WO2008030367A2 - Selective myostatin inhibitors - Google Patents

Abstract

Provided are mutant follistatins that act as selective myostatin inhibitors, as well as methods of using the same to treat muscle wasting disorders.

Description

SELECTIVE MYOSTA TIN INHIBITORS

RELATED APPLICATIONS

This application claims priority to United States Provisional Patent Application Ser. No. 60/841 ,841 filed 01 September 2006 entitled SELECTIVE MYOSTATIN

INHIBITORS and is herein incorporated by reference in its entirety including all drawings and figures.

BACKGROUND OF THE INVENTION Muscle wasting and weakness are associated with muscle disorders, as well as immobility, chronic diseases such as cancer and AIDS, and normal aging (sarcopenia). Although muscle has its own progenitor cell for regeneration, lost muscle bulk and strength due to disease and injury are often never completely recovered. Therefore, treatments that can stimulate muscle growth and prevent muscle loss are likely to benefit a significant proportion of the population.

Myostatin acts on muscle to control its development. In adults, myostatin suppresses muscle growth. Follistatin is a 35 kD glycoprotein that is synthesized in many tissues and acts as a binding protein for activin and other members of the TGFβ superfamily such as myostatin and some bone morphogenetic proteins. Follistatin is one of several natural myostatin inhibitors, although its physiological role in muscle regulation is currently unknown. Nevertheless, administration of follistatin in muscle has been observed to lead to increased muscle mass, which is believed to be due to its binding and neutralization of myostatin. One of the difficulties of using follistatin as a therapeutic for increasing muscle growth is that follistatin binds other TGFβ ligands besides myostatin, for example, activin. Loss of activin activity in mice leads to numerous developmental defects and neonatal death. Activin also limits growth of many types of epithelial tissue, so that 1 inhibition of activin action through administration of follistatin could lead to abnormal growth of these tissues and, eventually, to cancer. There are currently no approved commercial pharmaceutical means for inhibiting myostatin activity, that do not simultaneously alter activin activity. Myostatin antibodies have been developed which bind and neutralize myostatin without binding other TGFb family ligands. However, antibodies may have certain drawbacks that might limit their utilityas therapeutics for muscle wasting disorders. Hence, additional selective myostatin inhibitors are needed which can effectively neutralize myostatin without significantly affecting related ligands like activin.

SUMMARY OF THE INVENTION

Provided are follistatin mutants that inhibit myostatin, but have substantially reduced activin activity. These novel selective myostatin inhibitors may be administered to increase muscle mass in subjects in need thereof. Thus, the inhibitors may be useful for treating diseases where muscle wasting and/or weakness is a symptom. It is also possible that the inhibitors could be used to increase muscle in athletes. These mutants may also be effective in animals to increase muscle mass for strength, or to increase their food value. Thus, compositions comprising the mutants may be used as therapeutic agents for muscle wasting disorders or for enhancement of muscle mass in humans or animals..

Accordingly, provided herein, among other things, are novel follistatin polypeptides and compositions thereof that are capable of stimulating muscle growth, as well as nucleic acids, vectors, host cells, etc. for expression and production of the same. Further, provided are novel methods of treating disorders where muscle wasting and/or weakness is a symptom, as well as novel methods of increasing muscle mass in a human subject or animal, using the follistatiri polypeptides and compositions thereof, optionally in conjunction with other modes of therapy or stimulation, as well as kits for the practice of the same.

These embodiments of the present invention, other embodiments, and their features and characteristics will be apparent from the description, drawings, and claims that follow. BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 depicts domains of follistatin (FST) 288. Shown are the N-terminal domain (FSND) (SEQ ID NO: 1), three FST domains (FSDs) 1-3 (SEQ ID NOs: 2-4) aligned at their cysteine residues, along with the two FSDs of the FST homolog FSTL3 (SEQ ID NOs 5-6) and SPARC/BM40 (SEQ ID NO: 7) The sequences are divided to show the N-terminal (EGF-like) and C-terminal (Kazal-like) subdomains. Shaded bars denote the conserved hydrophobic residues used in mutational analyses. The heparin- binding sequence (FSD-I) is underlined.

FIGURE 2 depicts SDS-PAGE of follistatin mutants silver stained to determine purity. Silver stained gel of mutants- dFSD2, FSD3/1/2, L191D and Y185A and control

FST 288 provides information on purity of the protein preparation, and the estimated percent purity was used to calculate the adjusted protein concentration in Table 2.

FIGURE 3 depicts a Western blot analysis of FST mutants. SDS-PAGE followed by Western blot analysis of FST mutants was used to confirm the concentration of the purified mutants. The numbers in the parenthesis indicates the amount (in μg) of total protein loaded per lane.

FIGURE 4 depicts an iodinated activin binding curve. Iodinated activin was added at increasing levels (10,000-80,000 cpm/well; 2 fold dilutions) onto 96-well plates with adsorbed wild type (WT: means unmutated) FST or mutant FSTs at 125 ng/well. As shown, mutant FSTs showed vastly reduced binding to activin relative to WT FST 288.

FIGURE 5 depicts a solid phase direct binding assay of FST to activin. Increasing amounts of WT FST or FST mutant proteins (l-30ng/wells) were added to activin adsorbed to 96 well plates and binding measured.

FIGURE 6 depicts a solid phase direct binding assay of FST to myostatin. Increasing amounts of WT F ST or FST mutant proteins (l-30ng/wells) were added to myostatin (FIGURE 5) adsorbed to 96 well plates and binding measured.

FIGURE 7 depicts the effect of the maximal dose of WT FST or FST mutants in inhibition of activin or myostatin.

FIGURE 8 depicts the effect of WT or mutant FST on activin or myostatin activity. Increasing doses of FST DNA were transfected into 293 cells. Inhibition of activin and myostatin was dose-dependent. A total of 9 clones were found to have greater myostatin inhibition activity compared to activin. Six examples are shown here.

FIGURE 9 depicts the sequences of wildtype FST288 and various mutants thereof. DETAILED DESCRIPTION OF THE INVENTION A. General

We have been engaged in structure function studies of follistatin (FST) and as part of these studies, made a large number of mutants to determine which parts of the molecule were necessary for activin binding. We found that residues in the N-domain and second follistatin domain (FSD2), and to a lesser extent, residues in the first follistatin domain (FSDl) were important or critical for activin binding. We also made mutants in which the order of the follistatin domains was altered, or where individual domains were deleted and/or replaced by multiple copies of another domain. All of these domain mutants had vastly reduced or undetectable activin binding, except for those in which the third follistatin domain (FSD3) was deleted. Thus, mutating certain critical residues, or changing FSD order or content, vastly reduced or eliminated activin binding. While several studies, including our own, have shown that follistatin also binds myostatin with relatively high affinity, it was always assumed that any TGFβ superfamily ligand would bind to roughly the same residues in follistatin that are occupied by activin. We systematically tested more than 30 mutants in activin and myostatin bioassays and found that most mutations which altered FSD2 had dramatic effects on its ability to inhibit activin activity, but had much less or no effect on myostatin bioactivity. We then selected the most potent representative mutants for further testing, including dFSD2, in which the FSD2 is deleted; FSD312, in which the - order of follistatin domains is altered with FSD3 coming before FSD 1 and 2; and Y185D and Ll 9 ID, in which residues in FSD2 were mutated to residues with a different charge or shape. We then tested each of these mutants for actual direct binding to activin versus myostatin. We found that binding of activin was reduced nearly 1000-fold in the dFSD2 mutant compared to wild type follistatin while myostatin binding activity was altered to a much smaller degree. This mutant confirmed our original hypothesis that follistatin mutants would differentially bind activin versus myostatin and demonstrates our invention. The other three potent mutants were also selective for myostatin, but not to the same degree. B. Definitions

For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are defined here.

The singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

The term "amino acid" is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.

"Antibody" is intended to include whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc.), and includes fragments thereof which are also specifically reactive with a vertebrate, e.g., mammalian, protein. Antibodies may be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. Thus, the term includes segments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein. Non-limiting examples of such proteolytic and/or recombinant fragments include Fab, F(ab')2, Fab', Fv, and single chain antibodies (scFv) containing a V[L] and/or V[H] domain joined by a peptide linker. The scFv's may be covalently or non-covalently linked to form antibodies having two or more binding sites. The subject invention includes polyclonal, monoclonal or other purified preparations of antibodies and recombinant antibodies.

"Biological sample" or "sample", refers to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample may be of any biological tissue or fluid. Frequently the sample will be a "clinical sample" which is a sample derived from a patient. Such samples include, but are not limited to, sputum, blood, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.

A "combinatorial library" or "library" is a plurality of compounds, which may be termed "members," synthesized or otherwise prepared from one or more starting materials by employing either the same or different reactants or reaction conditions at each reaction in the library. In general, the members of any library show at least some structural diversity, which often results in chemical diversity. A library may have anywhere from two different members to about 10⁸ members or more. In certain embodiments, libraries of the present invention have more than about 12, 50 and 90 members. In certain embodiments of the present invention, the starting materials and certain of the reactants are the same, and chemical diversity in such libraries is achieved by varying at least one of the reactants or reaction conditions during the preparation of the library. Combinatorial libraries of the present invention may be prepared in solution or on the solid phase. "Derived from" as that phrase is used herein indicates a peptide or nucleotide sequence selected from within a given sequence. A peptide or nucleotide sequence derived from a named sequence may contain a small number of modifications relative to the parent sequence, in most cases representing deletion, replacement or insertion of less than about 15%, preferably less than about 10%, and in many cases less than about 5%, of amino acid residues or base pairs present in the parent sequence. In the case of

DNAs, one DNA molecule is also considered to be derived from another if the two are capable of selectively hybridizing to one another.

