 75.7 ± 1.1
164.5 ± 2.0
109.6 ± 1.4







dom. neg. Act RIIB











C5 (n = 15)
 89.7 ± 2.8***
115.9 ± 4.0***
229.3 ± 6.5***
161.8 ± 4.7***


C27 (n = 5)
117.6 ± 10.9***
138.6 ± 12.3***
314.0 ± 27.7***
207.6 ± 18.3***


C11 (n = 3)
180.3 ± 38.9
208.7 ± 45.7
430.3 ± 72.2*
291.7 ± 48.8*







pro peptide











B32A (n = 9)
 78.8 ± 2.9***
100.1 ± 3.7***
206.0 ± 2.7***
138.9 ± 3.1***


B32B (n = 2)
131.0 ± 18.4
151.5 ± 23.3
315.5 ± 58.7
199.5 ± 24.7


B32A + B (n = 4)
109.3 ± 9.5*
132.8 ± 6.0**
270.8 ± 6.9***
177.0 ± 2.4***


B53 (n = 6)
134.7 ± 7.7***
148.2 ± 12.1***
303.8 ± 18.5***
212.8 ± 12.9***





*p < 0.05,


**p < 0.01,


***p < 0.001.


All animals (including controls) represent 4 month old offspring of transgenic founders (SJL/C57BL/6) mated with wild type C57Bl/6 mice.