"Derivative" refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence may include, for example, replacement of hydrogen by an alkyl, acyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived. "Diagnosis" or "diagnosing" as used herein includes diagnosis, prognosis, monitoring, characterizing, selecting or screening patients, including participants in clinical trials, and identifying patients at risk for or having a particular disorder or those most likely to respond to a particular therapeutic treatment, or for assessing or monitoring a patient's response to a particular therapeutic treatment. A "fusion protein" or "fusion polypeptide" refers to a chimeric protein as that term is known in the art and may be constructed using methods known in the art. In many examples of fusion proteins, there are two different polypeptide sequences, and in certain cases, there may be more. The sequences may be linked in frame. A fusion protein may include a domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", "intergenic", etc. fusion expressed by different kinds of organisms. In various embodiments, the fusion polypeptide may comprise one or more amino acid sequences linked to a first polypeptide. In the case where more than one amino acid sequence is fused to a first polypeptide, the fusion sequences may be multiple copies of the same sequence, or alternatively, may be different amino acid sequences. The fusion polypeptides may be fused to the N-terminus, the C-terminus, or the N- and C-terminus of the first polypeptide. Exemplary fusion proteins include polypeptides comprising a glutathione S-transferase tag (GST-tag), histidine tag (His-tag), an immunoglobulin domain or an immunoglobulin binding domain.

"Gene" or "recombinant gene" refers to a nucleic acid molecule comprising an open reading frame and including at least one exon and (optionally) an intron sequence. "Intron" refers to a DNA sequence present in a given gene which is spliced out during mRNA maturation.

"Gene construct" refers to a vector, plasmid, viral genome or the like which includes a "coding sequence" for a polypeptide or which is otherwise transcribable to a biologically active RNA (e.g., antisense, decoy, ribozyme, etc), may transfect cells, in certain embodiments mammalian cells, and may cause expression of the coding sequence in cells transfected with the construct. The gene construct may include one or more regulatory elements operably linked to the coding sequence, as well as intronic sequences, polyadenylation sites, origins of replication, marker genes, etc.

"Host cell" refers to a cell transduced with a specified transfer vector. The cell is optionally selected from in vitro cells such as those derived from cell culture, ex vivo cells, such as those derived from an organism, and in vivo cells, such as those in an organism. "Recombinant host cells" refers to cells which have been transformed or transfected with vectors constructed using recombinant DNA techniques. "Host cells" or "recombinant host cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. "Interact" is meant to include detectable interactions between molecules, such as may be detected using, for example, a hybridization assay. Interact also includes "binding" interactions between molecules. Interactions may be, for example, protein- protein, protein-nucleic acid, protein-small molecule or small molecule-nucleic acid in nature.

"Isolated", with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule. Isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. "Isolated" also refers to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.

"Label" and "detectable label" refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (e.g., biotin or haptens) and the like. "Fluorophore" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range.

Particular examples of labels which may be used under the invention include fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, NADPH, alpha- or beta-galactosidase and horseradish peroxidase.

The term "mammal" is known in the art, and exemplary mammals include humans, primates, bovines, porcines, canines, felines, and rodents (e.g., mice and rats).

The term "modulation", when used in reference to a functional property or biological activity or process (e.g., enzyme activity or receptor binding), refers to the capacity to either up regulate (e.g., activate or stimulate), down regulate (e.g., inhibit or suppress) or otherwise change a quality of such property, activity or process. The term "modulator" refers to a polypeptide, nucleic acid, macromolecule, complex, molecule, small molecule, compound, species or the like (naturally-occurring or non-naturally-occurring), or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues, that may be capable of causing modulation. Modulators may be evaluated for potential activity as inhibitors or activators (directly or indirectly) of a functional property, biological activity or process, or combination of them, (e.g., agonist, partial antagonist, partial agonist, inverse agonist, antagonist, anti-microbial agents, inhibitors of microbial infection or proliferation, and the like) by inclusion in assays. In such assays, many modulators may be screened at one time. The activity of a modulator may be known, unknown or partially known.

The term "muscle wasting disorder" refers to any disorder or condition in which muscle wasting or weakness is present as a symptom, including old age, lack of physical fitness and immobility.

"Nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. ESTs, chromosomes, cDNAs, mRNAs, and rRNAs are representative examples of molecules that may be referred to as nucleic acids." Nucleic acid corresponding to a gene" refers to a nucleic acid that may be used for detecting the gene, e.g., a nucleic acid which is capable of hybridizing specifically to the gene. The term "operably linked", when describing the relationship between two nucleic acid regions, refers to a juxtaposition wherein the regions are in a relationship permitting them to function in their intended manner. For example, a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences, such as when the appropriate molecules (e.g., inducers and polymerases) are bound to the control or regulatory sequence(s).

A "patient", "subject" or "host" to be treated by the subject method may mean either a human or non-human animal.

The phrase "pharmaceutically acceptable" refers to those compositions and dosages thereof within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically-acceptable carrier" means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body. The term "pharmaceutically acceptable carrier" refers to a carrier(s) that is "acceptable" in the sense of being compatible with the other ingredients of a composition and not deleterious to the recipient thereof. Some examples of materials which may serve as pharmaceutically-acceptable carriers include; (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. "Protein", "polypeptide" and "peptide" are used interchangeably herein when referring to a gene product, e.g., as may be encoded by a coding sequence. By "gene product" it is meant a molecule that is produced as a result of transcription of a gene. Gene products include RNA molecules transcribed from a gene, as well as proteins translated from such transcripts. The terms "polypeptide fragment" or "fragment", when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions may occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least 5, 6, S or 10 amino acids long, at least 14 amino acids long, at least 20, 30, 40 or 50 amino acids long, at least 75 amino acids long, or at least 100, 150, 200, 300, 500 or more amino acids long. A fragment can retain one or more of the biological activities of the reference polypeptide. Further, fragments can include a sub- fragment of a specific region, which sub-fragment retains a function of the region from which it is derived.

The term "purified" refers to an object species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). A "purified fraction" is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all species present. In making the determination of the purity of a species in solution or dispersion, the solvent or matrix in which the species is dissolved or dispersed is usually not included in such determination; instead, only the species (including the one of interest) dissolved or dispersed are taken into account. Generally, a purified composition will have one species that comprises more than about 80 percent of all species present in the composition, more than about 85%, 90%, 95%, 99% or more of all species present. The object species may be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single species. A skilled artisan may purify a polypeptide of the invention using standard techniques for protein purification in light of the teachings herein. Purity of a polypeptide may be determined by a number of methods known to those of skill in the art, including for example, amino-terminal amino acid sequence analysis, gel electrophoresis and mass-spectrometry analysis. "Recombinant protein", "heterologous protein" and "exogenous protein" are used interchangeably to refer to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. That is, the polypeptide is expressed from a heterologous nucleic acid.

The term "regulatory sequence" is a generic term used throughout the specification to refer to polynucleotide sequences, such as initiation signals, enhancers, regulators and promoters, that are necessary or desirable to affect the expression of coding and non-coding sequences to which they are operably linked. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in

Enzymology. Academic Press, San Diego, CA (1990), and include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. The nature and use of such control sequences may differ depending upon the host organism. In prokaryotes, such regulatory sequences generally include promoter, ribosomal binding site, and transcription termination sequences. The term "regulatory sequence" is intended to include, at a minimum, components whose presence may influence expression, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. In certain embodiments, transcription of a polynucleotide sequence is under the control of a promoter sequence (or other regulatory sequence) which controls the expression of the polynucleotide in a cell-type in which expression is intended. It will also be understood that the polynucleotide can be under the control of regulatory sequences which are the same or different from those sequences which control expression of the naturally-occurring form of the polynucleotide.

The term "sequence homology" refers to the proportion of base matches between two nucleic acid sequences or the proportion of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of sequence from a desired sequence (e.g., SEQ. ID NO: 1) that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are used more frequently, with 2 bases or less used even more frequently. The term "sequence identity" means that sequences are identical (i.e., on a nucleotide-by-nucleotide basis for nucleic acids or amino acid-by- amino acid basis for polypeptides) over a window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions at which the identical amino acids occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Methods to calculate sequence identity are known to those of skill in the art and described in further detail below.

"Small molecule" refers to a composition, which has a molecular weight of less than about 2000 kDa. Small molecules may be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon-containing) or inorganic molecules. As those skilled in the art will appreciate, based on the present description, libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, may be screened with any of the assays of the invention to identify compounds that modulate a bioactivity. The term "specifically hybridize's" refers to detectable and specific nucleic acid binding. Polynucleotides, oligonucleotides and nucleic acids of the invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. Stringent conditions may be used to achieve selective hybridization conditions as known in the art and discussed herein. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and nucleic acids of the invention and a nucleic acid sequence of interest will be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or more. In certain instances, hybridization and washing conditions are performed under stringent conditions according to conventional hybridization procedures and as described further herein.

As applied to proteins, the term "substantial identity" means that two protein sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, typically share at least about 70 percent sequence identity, alternatively at least about 80, 85, 90, 95 percent sequence identity or more. In certain instances, residue positions that are not identical differ by conservative amino acid substitutions.

"Therapeutic agent" or "therapeutic" refers to an agent capable of having a desired biological effect on a host. Chemotherapeutic and genotoxic agents are examples of therapeutic agents that are generally known to be chemical in origin, as opposed to biological, or cause a therapeutic effect by a particular mechanism of action, respectively. Examples of therapeutic agents of biological origin include growth factors, hormones, and cytokines. A variety of therapeutic agents are known in the art and may be identified by their effects. Certain therapeutic agents are capable of regulating red cell proliferation and differentiation. Examples include chemotherapeutic nucleotides, drugs, hormones, non-specific (non-antibody) proteins, oligonucleotides (e.g., antisense oligonucleotides that bind to a target nucleic acid sequence (e.g., mRNA sequence)), peptides, and peptidomimetics. The term "therapeutically effective amount" refers to that amount of a modulator, drug or other molecule which is sufficient to effect treatment when administered to a subject in need of such treatment. The therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

"Treatment" or "treating" refers to therapy, prevention and prophylaxis and particularly refers to the administration of medicine or the performance of medical procedures with respect to a patient, for either prophylaxis (prevention) or to cure the infirmity or malady in the instance where the patient is afflicted. The term "vector" refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked. One type of vector which may be used in accord with the invention is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Other vectors include those capable of autonomous replication and expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as

"expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA molecules which, in their vector form are not bound to the chromosome. Infectious expression vectors, such as recombinant baculoviruses, are used to express proteins in cultured cells. Other infectious expression vectors, such as recombinant adenoviruses and vaccinia viruses, are used as vaccines to express foreign antigens in vacinees. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto. Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.

C. Mutant Follistatin Polypeptides and Methods and Compositions of Producing Them Follistatin (FST) and follistatin-like 3 (FSTL3) are endogenous proteins that bind and inhibit myostatin [12]; furthermore, FSTL3 is associated with myostatin in serum [13]. Follistatin consists of a 63-residue N-terminal, followed by three successive domains of 73-75 amino acids containing ten cysteine residues termed FSD-I, FSD-2 and FSD-3 with a heparin binding site responsible for cell surface proteoglycan binding [14] located within FSDl (FIGURE 1). Three protein isoforms are generated from the single FST gene through alternative mRNA splicing (FST 288 and FST 315), and post- translatinal proteolytic processing (FST315 cleaved to FST303) [15]. FSTL3 is a FST homolog that contains the same domain structure as FST except that it has only 2 FST domains, but no heparin-binding site sequence, and thus lacks the cell surface binding ability of FST [16]. FST binds myostatin in vitro, and transgenically expressed FST binds and neutralizes myostatin in vivo. FST is also co-expressed with myostatin in somites, suggesting that FST may prevent myostatin-mediated inhibition of limb muscle development in chick embryos [12].

Using substantially identical techniques to those described in the Examples below, we identified mutants of FST 288 (wildtype FST 288 sequence SEQ ID NO: 8,

FIGURE 9 ) which bind and neutralize myostatin, but but have vastly reduced activin binding activity. Thus, provided are isolated, recombinant mutant follistatins that inihibit myostatin activity much more than they inhibit activin activity.

In certain embodiments, an isolated, recombinant mutant FST 288 that inhibits myostatin but does not alter activin activity may comprise a FST 288 sequence such as

SEQ ID NO: 8 lacking FSD2 (SEQ ID NO: 10, FIGURE 9).

In other embodiments, an isolated, recombinant mutant follistatin that inhibits myostatin much more than activin activity may comprise the following follistatin domains in the following order N-terminus to C-terminus: FST N-terminal domain (FSTN), FSDl, and any one additional follistatin domain (1, 2, or 3) but no other follistatin domain beyond that.

In still other embodiments, an isolated, recombinant mutant FST 288 that inhibits myostatin but has a much reduced ability to inhibit activin activity may comprise the following FST 288 domains in the following order N-terminus to C-terminus: FSD3, FSDl and FSD2 (SEQ ID NO: 9, FIGURE 9).

In still other embodiments, an isolated, recombinant mutant FST 288 that inhibits myostatin but does not alter activin activity comprises a sequence having at least about 95% identity to SEQ ID NOs: 1 and any amino acid except tyrosine (Y) at position 185 that has different shape or charge from tyrosine (for example, SEQ ID NO: 11 , FIGURE

9)-

For example, an isolated, recombinant mutant FST 288 that inhibits myostatin but has little effect on activin activity may comprise a sequence having at least about 95% identity to SEQ ID NOs: 1 and an aspartic acid (D) at position 185 rather than a tyrosine

(Y).

In still other embodiments, an isolated, recombinant mutant FST 288 that inhibits myostatin but does not alter activin activity comprises a sequence having at least about 95% identity to SEQ ID NOs: 1 and any amino acid except leucine (L) at position 191 that has different shape or charge from leucine (for example, SEQ ID NO: 12, FIGURE

9).

For example, an isolated, recombinant mutant FST 288 that inhibits myostatin but does not alter activin activity may comprise a sequence having at least about 95% identity to SEQ ID NOs: 1 and an aspartic acid (D) at position 191 rather than a leucine (L).

In other embodiments, the rearrangements and mutations described above for FST 288 may be made in any of the other follistatin isoforms, that is, FST303 and FST 315.

In still other embodiments, the rearrangements and mutations described above for FST 288 may be made in other mammalian species of follistatin. There are only three amino acid differences among the various species of follistatin, i.e., the follistatins among mammalian species are highly conserved, and thus the rearrangements and mutations described above would be expected to work in other species of follistatin.

In certain embodiments, the subject polypeptides may comprise a fusion protein of any of the above-described polypeptides containing at least one domain which increases its solubility and/or facilitates its purification, identification, detection, and/or delivery.

Exemplary domains, include, for example, glutathione S-transferase (GST), protein A, protein G, calmodulin-binding peptide, thioredoxin, maltose binding protein, HA, myc, poly arginine, poly His, poly His- Asp or FLAG fusion proteins and tags. Additional exemplary domains include domains that alter protein localization in vivo, such as signal peptides, type III secretion system-targeting peptides, transcytosis domains, nuclear localization signals, etc. In various embodiments, a polypeptide of the invention may comprise one or more heterologous fusions. Polypeptides may contain multiple copies of the same fusion domain or may contain fusions to two or more different domains. The fusions may occur at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or at both the N- and C-terminus of the polypeptide. Linker sequences between a polypeptide of the invention and the fusion domain may be included in order to facilitate construction of the fusion protein or to optimize protein expression or structural constraints of the fusion protein. In another embodiment, the polypeptide may be constructed so as to contain protease cleavage sites between the fusion polypeptide and polypeptide of the invention in order to remove the tag after protein expression or thereafter. Examples of suitable endoproteases, include, for example, Factor Xa and TEV proteases. In another embodiment, the subject polypeptides may be modified so that the rate of traversing the cellular membrane is increased. For example, the polypeptide may be fused to a second peptide which promotes "transcytosis," e.g., uptake of the peptide by cells. The peptide may be a portion of the HIV transactivator (TAT) protein, such as the fragment corresponding to residues 37-62 or 48-60 of TAT, portions which have been observed to be rapidly taken up by a cell in vitro (Green and Loewenstein, (1989)

Cell 55:1179-1188). Alternatively, the internalizing peptide may be derived from the Drosophila antennapedia protein, or homologs thereof. The 60 amino acid long homeodomain of the homeo-protein antennapedia has been demonstrated to translocate through biological membranes and can facilitate the translocation of heterologous polypeptides to which it is coupled. Thus, polypeptides may be fused to a peptide consisting of about amino acids 42-58 of Drosophila antennapedia or shorter fragments for transcytosis (Derossi et al. (1996) J Biol Chem 271:18188-18193; Derossi et al. (1994) J Biol Chem 269:10444-10450; and Perez et al. (1992) J Cell Sci 102:717-722). The transcytosis polypeptide may also be a non-naturally-occurring membrane- translocating sequence (MTS), such as the peptide sequences disclosed in U.S. Patent

No. 6,248,558.

In another embodiment, truncated polypeptides may be prepared. Truncated polypeptides have from 1 to 20 or more amino acid residues removed from either or both the N- and C-termini. Such truncated polypeptides may prove more amenable to expression, purification or characterization than the full-length polypeptide. In addition, the use of truncated polypeptides may also identify stable and active domains of the full-length polypeptide that may be more amenable to characterisation or incorporation into a pharmaceutical composition.

It is also possible to modify the structure of the polypeptides of the invention for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelf life, resistance to proteolytic degradation in vivo, etc.). Such modified polypeptides, when designed to retain at least one activity of the naturally-occurring form of the protein, are considered "functional equivalents" of the polypeptides described in more detail herein. Such modified polypeptides may be produced, for instance, by amino acid substitution, deletion, or addition, which substitutions may consist in whole or part by conservative amino acid substitutions.

For instance, it is reasonable to expect that an isolated conservative amino acid substitution, such as replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, will not have a major affect on the biological activity of the resulting molecule. Whether a change in the amino acid sequence of a polypeptide results in a functional homolog may be readily determined by assessing the ability of the variant polypeptide to produce a response similar to that of the wild-type protein. Polypeptides in which more than one replacement has taken place may readily be tested in the same manner.