EXAMPLE 15
FLRG Acts in a Dose-Dependent Manner
This example presents data showing that FLRG, like follstatin, can promote muscle growth when expressed as a transgene in skeletal muscle and that both of these molecules appear to act by blocking not only myostatin but also other ligands with similar activity to myostatin. By combining the follstatin transgene with a myostatin null mutation, mice with quadrupled muscle mass were generated, which represents yet another doubling of muscle mass compared to mice only lacking myostatin. These studies demonstrate that muscle mass in mice is controlled by multiple members of the transforming growth factor-β superfamily acting in concert. A similar conclusion was reached in an earlier study in which we demonstrated that administration of a soluble form of the ACVR2B receptor to wild type could cause more extensive muscle growth than what had been observed previously using myostatin-specific inhibitors and that this soluble receptor could also increase muscle growth even in mice completely lacking myostatin. The studies presented herein thus demonstrate that the capacity for promoting muscle growth by targeting this general signaling pathway is far greater than previously appreciated.
Mstn mutant and F66 transgenic mice were backcrossed at least six times onto a C57 BL/6 background prior to analysis. All analysis was carried out on 10 week old mice. For measurement of muscle weights, individual muscles from both sides of the animal were dissected, and the average weight was used for each muscle. For morphometric analysis, the gastrocnemius and plantars muscles were sectioned serially to their widest point using a cryostat, and fiber diameters were measured (as the shortest distance across the fiber passing through the midpoint) from hematoxylin and eosin stained sections. Measurements were carried out on 250 fibers per animal, and all data for a given genotype were pooled.
To determine whether FLRG can have similar effects to follstatin in vivo, a construct in which the FLRG coding sequence was placed downstream of a myosin light chain promoter/enhancer was generated. From pronuclear injections of this construct, a total of four transgenic mouse lines (Z111A, Z111B, Z116A, and Z116B) were obtained containing independently segregating insertion sites. Each of these four transgenic lines was backcrossed at least six times to C57 BL/6 mice prior to analysis in order to control for genetic background effects. Northern analysis revealed that in three of these lines the transgene was expressed in skeletal muscles but not in any of the non-skeletal muscle tissues examined (FIG. 1); in the fourth line, Z111B, the expression of the transgene was below the level of detection in these blots. As shown in Table 4, all four lines exhibited significant increases in muscle weights compared to wild type control mice. These increases were observed in all four muscles that were examined as well as in both sexes. Moreover, the rank order of magnitude of these increases correlated with the rank order of expression levels of the transgene; in the highest-expressing line, Z116A, muscle weights were increased by 57-81% in females and 87-116% in males compared to wild type mice. Hence, FLRG is capable of increasing muscle growth in a dose-dependent manner when expressed as a transgene in skeletal muscle.
EXAMPLE 16
FLRG Suppresses Additional Ligands
To determine whether the FLRG transgene was causing increased muscle growth by blocking myostatin activity, the effect of combining the FLRG transgene with a loss-of-function mutation in the myostatin gene was examined. A number of female Z116A transgenic mice that were heterozygous for the myostatin mutation were obtained. As shown in Table 4, these mice exhibited further increases in muscle weights compared to Z116A mice that were wild type for myostatin. Most importantly, in two of the muscles that were examined (quadriceps and gastrocnemius) the observed increases were also greater than those seen in Mstn−/− mice lacking the transgene. Based on this finding, it appears that myostatin is not the sole target for FLRG in the transgenic mice and, therefore, that additional ligands must be capable of suppressing muscle growth in vivo.
A similar set of experiments utilizing follstatin transgenic mice was performed. The results demonstrated that these additional ligands do play a major role in suppressing muscle growth. In previous studies, several transgenic founders expressing follstatin from a myosin light chain promoter/enhancer were generated. A transgenic line was established from one of these founders (F66), which was extensively backcrossed to C57 BL/6 mice for subsequent analysis. In this line, the transgene was most likely located on the Y chromosome, as the transgene was transmitted to all of the male offspring and none of the female offspring. F66 transgenic mice were mated with Mstn mutant mice, and F66/Mstn+/− males were then mated with either Mstn+/− or Mstn−/− females. As shown in Table 4, the presence of one or two Mstn mutant alleles in combination with the F66 transgene resulted in increasingly more muscle mass than seen in F66 transgenic mice that were wild type for Mstn. Moreover, muscle weights in either F66/Mstn+/− or F66/Mstn−/− mice were dramatically higher than in Mstn−/− mice lacking the F66 transgene. In the most extreme case, muscle weights in F66/Mstn−/− mice were increased by 250-350% from those seen in wild type mice (FIGS. 4A and 5). Hence, the presence of the F66 transgene in a Mstn−/− background caused yet another doubling of muscle weights, resulting in mice with approximately quadruple the normal amount of muscle. These findings demonstrate that like FLRG, follstatin must be exerting its effect on muscle growth by targeting other ligands in addition to myostatin and that the effect of blocking these other ligands is comparable in magnitude to that resulting from loss of myostatin.
It has been shown that the increase in muscle mass in Mstn−/− mice results from a combination of increased fiber numbers and increased fiber sizes. To determine whether the same is true for the additional muscle mass seen upon introduction of the F66 transgene, morphometric analysis of the gastrocnemius/plantaris muscles was performed. As shown in Table 5 and FIG. 4B, total fiber number and mean fiber diameter were increased by about 48% and 19%, respectively, in Mstn−/− mice compared to wild type mice. As the cross-sectional area of the muscle would be expected to be roughly proportional to the square of the diameter, increased fiber diameter in Mstn−/− mice would correspond to an approximately 43% increase in fiber mass. Hence, muscle fiber hyperplasia and hypertrophy appear to contribute roughly equally to give the overall doubling of gastrocnemius/plantaris mass in Mstn−/− mice. In contrast, a similar analysis of F66 transgenic mice revealed that although total fiber number was increased slightly (16%), the overall increase in gastrocnemius/plantaris mass resulted almost entirely from muscle fiber hypertrophy (93% increase in cross-sectional area). In mice in which the F66 transgene was combined with the Mstn null mutation, the two phenotypes appeared to be additive; that is, the quadrupling of muscle mass in F66/Mstn−/− mice resulted from an approximately 73% increase in fiber number and 117% increase in fiber cross-sectional area. These results suggest that the additional muscle mass induced by follistatin in Mstn null mice results from inhibition of additional ligands that act predominantly to regulate muscle fiber growth.
In these experiments, a consistent finding was that muscle weights were higher in animals of the same genotype if they arose from crosses in which the mother had fewer functional Mstn alleles (Table 4). This maternal effect was observed to some extent in all of the muscles examined but was most pronounced in the quadriceps and gastrocnemius. For example, muscle weights of F66/Mstn+/+ males obtained from crosses with Mstn+/− females were higher than those of F66/Mstn+/+ males obtained from crosses with Mstn+/+ females. Similarly, muscle weights of F66/Mstn+/− males obtained from crosses with Mstn−/− females were higher than those of F66/Mstn+/− males obtained from crosses with Mstn+/− females. The most dramatic effects were observed in F66/Mstn−/− mice obtained from crosses with Mstn−/− females, in which muscle weights were approximately quadrupled compared to wild type mice.
To determine whether this maternal effect was specific to the presence of the F66 transgene, a variety of crosses of Mstn mutant mice lacking the transgene was performed. As shown in Table 6, the maternal effect on muscle weights was observed in these crosses as well. In virtually every case, mice with identical genotypes exhibited higher muscle weights if the mother had fewer functional Mstn alleles. The most clear cut results were obtained in analyses of Mstn+/− offspring derived from crosses of Mstn+/+ males with Mstn−/− females, which showed significantly higher muscle weights than Mstn+/− offspring derived from crosses of Mstn−/− males with Mstn+/+ females. Hence, the maternal effect on muscle mass was not dependent on the presence of the F66 transgene.
EXAMPLE 17
Effect of Transferring Neonates to Foster Mothers
Conceivably, the maternal effect observed above could result from transfer of myostatin or a downstream mediator either prenatally from the maternal to fetal circulations or postnatally from the mother to the offspring during nursing; in this respect, myostatin mRNA has been reported to be expressed in the mammary gland of lactating sheep. To distinguish these two possibilities, the effect of transferring neonates obtained from crosses with mothers of one Mstn genotype to foster mothers of a different Mstn genotype was analyzed. In these experiments, all transfers were carried out using neonatal mice less than 24 hours old to mothers that had delivered their own litters also within the previous 24 hours. In order to control for effects of the transfer process per se, transfers of neonates obtained from crosses with mothers of one Mstn genotype to foster mothers of the same Mstn genotype was also carried out. As shown in Table 7, mice of a given genotype and parentage exhibited comparable muscle weights regardless of the genotype of the foster mothers. Hence, if there is a mediator of muscle mass that is transferred through the milk, any resultant effects on muscle mass was undetectable in these experiments. Taken together, these results suggest that the maternal effect on muscle mass results most likely from prenatal transfer of some mediator from mother to fetus, perhaps myostatin itself.