Protein homologs may be generated combinatorially. In a representative embodiment of this method, the amino acid sequences for a population of protein homologs are aligned, preferably to promote the highest homology possible. Such a population of variants may include, for example, homologs from one or more species, or homologs from the same species but which differ due to mutation. Amino acids which appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences. In certain embodiments, the combinatorial library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential protein sequences. For instance, a mixture of synthetic oligonucleotides may be enzymatically ligated into gene sequences such that the degenerate set of potential nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display).

There are many ways by which the library of potential homologs may be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence may be carried out in an automatic DNA synthesizer, and the synthetic genes may then be ligated into an appropriate vector for expression. One purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential protein sequences. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed.

AG Walton, Amsterdam: Elsevierpp. 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11 :477). Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S. Patent Nos: 5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis may be utilized to generate a combinatorial library. For example, protein homologs may be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994)J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838; and Cunningham et al., (1989) Science 244:1081-1085), by linker scanning mutagenesis (Gustin et al., (1993) Virology

193:653-660; Brown et al., (1992) MoI. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science 232:316); by saturation mutagenesis (Meyers et al., (1986) Science 232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell MoI Biol 1 :11-19); or by random mutagenesis (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al., (1994) Strategies in MoI Biol

7:32-34). Linker scanning mutagenesis, particularly in a combinatorial setting, is an attractive method for identifying truncated forms of proteins that are bioactive. Another aspect of the invention relates to polypeptide fragments derived from the full-length polypeptides of the invention. Fragments of the polypeptides may be produced using standard polypeptide synthesis methods as will be known to one of skill in the art. Alternatively, such polypeptide fragments, as well as the subject polypeptides, may be produced using recombinant techniques. Chemical synthesis of polypeptides of the invention may be carried out using a variety of art recognized methods, including stepwise solid phase synthesis, semi-synthesis through the conformationally-assisted re-ligation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site. Full length ligation products are chemically identical to proteins produced by cell free synthesis. Full length ligation products may be refolded and/or oxidized, as allowed, to form native disulfide- containing protein molecules, (see e.g., U.S. Patent Nos. 6,184,344 and 6,174,530; and T. W. Muir et al., Curr. Opin. Biotech. (1993): vol. 4, p 420; M. Miller, et al., Science (1989): vol. 246, p 1149; A. Wlodawer, et al., Science (1989): vol. 245, p 616; L. H. Huang, et al., Biochemistry (1991): vol. 30, p 7402; M. Schnolzer, et al., Int. J. Pept. Prot. Res. (1992): vol. 40, p 180-193; K. Rajarathnam, et al., Science (1994): vol. 264, p

90; R. E. Offord, "Chemical Approaches to Protein Engineering", in Protein Design and the Development of New therapeutics and Vaccines, J. B. Hook, G. Poste, Eds., (Plenum Press, New York, 1990) pp. 253-282; C. J. A. Wallace, et al., J. Biol. Chem. (1992): vol. 267, p 3852; L. Abrahmsen, et al., Biochemistry (1991): vol. 30, p 4151; T. K. Chang, et al., Proc. Natl. Acad. Sci. USA (1994) 91 : 12544-12548; M. Schnlzer, et al., Science (1992): vol., 3256, p 221; and K. Akaji, et al., Chem. Pharm. Bull. (Tokyo) (1985) 33: 184).

The present invention also provides isolated nucleic acid sequences that encode all or a substantial portion of the amino acid sequences set forth in SEQ ID NOs: δthrough 12 or other polypeptides of the invention described above, as well as vectors, host cells, and cultures for the expression and production thereof or for gene therapy methods. Isolated nucleic acids which differ from the nucleic acids of the invention due to degeneracy in the genetic code are also within the scope of the invention. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent" mutations which do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the polypeptides of the invention will exist.

One skilled in the art will appreciate that these variations in one or more nucleotides (from less than 1% up to about 3 or 5% or possibly more of the nucleotides) of the nucleic acids encoding a particular protein of the invention may exist among a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.

Bias in codon choice within genes in a single species appears related to the level of expression of the protein encoded by that gene. Accordingly, the invention encompasses nucleic acid sequences which have been optimized for improved expression in a host cell by altering the frequency of codon usage in the nucleic acid sequence to approach the frequency of preferred codon usage of the host cell. Due to codon degeneracy, it is possible to optimize the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide.

Expression vehicles for production of a recombinant polypeptide include plasmids and other vectors. For instance, suitable vectors for the expression of a polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli. In one aspect of the invention, the subject nucleic acid is provided in a vector comprising a nucleotide sequence encoding a polypeptide of the invention, and operably linked to at least one regulatory sequence. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. The vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should be considered. Such vectors may be administered in any biologically effective carrier, e.g., any formulation or composition capable of effectively transfecting cells either ex vivo or in vivo with genetic material encoding a polypeptide. Approaches include insertion of the nucleic acid in viral vectors including recombinant retroviruses, adenoviruses, adeno-associated viruses, human immunodeficiency viruses, and herpes simplex viruses- 1, or recombinant bacterial or eukaryotic plasmids. Viral vectors may be used to transfect cells directly; plasmid DNA may be delivered alone with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers. Nucleic acids may also be directly injected. Alternatively, calcium phosphate precipitation may be carried out to facilitate entry of a nucleic acid into a cell. The subject nucleic acids may be used to cause expression and over- expression of polypeptide of interest in cells propagated in culture, e.g. to produce proteins or polypeptides. This invention also pertains to a host cell transfected with a recombinant gene in order to express a polypeptide of the invention. The host cell may be any prokaryotic or eukaryotic cell. For example, a gene comprising a polypeptide of interest may be expressed in bacterial cells, such as E. coli, insect cells (baculovirus), yeast, insect, plant, or mammalian cells. In those instances when the host cell is human, it may or may not be in a live subject. Other suitable host cells are known to those skilled in the art. Additionally, the host cell may be supplemented with tRNA molecules not typically found in the host so as to optimize expression of the polypeptide. Other methods suitable for maximizing expression of the polypeptide are known to those in the art. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. A polypeptide may be secreted and isolated from a mixture of cells and medium comprising the polypeptide. Alternatively, a polypeptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A polypeptide may be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and affinity purification with antibodies specific for particular epitopes or with the ligand of a fusion tag.

Generally, a nucleic acid encoding a polypeptide of the invention is introduced into a host cell, such as by transfection or infection, and the host cell is cultured under conditions allowing expression of the polypeptide. Methods of introducing nucleic acids into prokaryotic and eukaryotic cells are well known in the art. Suitable media for mammalian and prokaryotic host cell culture are well known in the art. In some instances, the nucleic acid encoding the subject polypeptide is under the control of an inducible promoter, which is induced once the host cells comprising the nucleic acid have divided a certain number of times. For example, where a nucleic acid is under the control of a beta-galactose operator and repressor, isopropyl beta-D- thiogalactopyranoside (IPTG) is added to the culture when the bacterial host cells have attained a density of about OD600 0.45-0.60. The culture is then grown for some more time to give the host cell the time to synthesize the polypeptide. Cultures are then typically frozen and may be stored frozen for some time, prior to isolation and purification of the polypeptide. Thus, a nucleotide sequence encoding all or part of a polypeptide of the invention may be used to produce a recombinant form of a protein via microbial or eukaryotic. cellular processes. Ligating the sequence into a polynucleotide construct, such as an expression vector, and transforming, infecting, or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures. Similar procedures, or modifications thereof, may be employed to prepare recombinant polypeptides by microbial means or tissue-culture technology in accord with the subject invention.

Other embodiments of nucleic acid sequences encoding the polypeptides of the invention, as well as vectors, host cells, and cultures thereof are further described below.

In another embodiment, the nucleic acid encoding a polypeptide of the invention is operably linked to a bacterial promoter, e.g., the anaerobic E. coli, NirB promoter or the E. coli lipoprotein lip promoter, described, e.g., in Inouye et al. (1985) Nucl. Acids Res. 13:3101; Salmonella pagC promoter (Miller et al., supra), Shigella ent promoter (Schmitt and Payne, J. Bacteriol. 173:816 (1991)), the tet promoter on TnIO (Miller et al., supra), or the ctx promoter of Vibrio cholera. Any other promoter can be used in the invention. The bacterial promoter can be a constitutive promoter or an inducible promoter. An exemplary inducible promoter is a promoter which is inducible by iron or in iron-limiting conditions. In fact, some bacteria, e.g., intracellular organisms, are believed to encounter iron-limiting conditions in the host cytoplasm. Examples of iron- regulated promoters of FepA and TonB are known in the art and are described, e.g., in the following references: Headley, V. et al. (1997) Infection & Immunity 65:818; Ochsner, U.A. et al. (1995) Journal of Bacteriology 177:7194; Hunt, M.D. et al. (1994) Journal of Bacteriology 176:3944; Svinarich, D.M. and S. Palchaudhuri. (1992) Journal of Diarrhoeal Diseases Research 10:139; Prince, R. W. et al. (1991) Molecular Microbiology 5:2823; Goldberg, M.B. et al. (1990) Journal of Bacteriology 172:6863; de Lorenzo, V. et al. (1987) Journal of Bacteriology 169:2624; and Hantke, K. (1981) Molecular & General Genetics 182:288.

In another embodiment, a signal peptide sequence is added to the construct, such that the polypeptide is secreted from cells. Such signal peptides are well known in the art.