TABLE 4







Muscle weights of FLRG and follistatin transgenic mice.












pectoralis
triceps
quadriceps
gastrocnemius











FEMALES












Mstn+/+
(n = 22)
 47.3 ± 0.8
 68.2 ± 1.1
142.8 ± 1.7
 95.9 ± 1.3


Mstn+/−
(n = 15)
 63.0 ± 0.8a
 90.0 ± 1.5a
176.6 ± 2.4a
122.8 ± 1.6a


Mstn−/−
(n = 22)
105.7 ± 3.4a
148.7 ± 3.7a
266.9 ± 6.7a
181.1 ± 3.8a


TG Z111B
(n = 11)
 53.5 ± 1.5b
 75.3 ± 2.2b
151.2 ± 4.2
102.7 ± 2.8c


TG Z116B
(n = 15)
 64.9 ± 1.2a
 98.3 ± 2.0a
200.2 ± 3.8a
141.5 ± 3.1a


TG Z111A
(n = 12)
 69.8 ± 3.1a
105.4 ± 4.6a
223.9 ± 8.3a
160.6 ± 6.7a


TG Z116A
(n = 11)
 74.4 ± 2.1a
116.6 ± 3.8a
236.8 ± 5.8a
173.6 ± 5.0a


TG Z116A, Mstn+/−
(n = 5)
 92.2 ± 6.7
149.2 ± 5.9
289.6 ± 14.0e
222.6 ± 10.4d







MALES












Mstn+/+
(n = 19)
 73.5 ± 1.3
 91.5 ± 1.6
190.0 ± 3.2
129.4 ± 1.7


Mstn+/−
(n = 13)
 94.3 ± 2.0f
127.1 ± 2.6f
243.2 ± 5.4f
167.5 ± 3.2f


Mstn−/−
(n = 10)
190.8 ± 7.1f
236.1 ± 5.2f
390.1 ± 9.4f
272.6 ± 4.9f


TG Z111B
(n = 10)
 78.5 ± 1.8h
 99.4 ± 2.2g
199.9 ± 3.9
135.0 ± 2.9


TG Z116B
(n = 11)
 98.6 ± 3.9f
131.1 ± 4.3f
267.1 ± 8.5f
188.8 ± 5.3f


TG Z111A
(n = 9)
113.7 ± 6.4f
156.4 ± 9.5f
307.4 ± 15.5f
221.3 ± 10.4f


TG Z116A
(n = 11)
137.3 ± 6.7f
196.5 ± 5.9f
370.5 ± 14.0f
279.5 ± 10.4f







F66, Mstn+/+












from Mstn+/+ mother
(n = 20)
121.9 ± 2.3f
182.6 ± 5.0f
440.6 ± 11.1f
295.3 ± 5.6f


from Mstn+/− mother
(n = 23)
126.5 ± 2.6
186.6 ± 4.5
480.7 ± 11.6i
314.7 ± 6.7i







F66, Mstn+/−












from Mstn+/− mother
(n = 12)
185.4 ± 6.1j
307.2 ± 8.9j
583.7 ± 19.2j
384.3 ± 10.9j


from Mstn−/− mother
(n = 11)
200.3 ± 5.9
306.5 ± 9.6
637.4 ± 12.5l
439.3 ± 9.8k







F66, Mstn−/−












from Mstn+/− mother
(n = 14)
208.1 ± 7.7k
383.7 ± 9.2k
619.7 ± 16.0k
492.1 ± 13.4k


from Mstn−/− mother
(n = 15)
320.1 ± 9.0n,o
412.1 ± 4.5n,o
668.9 ± 8.2m,o
529.6 ± 10.1m,o





ap < 0.001 vs. line 1,


bp < 0.01 vs. line 1,


cp < 0.05 vs. line 1,


dp < 0.001 vs. line 3,


ep < 0.05 vs. line 3,


fp < 0.001 vs. line 9,


gp < 0.01 vs. line 9,


hp < 0.05 vs. line 9,


ip < 0.05 vs. line 16,


jp < 0.001 vs. line 17,


kp < 0.001 vs. line 18,


lp < 0.05 vs. line 18,


mp < 0.01 vs. line 20,


np < 0.05 vs. line 20,


op < 0.001 vs. line 11.













TABLE 5







Morphometric analysis of gastrocnemius/plantaris muscles.

















Relative




Relative
Mean fiber
Relative
cross-



Total fiber
fiber
diameter
fiber
sectional



number
number
(μm)
diameter
areaa

















Mstn+/+
(n = 4)
 8451 ± 505
1.00
41.3 ± 1.0
1.00
1.00


Mstn−/−
(n = 4)
12488 ± 1251b
1.48
49.3 ± 2.2b
1.19
1.43


F66, Mstn+/+
(n = 3)
 9838 ± 84b
1.16
57.4 ± 1.1c
1.39
1.93


F66, Mstn−/−
(n = 2)
14593 ± 849b
1.73
60.8 ± 1.1c
1.47
2.17





acalculated as relative fiber diameter,


bp < 0.05 vs. Mstn+/+,


cp < 0.001 vs. Mstn+/+













TABLE 6







Muscle weights (mg) of Mstn mutant mice.