In one embodiment, the powerful phage T5 promoter, that is recognized by E. coli RNA polymerase is used together with a lac operator repression module to provide tightly regulated, high level expression or recombinant proteins in E. coli. In this system, protein expression is blocked in the presence of high levels of lac repressor.

In one embodiment, the DNA is operably linked to a first promoter and the bacterium further comprises a second DNA encoding a first polymerase which is capable of mediating transcription from the first promoter, wherein the DNA encoding the first polymerase is operably linked to a second promoter. In a preferred embodiment, the second promoter is a bacterial promoter, such as those delineated above. In an even more preferred embodiment, the polymerase is a bacteriophage polymerase, e.g., SP6, T3, or T7 polymerase and the first promoter is a bacteriophage promoter, e.g., an SP6, T3, or T7 promoter, respectively. Plasmids comprising bacteriophage promoters and plasmids encoding bacteriophage polymerases can be obtained commercially, e.g., from Promega Corp. (Madison, Wis.) and InVitrogen (San Diego, Calif.), or can be obtained directly from the bacteriophage using standard recombinant DNA techniques (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, 1989). Bacteriophage polymerases and promoters are further described, e.g., in the following references: Sagawa, H. et al. (1996) Gene 168:37; Cheng, X. et al. (1994) PNAS USA 91:4034; Dubendorff, J. W. and F.W. Studier (1991) Journal of Molecular Biology 219:45: Bujarski, JJ. and P. Kaesberg (1987) Nucleic Acids Research 15:1337; and Studier, F.W. et al. (1990) Methods in Enzvmology 185:60). Such plasmids can further be modified according to the specific embodiment of the invention.

In another embodiment, the bacterium further comprises a DNA encoding a second polymerase which is capable of mediating transcription from the second promoter, wherein the DNA encoding the second polymerase is operably linked to a third promoter. In a preferred embodiment, the third promoter is a bacterial promoter. However, more than two different polymerases and promoters could be introduced in a bacterium to obtain high levels of transcription. The use of one or more polymerase for mediating transcription in the bacterium can provide a significant increase in the amount of polypeptide in the bacterium relative to a bacterium in which the DNA is directly under the control of a bacterial promoter. The selection of the system to adopt will vary depending on the specific use of the invention, e.g., on the amount of protein that one desires to produce. When using a prokaryotic host cell, the host cell may include a plasmid which expresses an internal T7 lysozyme, e.g., expressed from plasmid pLysSL (see Examples). Lysis of such host cells liberates the lysozyme which then degrades the bacterial membrane.

Other sequences that may be included in a vector for expression in bacterial or other prokaryotic cells include a synthetic ribosomal binding site; strong transcriptional terminators, e.g., t0 from phage lambda and t4 from the rrnB operon in E. coli, to prevent read through transcription and ensure stability of the expressed polypeptide; an origin of replication, e.g., CoIEl; and beta-lactamase gene, conferring ampicillin resistance. Other host cells include prokaryotic host cells. Even more preferred host cells are bacteria, e.g., E. coli. Other bacteria that can be used include Shigella spp., Salmonella spp., Listeria spp., Rickettsia spp., Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp., and Erysipelothrix spp. Most of these bacteria can be obtained from the American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, VA 20110-2209).

A number of vectors exist for the expression of recombinant proteins in yeast. For instance, YEP24, YIP5, YEP51 , YEP52, pYES2, and YRP 17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al., (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83). These vectors may replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin may be used.

In certain embodiments, mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, _PTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-I), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to express the recombinant protein by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pFastBac-derived vectors.

In another variation, protein production may be achieved using in vitro translation systems. In vitro translation systems are, generally, a translation system which is a cell- free extract comprising at least the minimum elements necessary for translation of an

RNA molecule into a protein. An in vitro translation system typically comprises at least ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex, comprising the cap-binding protein (CBP) and eukaryotic initiation factor 4F (eIF4F). A variety of in vitro translation systems are well known in the art and include commercially available kits.

Examples of in vitro translation systems include eukaryotic lysates, such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis.; Stratagene, La Jolla, Calif.; Amersham, Arlington Heights, 111.; and GE3CO/BRL, Grand Island, N. Y. In vitro translation systems typically comprise macromolecules, such as enzymes, translation, initiation and elongation factors, chemical reagents, and ribosomes. In addition, an in vitro transcription system may be used. Such systems typically comprise at least an RNA polymerase holoenzyme, ribonucleotides and any necessary transcription initiation, elongation and termination factors. An RNA nucleotide for in vitro translation may be produced using methods known in the art. In vitro transcription and translation may be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs. When expression of a carboxy terminal fragment of a polypeptide is desired, i.e. a truncation mutant, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment comprising the desired sequence to be expressed. It is well known in the art that a methionine at the N-terminal position may be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat et al., (1987) J. Bacteriol. 169:751-757) and

Salmonella typhimurium and its in vitro activity has been demonstrated on recombinant proteins (Miller et al., (1987) PNAS USA 84:2718-1722). Therefore, removal of an N- terminal methionine, if desired, may be achieved either in vivo by expressing such recombinant polypeptides in a host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e.g., procedure of Miller et al.).

In cases where plant expression vectors are used, the expression of a polypeptide may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature. 310:511- 514), or the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO J.. 6:307- 311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO

(Coruzzi et al., 1994, EMBO J.. 3:1671-1680; Broglie et al., 1984, Science. 224:838- 843); or heat shock promoters, eg., soybean hsp 17.5-E or hsp 17.3-B (Gurley et al., 1986, MoI. Cell. Biol.. 6:559-565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors; direct DNA transformation; microinjection, electroporation, etc. For reviews of such techniques see, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, New York, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9. An alternative expression system which can be used to express a polypeptide is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The PGHS-2 sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of the coding sequence will result in inactivation of the polyhedrin gene and production of non- occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, (e.g., see Smith et al., 1983, J.

Virol.. 46:584, Smith, U.S. Pat. No. 4,215,051).

Tn a specific embodiment of an insect system, the DNA encoding the subject polypeptide is cloned into the pBlueBacIII recombinant transfer vector (Invitrogen,, San Diego, Calif.) downstream of the polyhedrin promoter and transfected into Sf9 insect cells (derived from Spodoptera frugiperda ovarian cells, available from Invitrogen, San

Diego, Calif.) to generate recombinant virus. After plaque purification of the recombinant virus high-titer viral stocks are prepared that in turn would be used to infect Sf9 or High FiveTM (BTI-TN-5B1-4 cells derived from Trichoplusia ni egg cell homogenates; available from Invitrogen, San Diego, Calif.) insect cells, to produce large quantities of appropriately post-translationally modified subject polypeptide.

Although it is possible that these cells themselves could be directly useful for drug assays, the subject polypeptides prepared by this method can be used for in vitro assays. In another embodiment, the subject polypeptides are prepared in transgenic animals, such that in certain embodiments, the polypeptide is secreted, e.g., in the milk of a female animal.

Viral vectors may also be used for efficient in vitro introduction of a nucleic acid into a cell. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, polypeptides encoded by genetic material in the viral vector, e.g., by a nucleic acid contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into mammals. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. A major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild- type virus in the cell population. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271). Thus, recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding one of the antisense E6AP constructs, rendering the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al.

(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZEP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include Crip, Cre, 2 and Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. ScL USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145:

Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043: Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115: U.S. Patent No.

4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). In choosing retroviral vectors as a gene delivery system for nucleic acids encoding the subject polypeptides, it is important to note that a prerequisite for the successful infection of target cells by most retroviruses, and therefore of stable introduction of the genetic material, is that the target cells must be dividing. Tn general, this requirement will not be a hindrance to use of retroviral vectors. In fact, such limitation on infection can be beneficial in circumstances wherein the tissue (e.g., nontransformed cells) surrounding the target cells does not undergo extensive cell division and is therefore refractory to infection with retroviral vectors.

Furthermore, it has been shown that it is possible to limit the infection spectrum of retroviruses and consequently of retroviral-based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example, PCT publications WO93/25234, WO94/06920, and WO94/11524). For instance, strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) PNAS 86:9079-9083; Man et al. (1992) J. Gen Virol 73:3251-3255; and Goud et al.

(1983) Virology 163:251-254); or coupling cell surface ligands to the viral env proteins (Neda et al. (1991) J Biol Chem 266:14143-14146). Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g., lactose to convert the env protein to an asialoglycoprotein), as well as by generating chimeric proteins (e.g., single-chain antibody/env chimeric proteins). This technique, while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector in to an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences which control expression of the genetic material of the retroviral vector.

Another viral gene delivery system utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactive in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434: and Rosenfeld et al. (1992) Cell 68:143-155). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are well known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are capable of infecting non-dividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Ouantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and, as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral El and

E3 genes but retain as much as 80% of the adenoviral genetic material (see, for example, Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham et al. in Methods in Molecular Biology, EJ. Murray, Ed. (Humana, Clifton, NJ, 1991) vol. 7. pp. 109-127). Expression of the inserted genetic material can be under control of, for example, the ElA promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences.

Yet another viral vector system useful for delivery of genetic material encoding the subject polypeptides is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. Curr. Topics in Micro, and Immunol. (1992) 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. MoI. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors comprising as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) MoI. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) MoI. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) MoI. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

In particular, a AAV delivery system suitable for targeting muscle tissue has been developed by Gregorevic, et al. , Nat Med. 2004 Aug;10(8):828-34. Epub 2004 JuI 25, which is able to 'home-in' on muscle cells and does not trigger an immune system response. The delivery system also includes use of a growth factor, VEGF, which appears to increase penetration into muscles of the gene therapy agent.

Other viral vector systems may be derived from herpes virus, vaccinia virus, and several RNA viruses.

In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of nucleic acids encoding the subject polypeptides, e.g. in a cell in vitro or in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of genetic material by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, polylysine conjugates, and artificial viral envelopes.

In a representative embodiment, genetic material can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and, optionally, which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551 ; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075). For example, lipofection of papilloma-infected cells can be carried out using liposomes tagged with monoclonal antibodies against PV-associated antigen (see Viae et al. (1978) J Invest Dermatol 70:263-266; see also Mizuno et al. (1992) Neurol. Med. Chir. 32:873-876). In yet another illustrative embodiment, the gene delivery system comprises an antibody or cell surface ligand which is cross-linked with a gene binding agent such as polylysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180). For example, genetic material encoding the subject chimeric polypeptides can be used to transfect hepatocytic cells in vivo using a soluble polynucleotide carrier comprising an asialoglycoprotein conjugated to a polycation, e.g., polylysine (see U.S. Patent 5,166,320). It will also be appreciated that effective delivery of the subject nucleic acid constructs via mediated endocytosis can be improved using agents which enhance escape of the gene from the endosomal structures. For instance, whole adenovirus or fusogenic peptides of the influenza HA gene product can be used as part of the delivery system to induce efficient disruption of DNA-comprising endosomes (Mulligan et al. (1993) Science 260-926; Wagner et al. (1992) PNAS 89:7934; and Christiano et al. (1993) PNAS 90:2122). D. Pharmaceutical Compositions

The present invention also provides pharmaceutical compositions comprising a therapeutically effective amount of the polypeptides and nucleic acids described above. In one embodiment, the pharmaceutical composition comprises any of the isolated, recombinant mutant follistatin polypeptides described in the preceding section. In certain embodiments, the polypeptide is a fusion polypeptide comprising a mutant follistatin polypeptide and a polypeptide that aids in localizing or delivering the mutant follistatin polypeptide. In another embodiment, the pharmaceutical composition comprises an isolated, purified nucleic acid encoding a mutant follistatin polypeptide described above. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Further, the invention provides devices for administering the pharmaceutical compositions, for example, devices for intravenous, intraperitoneal, or subcutaneous injection

The compositions of the present invention may be administered by various means, depending on their intended use, as is well known in the art. For example, if compositions of the present invention are to be administered orally, they may be formulated as tablets, capsules, granules, powders or syrups. Alternatively, formulations of the present invention may be administered parenterally as injections (intravenous, intrathecal, intraperitoneal or subcutaneous), drop infusion preparations or suppositories. For application by the ophthalmic mucous membrane route, compounds of the present invention may be formulated as eyedrops or eye ointments. These formulations may be prepared by conventional means, and, if desired, the compounds may be mixed with any conventional additive, such as an excipient, a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent or a coating agent.

In formulations of the subject invention, wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may be present in the formulated agents.

Subject compositions may be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of agent that may be combined with a carrier material to produce a single dose vary depending upon the subject being treated, and the particular mode of administration.

Methods of preparing these formulations include the step of bringing into association agents of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association agents with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), each containing a predetermined amount of a compound thereof as an active ingredient. Compounds of the present invention may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the coordination complex thereof is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the supplement or components thereof moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the compound, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Suspensions, in addition to compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, micro crystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a coordination complex of the present invention with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent. Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for transdermal administration of a supplement or component includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. For transdermal administration of transition metal complexes, the complexes may include lipophilic and hydrophilic groups to achieve the desired water solubility and transport properties.

The ointments, pastes, creams and gels may contain, in addition to a supplement or components thereof, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays may contain, in addition to a supplement or components thereof, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Compounds of the present invention may alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A non-aqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers may be used because they minimize exposing the agent to shear, which may result in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the compound together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include non-ionic surfactants (T weens, Plυronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more components of a supplement in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

In another aspect, the present invention provides methods of stimulating muscle growth comprising administering the pharmaceutical composition to a subject. For example, stimulating muscle growth may be used to treat a subject that has a muscle wasting disorder. Accordingly, the present invention provides methods of treating a subject having a muscle wasting disorder comprising administering a pharmaceutical composition comprising a polypeptide or polynucleotide of the invention to a subject. In certain embodiments, the muscle wasting disorder is cancer. In other embodiments, the muscle wasting disorder is AIDS. In other embodiments, the muscle wasting disorder is sarcopenia. Such administration may be, for example, intrathecal, peripheral, systemic, or local.

In certain embodiments, the dosage of the subject pharmaceutical compositions will generally be in the range of about 0.01 ng to about 10 g per kg body weight, specifically in the range of about 1 ng to about 0.1 g per kg, and more specifically in the range of about 100 ng to about 10 mg per kg.

An effective dose or amount, and any possible effects on the timing of administration of the formulation, may need to be identified for any particular compound of the present invention. This may be accomplished by routine experiment as described herein, using one or more groups of animals (preferably at least 5 animals per group), or in human trials if appropriate. The effectiveness of any compound and method of treatment or prevention maybe assessed by administering the supplement and assessing the effect of the administration by measuring one or more indices associated with the neoplasm of interest, and comparing the post-treatment values of these indices to the values of the same indices prior to treatment.

The precise time of administration and amount of any particular compound that will yield the most effective treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a particular compound, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), route of administration, and the like. The guidelines presented herein may be used to optimize the treatment, e.g., determining the optimum time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing.

While the subject is being treated, the health of the patient may be monitored by measuring one or more of the relevant indices at predetermined times during a 24-hour period. Treatment, including supplement, amounts, times of administration and formulation, may be optimized according to the results of such monitoring. The patient may be periodically reevaluated to determine the extent of improvement by measuring the same parameters, the first such reevaluation typically occurring at the end of four weeks from the onset of therapy, and subsequent reevaluations occurring every four to eight weeks during therapy and then every three months thereafter. Therapy may continue for several months or even years, with a minimum of one month being a typical length of therapy for humans. Adjustments to the amount(s) of agent administered and possibly to the time of administration may be made based on these reevaluations.

Treatment may be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage may be increased by small increments until the optimum therapeutic effect is attained,

The combined use of several compounds of the present invention, or alternatively other chemotherapeutic agents, may reduce the required dosage for any individual component because the onset and duration of effect of the different components may be complimentary. In such combined therapy, the different active agents may be delivered together or separately, and simultaneously or at different times within the day.

Toxicity and therapeutic efficacy of subject compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 and the ED50. Compositions that exhibit large therapeutic indices are preferred. Although compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets the compounds to the desired site in order to reduce side effects.

The data obtained from the cell culture assays and animal studies may be used in formulating a range of dosage for use in humans. The dosage of any supplement, or alternatively of any components therein, lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For agents of the present invention, the therapeutically effective dose may be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information may be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

The mutant follistatin polypeptides and compositions thereof described herein can be used in combination with other therapies to promote muscle growth.

Mutant follistatin polypeptide treatment, for example, can be combined with physical therapy, by which a subject receives training to perform a particular motion. The physical therapy causes stimulation of muscles and can thereby strengthen and reinforce muscles. The physical therapy can be administered by a human therapist, by continuous passive motion machines, or by robots.

Other forms of therapy that may be combined with mutant follistatin polypeptide therapy include drug therapy. Drugs typically used to treat muscle wasting or enhance muscle growth include, but are not limited to, anabolic steroids (for example, tetrahydrogestrinone, dehydrochlormethyltestosterone, metandienone, methyltestosterone, nandrolone, oxandrolon, oxymetholone and stasnozolol), creatine, androstenedione, testosterone, growth hormones, insulin-like growth factor, andgrowth hormone releasing hormone.

E. Kits

The present invention provides kits for treating muscle wasting disorders in a subject in need thereof. For example, a kit may also comprise one or more polypeptides or nucleic acids of the present invention, or a pharmaceutical composition thereof. Kit components may be packaged for either manual or partially or wholly automated practice of the foregoing methods. In other embodiments involving kits, this invention contemplates a kit including compositions of the present invention, and optionally instructions for their use. In other embodiments, a kit may further comprise controls, reagents, buffers, and/or instructions for use. Such kits may have a variety of uses, including, for example, imaging, diagnosis, therapy, and other applications.

EXEMPLIFICATION The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references including literature references, issued patents, published or non published patent applications as cited throughout this application are hereby expressly incorporated by reference. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. (See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent

No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian

Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); , VoIs. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Example 1: Identification of Follistatin Mutations that Antagonize Myostatin but not Activin A In a prior study, we tested the biological activities of myostatin and activin with respect to FST mutants. We found that FST mutants with domain 2 interruptions elicit greater inhibition of myostatin activity than activin, suggesting that domain 2 is more important for activin binding and inhibition. In order to test whether these differences in biological activities are in fact due to differential binding, we examined four FST mutants with the largest difference between myostatin and activin inhibition in a prior study.