pectoralis
triceps
quadriceps
gastrocnemius











FEMALES


Mstn+/+












male+/+ × female+/+
(n = 22)
 47.3 ± 0.8
 68.2 ± 1.1
142.8 ± 1.7
 95.9 ± 1.3


male+/− × female+/−
(n = 15)
 51.2 ± 1.4a
 70.7 ± 1.4
147.9 ± 3.2
101.3 ± 2.3a







Mstn+/−












male−/− × female+/+
(n = 19)
 59.8 ± 1.1
 84.3 ± 1.6
165.4 ± 2.6
113.4 ± 1.3


male+/− × female+/−
(n = 15)
 63.0 ± 0.8b
 90.0 ± 1.5b
176.6 ± 2.4c
122.8 ± 1.6d


male+/+ × female−/−
(n = 19)
 65.3 ± 2.5c
 93.7 ± 2.8d
181.3 ± 5.0d
123.6 ± 3.2d







Mstn−/−












male+/− × female+/−
(n = 10)
105.7 ± 3.4
148.7 ± 3.7
266.9 ± 6.7
181.1 ± 3.8


Male−/− × female−/−
(n = 19)
110.6 ± 1.9
156.9 ± 3.1
278.6 ± 4.9
192.5 ± 3.6e







MALES


Mstn+/+












male+/+ × female+/+
(n = 19)
 73.5 ± 1.3
 91.5 ± 1.6
190.0 ± 3.2
129.4 ± 1.7


male+/− × female+/−
(n = 13)
 79.1 ± 2.2f
 99.4 ± 3.4f
198.5 ± 4.9
137.0 ± 3.0f







Mstn+/−












male−/− × female+/+
(n = 28)
 93.6 ± 1.9
120.1 ± 2.3
230.0 ± 4.0
158.8 ± 2.4


male+/− × female+/−
(n = 13)
 94.3 ± 2.0
127.1 ± 2.6
243.2 ± 5.4
167.5 ± 3.2g


male+/+ × female−/−
(n = 21)
101.7 ± 1.7h
133.4 ± 2.2i
252.5 ± 4.1i
168.2 ± 2.5h







Mstn−/−












male+/− × female+/−
(n = 10)
190.8 ± 7.1
236.1 ± 5.2
390.1 ± 9.4
272.6 ± 4.9


Male−/− × female−/−
(n = 17)
193.7 ± 3.6
240.7 ± 3.3
397.3 ± 5.6
277.7 ± 4.1





ap < 0.05 vs. line 1,


bp < 0.05 vs. line 3,


cp < 0.01 vs. line 3,


dp < 0.001 vs. line 3,


ep < 0.05 vs. line 6,


fp < 0.05 vs. line 8,


gp < 0.05 vs. line 10,


hp < 0.01 vs. line 10,


ip < 0.001 vs. line 10.













TABLE 7







Muscle weights (mg) of mice transferred to foster mothers at birth.












pectoralis
triceps
quadriceps
gastrocnemius











Mstn+/+ females












fostered to Mstn+/+
(n = 8)
 47.8 ± 1.4
 66.0 ± 1.1
143.3 ± 4.2
 97.0 ± 2.5


mother


fostered to Mstn−/− mother
(n = 19)
 47.9 ± 0.9
 66.8 ± 0.7
143.2 ± 1.9
 97.1 ± 1.2







Mstn+/+ males












fostered to Mstn+/+
(n = 10)
 73.7 ± 2.5
 92.9 ± 3.0
191.0 ± 7.4
129.3 ± 4.3


mother


fostered to Mstn−/− mother
(n = 15)
 74.5 ± 1.6
 91.5 ± 1.8
192.8 ± 3.6
129.6 ± 1.9







Mstn−/− females












fostered to Mstn+/+
(n = 13)
115.9 ± 2.9
156.6 ± 3.3
280.8 ± 7.2
191.8 ± 4.2


mother


fostered to Mstn−/− mother
(n = 11)
110.5 ± 1.9
150.5 ± 2.1
281.0 ± 4.8
194.5 ± 2.9







Mstn−/− males












fostered to Mstn+/+
(n = 15)
189.5 ± 5.0
215.9 ± 5.3
376.2 ± 7.2
260.3 ± 4.8


mother


fostered to Mstn−/− mother
(n = 14)
193.5 ± 3.7
218.4 ± 4.5
385.4 ± 7.3
267.2 ± 5.0









The finding that myostatin is not the sole regulator of muscle mass in mice raises the question as to whether targeting myostatin alone will be the most effective strategy for manipulating this signaling pathway in humans. In this respect, it is known that the circulating levels of myostatin protein in humans are considerably lower than in mice8, 13, raising the possibility that the balance of the relative roles played by myostatin and by these other regulators may have shifted further away from myostatin in humans compared to mice.
Although the invention has been described with reference to the above examples, 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

1. A method of increasing muscle tissue growth in a subject comprising:
(a) administering to the subject a therapeutically effective amount of a Follistatin-Like Related Gene (FLRG) polynucleotide or polypeptide in the absence of other agents;
(b) administering to a corresponding subject a specific inhibitor of myostatin expression or activity in combination with the FLRG polynucleotide or polypeptide;
(c) comparing muscle tissue growth in the subject of (a) to muscle tissue growth of the corresponding subject of (b) to determine whether there is an increase in muscle tissue growth in the corresponding subject of (b) greater than that in the subject of (a); and
(d) further administering to the subject of (a) a specific inhibitor of myostatin expression or activity wherein the muscle tissue mass in the corresponding subject of (b) is greater than that in the subject of (a), thereby increasing muscle tissue growth in the subject.

2. The method of claim 1, wherein the subject is a mammal.

3. The method of claim 2, wherein the mammal is ovine, porcine, bovine, murine or human.

4. The method of claim 1, wherein the muscle tissue growth of the subject is at least two-fold greater than the muscle tissue growth of a corresponding subject not treated with the specific inhibitor of myostatin expression or activity, or the FLRG polynucleotide or polypeptide.