Mutants were expressed in human embryonic kidney (HEK 293) cells and we confirmed these mutants' preferential binding to myostatin over activin through binding assays. The assays assess the relative ability of mutant FSTs to bind activin or myostatin. Our results demonstrate that the FST mutants continue to bind to myostatin and inhibit myostatin's activity, athough to a lesser degree, when compared to wild type FST. However, these mutants all had drastically reduced activin binding and inhibitory activity compared to WT FST288. This differential binding suggests that mutations in domain 2 interrupted the binding of activin; thus it may not affect activin's concurrent signaling pathway and biological activity. Meanwhile, the disruption in domain 2 does not significantly affect myostatin's binding since these mutants continue to bind and inhibit myostatin's actions. These differential binding properties suggests that the ability of WT FST to bind both activin and myostatin can be circumvented, thus rendering our mutant FST proteins, or analogs based on our mutations which further increase this difference between activin and myostatin inhibition, potential novel therapeutic agents for enhanced muscle development.

Prior studies have shown that activin inhibition requires all FSD domains in FST except FSD3 whereas inhibition of myostatin action requires the N-domain and FSD-I of FST for inhibition [14]. In an attempt to inhibit myostatin without altering activin activity, we examined FST mutants that inhibit myostatin bioactivity 10-100 fold more than they inhibit activin bioactivity (data not shown). Results from four mutants that have this characteristic are as shown in Table 2 along with their inhibitory activity for myostatin or activin (relative to WT FST). Mutant dFSD2 has a deleted FSD2, mutant FSD 3/1/2 has transposed domains with FSD3 coming before FSD 1 and 2, and mutant L191D and Y185 are point mutations in the hydrophobic regions of domain 2.

We hypothesized that the activity differences observed in these four mutants are due to differential binding of mutant FST between activin and myostatin. In this study, we tested this differential binding with a solid phase direct binding assay. Indeed, those mutants that were poor inhibitors of activin bioactivity were also weak binders of activin. Since activin and myostatin are so closely related, inhibition of myostatin must be specific in order to be therapeutically beneficial. An ideal myostatin inhibitor, such as the mutant FST suggested in this study, should have minimal influence on activin action at doses that are therapeutic for inhibiting activin in order to minimize any side effects that might manifest in vivo.

Methods

Materials

Activin A was purchased from R&D Systems (Minneapolis, MN), and myostatin (GDF-8) was purchased from Cell Sciences (Canton, MA). Mutations were introduced in the FST cDNA as previously discussed [14] and mutant sequences were verified by bidirectional sequencing. Radioiodine was obtained from New England Nuclear Corp. (Boston, MA), and all electrophoresis materials were purchased from Bio-Rad (Hercules, CA). DNA Purification

A single bacterial colony with mutant or wild type FST 288 myc-His expression vector was inoculated in 200 ml of LB medium (plus Ampicillin at 75 μg/ml), and the culture was shaken at 37°C and 220-RPM overnight. The culture was collected by centrifugation at 6000-x g for 15 minutes. Plasmid DNA was then purified using NucleoBond® High Copy Plasmid Purification for Maxi (BD Biosciences Clontech,

Palo Alto, CA) according to the manufacturer's protocol.

Protein Production and Purification

Human embryonic kidney FreeStyle™ 293-F cells were maintained in 30ml of FreeStyleTM 293 Expression Medium (Life Technologies, Inc., Rockville, MD) to a density of 1 x 104 cells / ml with a minimum of 85% viability. 24 hrs prior to transfection, cells were cultured at density of 1.8 x 104 cells/ml and medium was changed 2 hrs before transfection. For every 30ml of culture, 35 μg of purified plasmid DNA was transfected using 40 μl of 293fectin™ Reagent and 2 ml of GIBCO™ Opti- MEM® (Life Technologies, Inc., Rockville, MD) according to the manufacturer's protocol. The culture was incubated for 72 hrs at 37°C, after which the culture was centrifuged at 3500 RPM for 15 minutes. The supernatant was collected and FST isolated from medium by binding to nickel-Sephoraose affinity columns (Qiagen, Valencia, CA) via the C-terminal poly-His tag using a peristaltic pump at 4°C overnight. Following stepwise elution with imidazole, (1 ImI of 30OmM imidazole eluted at pH 6.8) eluted protein was concentrated and exchanged into Dulbecco's PBS using Amicon Ultra-15 Centrifugal Filter Unit (Amicon, Bedford, MA). Quantitation of Purified Protein The protein concentration of the wild type FST and mutant FSTs were determined by three different assays. First, the Bradford Protein Assay (Bio-Rad, Hercules, CA) was used to determine total protein in the eluate. Second, after SDS-PAGE, silver staining (Biorad) of the gel provided purity information of the different protein preparations. The staining procedure was modified such that for each gel, the Fixative Enhance Solution was prepared using 25 ml of Reagent Grade Methanol, 5 ml of

Reagent Grade Acetic Acid, 5 ml of Fixative Enhance Concentrate, and 15 ml of Deionized Distilled Water. For the Staining Solution, 17.5 ml of deionized water, 2.5 ml of Silver Complex Solution, 2.5 ml of Reduction Moderator Solution, 2.5 ml of Image Development Reagent, and 25 ml of Development Accelerator Concentrate were used instead. The developed gel was then analyzed in conjunction to Bradford Assay to estimate the purity of the protein. To confirm the identity of the FST bands and relative amounts of FST in each preparation, we performed Western Blotting of the FST preparations after SDS-PAGE. After electrophoresis, the gel was transferred onto Nitrocellulose membrane; anti-myc antibody (clone 4A6, Upstate Biologicals, Lake Placid, NY) was used at a final dilution of 1 : 1000 in 4% dry milk/TBS/0.2% Tween-20.

Goat Anti-Mouse IgG (Jackson Immuno Research, West Grove, PA) was used at a final dilution of 1:15,000 as the secondary antibody in 4% dry milk/TBS/0.2% Tween-20. The concentration of the FST proteins were then compared to a sample of wild type FST with a known concentration. Solid Phase Radioligand Binding Assay

Activin was labeled with ¹²⁵I using the method described previously in Schneyer et al. [24] and myostatin was radioiodinated by using the chloramine T method [26] Purified wild type and mutant FSTs were plated onto 96- well Immulon-2 plates (Dynatech Laboratories, Chantilly, VA) in 0.1 M carbonate buffer (pH 9.6) overnight at 4⁰C in a density of 125ng/50μl/well [24]. Each well was aspirated and then blocked with 200 μl of blocking buffer (PBS/0.05% Tween-20/3%BSA) for at least 2 hours, or overnight at 4°C. After three washes in radioimmuno-assay (RIA) buffer (0.01M PBS / 0.1% Gelatin. pH 7.4), 100μl of RIA buffer was added to all wells. For Non-Specific

Binding (NSB), unlabeled activin (50ng) was added to the wells. (¹²⁵I)-labeled Activin was diluted in RIA buffer and 50ul was added to all wells. The plate was incubated for 2 hours at room temperature. After three washes in RIA buffer, the wells were aspirated and counted in a gamma counter. Although we originally intended to test radiolabeled myostatin in the assay as well, we have not yet been successful in producing functional, radiolabeled myostatin. Solid Phase Direct Binding Assay

Myostatin or activin was plated onto 96-well Immulon-2 plates (Dynatech Laboratory, Chantilly, VA) in carbonate buffer overnight at 4°C in a density of 50ng/50μl/well [24]. Each well was aspirated and blocked with 200 μl of blocking buffer for at least 2 hours. NSB wells were left in blocking solution and all other wells were washed three times using Tween Tris Buffered Saline (TTBS) (TBS / 0.05% Tween-20). Increasing concentrations of FST WT or mutants, diluted in TBS, were added to the well for 1 hr at room temperature. After washing, lOOul of anti-myc antibody (clone 4A6, Upstate Biologicals, Lake Placid, NY) was used at a final dilution of 1 :500 in TBS/0.1% BSA and incubated for one hour at room temperature. After three washes of TTBS, Goat Anti-Mouse IgG- Alkaline Phosphatase (Jackson Immuno Research, West Grove, PA) was used at a final dilution of 1 :500 as the secondary antibody in TBS/0.1% BSA. The plate was incubated for one hour at room temperature and washed three times with TTBS. Nitrophenol Phosphate (.NP) (Sigma, St. Louis,

MO) was dissolved in 0.1M Glycine Buffer with ImM MgC12 and ImM ZnC12 (pH 10.4). 200ul was added to each well for thirty minutes in room temperature. The plate was analyzed on a microplate reader at 405 nm. Results Twenty-six FST mutants were previously identified to have reduced activin inhibiting activity relative to wild type [14]. We have further demonstrated that seven of these mutants (dFSD2, FSD 1/1/3, FSD3/1/2, FSD2/1/3, Y185A, Y185D, and L191D) have either equivalent or slightly diminished myostatin inhibiting activity (unpublished observations). We hypothesized that the diminished activin inhibition with constant myostatin inhibition of the mutants was due to differential binding of activin versus myostatin. We tested this hypothesis with four mutants (dFSD2, FSD3/1/2, Yl 85A, and Ll 9 ID), which had the greatest differences between myostatin and activin inhibition by assessing binding of activin and myostatin to mutant and wild type FST proteins.

As mentioned above, mutant dFSD2 is a deletion of FST domain 2 from WT FST 288; mutant FSD 3/1/2 demonstrates the effect of transposition of FSD3 before FSDl and 2 within the otherwise intact FST-288 molecule; and mutants L191D and Yl 85 A are point mutations in the C-terminal sub domain of FST domain 2. These point mutations are located in conserved residues across domains, which may be important structurally for maintaining domain conformation or stability.