5. A method of increasing the growth of muscle tissue in a subject comprising administering to the subject a therapeutically effective amount of an inhibitor of myostatin expression or activity in combination with a therapeutically effective amount of an FLRG polynucleotide or polypeptide either prior to, simultaneously with or following the inhibitor of myostatin expression or activity, thereby increasing muscle tissue growth in the subject.

6. The method of claim 5, wherein the inhibitor of myostatin expression or activity is a myostatin prodomain or a functional peptide portion thereof, comprising amino acid residues from 20 to 262 of a promyostatin polypeptide as set forth in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20.

7. The method of claim 5, wherein the inhibitor of myostatin expression or activity is an antibody, or functional fragment thereof, which binds to myostatin.

8. The method of claim 7, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

9. The method of claim 8, wherein the monoclonal antibody is a humanized monoclonal antibody or a chimeric monoclonal antibody or fragment thereof.

10. The method of claim 5, wherein the inhibitor of myostatin expression or activity is an antisense or interfering RNA nucleic acid.

11. The method of claim 5, wherein the inhibitor of myostatin expression or activity is a polynucleotide encoding a dominant negative myostatin polypeptide or a polynucleotide encoding a truncated myostatin polypeptide.

12. The method of claim 5, wherein the muscle tissue growth is at least two-fold greater than the muscle tissue growth of a corresponding subject not treated with the specific inhibitor of myostatin expression or activity, or the FLRG polynucleotide or polypeptide.

13. The method of claim 5, wherein the subject is a mammal.

14. The method of claim 13, wherein the mammal is ovine, porcine, bovine, murine, or human.

15. 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 comprising:
(a) contacting a muscle cell of a subject in need thereof with an FLRG polynucleotide or polypeptide, and
(b) contacting a muscle cell of a corresponding subject with a specific inhibitor of myostatin expression or activity in combination with the FLRG polynucleotide or polypeptide;
(c) comparing muscle tissue growth in the subject of (a) to muscle tissue growth of the corresponding subject of (b) to determine whether there is an increase in muscle tissue growth in the corresponding subject of (b) greater than that in the subject of (a); and
(d) further contacting the muscle cell of (a) with a specific inhibitor of myostatin expression or activity wherein the muscle tissue mass in the corresponding subject of (b) is greater than that in the subject of (a), thereby ameliorating the severity of the pathologic condition.

16. The method of claim 15, wherein the pathological condition is a wasting disorder.

17. The method of claim 16, wherein the wasting disorder is selected from the group consisting of cachexia and anorexia.

18. The method of claim 16, wherein the wasting disorder is selected from the group consisting of a muscular dystrophy and a neuromuscular disease.

19. The method of claim 15, wherein the pathological condition is a metabolic disorder.

20. The method of claim 19, wherein the metabolic disorder is selected from the group consisting of obesity and type II diabetes.

21. 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 comprising contacting a muscle cell of a subject in need thereof with an inhibitor of myostatin expression or activity in combination with an FLRG polynucleotide or polypeptide either prior to, simultaneous with or following the inhibitor of myostatin expression or activity.

22. The method of claim 21, wherein the inhibitor of myostatin expression or activity is a myostatin prodomain or a functional peptide portion thereof, comprising amino acid residues from 20 to 262 of a promyostatin polypeptide as set forth in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20.

23. The method of claim 21, wherein the inhibitor of myostatin expression or activity is an antibody, or functional fragment thereof, which binds to myostatin.

24. The method of claim 21, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

25. The method of claim 24, wherein the monoclonal antibody is a humanized monoclonal antibody or a chimeric monoclonal antibody or fragment thereof.

26. The method of claim 21, wherein the inhibitor of myostatin expression or activity is an antisense or interfering RNA nucleic acid.

27. The method of claim 21, wherein the inhibitor of myostatin expression or activity is a polynucleotide encoding a dominant negative myostatin polypeptide or a polynucleotide encoding a truncated myostatin polypeptide.

28. The method of claim 21, wherein the pathological condition is a wasting disorder.

29. The method of claim 28, wherein the wasting disorder is selected from the group consisting of cachexia and anorexia.

30. The method of claim 28, wherein the wasting disorder is selected from the group consisting of a muscular dystrophy and a neuromuscular disease.

31. The method of claim 21, wherein the pathological condition is a metabolic disorder.

32. The method of claim 31, wherein the metabolic disorder is selected from the group consisting of obesity and type II diabetes.