Bradford assay provided the overall protein concentration of the mutant and WT FST (Table 1). These concentrations were then adjusted according to their purity based on silver stained gel (FIGURE 2). The molecular weight of WT FST is approximately

38kDa, and often displays as a double band on SDS-PAGE analysis. After accounting for the presence of other bands, we estimated the purity of the protein preparations, then calculated and tabulated the adjusted concentration for each preparation (Table 1). The final concentrations of each mutant were then confirmed by Western blot analysis against a control sample of FST with a known concentration (FIGURE 3). Alternate amounts of 30μg and 7.5μg of each protein preparation as well as the control were tested because bands with strong intensity (too much protein) might appear too dark thus difficult to compare quantitatively. The double bands around 38-kDa confirm the presence of the FST 288 protein. Protein from mutant dFSD2 showed a lower molecular weight, which could be explained by the domain 2 deletion. Lanes with

7.5μg of proteins were compared across the preparations and they showed approximately the same intensity relative to each other as well as the control, confirming that the adjusted concentration for the protein preparations were close to the true values. Thus far, we have demonstrated that the protein preparations are in fact FST, shown by the correct bands in both silver stained gel and Western blot analyses; moreover, we determined their concentrations based on their purity, which was confirmed by Western blot analyses. This provides significant quantitative information necessary for the binding assays described below.

Table 1. Protein concentrations of FST mutants. Protein concentration of mutants- dFSD2, FSD3/1/2, L191D and Yl 85 A and control FST 288 were obtained using

Bradford Assay. Final concentration was adjusted based on the purity of each sample as determined by silver stained gel in FIGURE 2.

Differential binding of mutant and WT FST between activin and myostatin was first tested using solid phase radiolabeled binding assay. The results demonstrated that at increasing levels of iodinated activin (10,000-80,000 cpm/well; 2 fold dilutions), there is very low or no binding to the FST mutants (at 125ng/well), while wild type FST protein bound more than 30% of added radiolabeled activin (FIGURE 4). We attempted to iodinate myostatin to examine its binding to FST mutants but were unable to obtain active ¹²⁵I-myostatin using several different protocols. Since radiolabeled activin did not detectably bind to the FST mutants, we could not explore myostatin's ability to compete with activin either.

To verify differential binding of the mutants to myostatin and activin, we compared the binding of increasing amounts of wild type FST or FST mutant protein (1- 30ng/wells) with either activin or myostatin adsorbed to 96 well plates. As expected, WT FST, but not FST mutants bound to solid phase activin (FIGURE 5). On the other hand, both WT FST and FST mutants bound to myostatin; however, the mutants bound at slightly diminished affinities (FIGURE 6).

Table 2. Differential Binding Verses. Differential Activity of FST Mutants to Myostatin and Activin. Relative binding was determined by solid phase direct binding assay. Relative activity is from unpublished data from our laboratory. In agreement with the activity data, all mutants bind myostatin better than activin.

Relative to WT FST, mutant dFSD2, FSD 3/1/2, L191D, and Y185 bound to myostatin with approximately 80%, 50%, 30%, and 20% affinities, respectively. These results agreed with the differential activin and myostatin inhibiting activity data obtained earlier, which showed a greater overall myostatin than activin inhibition with these four mutants relative to the WT FST (Table 2). However, the earlier observation showed that for these mutants, there were at least 25% bioactivities relative to WT, and our current study did not detect any binding between these mutants and activin. Nevertheless, this does not necessarily mean that the mutants do not bind activin; rather it is possible that the binding is beneath the minimum threshold for detection in the direct binding assays, and they might be less sensitive than the biological activity assay. Discussion

Effect of FST Domain Deletion Deletion of FST domain 2 from WT FST 288 yielded proteins with significantly diminished activin binding (approximately 5% of the WT FST) (FIGURE 4). It has been previously shown that removal of both FST domains 2 and 3 leaving only the N- domain and FST domain 1 markedly diminished activin inhibition activity, demonstrating the need for more than one FST domain for activity [14]. Earlier data from our laboratory showed that in addition to the N-domain, both FST domains 1 and 2 are required for activin inhibition. Hence, our current data agreed with these previous findings and further demonstrated that the presence of the N-domain and both FST domains 1 and 2 are necessary for activin binding, while removing domain 2 disrupts the ability of FST to bind to activin and inhibit its activity.

In contrast to activin, deletion of FST domain 2 (FIGURE 6) had relatively little effect on myostatin binding (approximately 80% of WT FST). This agrees with our earlier observation that showed essentially WT inhibition (approximately 83% of WT FST) after deletion of domain 2 (Table 2). As with activin, it has been previously shown that removal of both FST domains 2 and 3, leaving only the N-domain and FST domain 1 also markedly diminished myostatin binding activity, emphasizing the need for more than one FST domain for myostatin binding activity as well [12]. Hence, our present data suggested that in addition to the N-terminal domain, FST domain 1 and an additional domain (1 , 2, or 3) are required for myostatin binding. The additional domain might be present for spatial reasons but it does not appear to be required for inhibition.

Exchange of FST Domains

The effect of transposition of FST domains within the otherwise intact FST-288 molecule was studied using a series of exchange mutations. FST mutant FSD 3/1/2, in which domain 3 separated the N domain from FST domains 1 and 2, had significantly reduced activin binding (approximately <5% of WT FST) and markedly impaired activin inhibiting activity (35% of WT FST). Meanwhile, mutants with reversal of FST domains 1 and 2 (FSD 2/1/3), or provision of two copies of FST domain 1 (FSD 1/1/3) and two copies of FST domain 2 (FSD 2/2/3) all showed diminished activin inhibiting activity (unpublished data). Thus, the sequential order of FST domains and their orientation relative to the N domain is essential to activin binding and inhibition by FST.

While mutant FSD 3/1/2 significantly affected activin binding, binding to myostatin was affected only slightly (approximately 50% of WT FST). Previously, mutants with reversal of FST domains 1 and 2 (FSD 2/1/3), or provision of two copies of FST domain 1(FSD 1/1/3) have been shown to generate proteins that retained myostatin inhibiting activity (90% and 98% of WT, respectively) (unpublished data). In addition, FSD 2/2/3 markedly impaired myostatin inhibiting activity (50% of WT). Thus, while activin binding by FST requires strict sequential order of FST domains and their correct orientation relative to the N-domain, these factors are not essential for myostatin binding and inhibition. Therefore, myostatin binding requires only the presence of the 5 N-domain, FST domain 1 and any other FST domain, in any order.

Point Mutations within FST Domains 1 and 2

Within the FST domains, several hydrophobic or neutral residues are conserved across domains [14], suggesting that these hydrophobic residues might be important structurally for maintaining domain conformation or stability (FIGURE 6). In FST,

10. these residues include Tyr-185 and Leu-191 in the C-terminal sub domain of FST domain 2. Mutation of Tyr-185 to Ala (Yl 85A) and Leu-191 to Asp (L191D) in FST domain 2 demonstrated significantly diminished activin binding (approximately <1% and <5% of WT FST, respectively). Previous data showed that mutation of Tyr-185 to Asp also demonstrated significantly impaired activin inhibition but mutation of Leu-191

15 to Ala retained most of its activin inhibiting activity (approximately 95% of WT FST)

(unpublished data). These results suggested that the Tyr-185 residue is likely to be the more important site and serves as a specific binding site for activin, or a stabilizer of FST domain 2. Thus, point mutations in the hydrophobic site of domain 2 disrupted activin binding.

20 Mutations in the hydrophobic regions of domain 2 did inhibit myostatin binding but not to the extent of activin binding, with Yl 85 A and Ll 9 ID retaining approximately 20% and 30% of WT FST binding. This finding concurs with the effect of outright domain2 deletion (dFSD2 mutant), which we showed earlier to retain its myostatin binding. On the other hand, activin binding was significantly diminished by domain 2

25 mutations (Y185A, Y185D, and L191D), suggesting that these amino acids are likely to stabilize the domain and therefore mutations at these structural positions will show similar properties as FST mutant with a domain 2 deletion. Final Conclusions To summarize our findings, activin and myostatin are bound by FST in partially

30 distinct sites so that mutations or deletion of FSD2 affect activin binding much more than myostatin binding. For activin binding, the FST molecule must have a continuous sequence of N-terminal domain and the first two FST domains in the correct sequential order. Thus each domain appears to contribute uniquely to activin binding, possibly through interactions with essential hydrophobic determinants in the N-terminal domain. For myostatin binding, the minimal requirements are the N-terminal domain and FST domain 1 , and an additional domain (FSDl , 2, or 3). It is possible for FST with rearranged domains to bind to myostatin, however our data demonstrated somewhat diminished binding for such rearrangement (FSD 3/1/2 mutant). Mutation in the hydrophobic region of the domain 2 (Ll 91D and Yl 85A) mutants also demonstrated minimal activin binding and substantial myostatin binding but the greatest binding differences were seen in FST mutants with domain 2 deletion (dFSD 2 mutant). This mutant displayed <10% activin binding and >80% myostatin binding relative to WT FST. Such differential binding properties can be useful in the development of therapeutics aiming to increase muscle mass by neutralizing myostatin, without the detrimental side effects of concurrent activin inhibition. A compound that mimics the characteristics of the FST mutant dFSD2 could possibly serve as an effective treatment for muscle-wasting by improving muscle development. For patients who suffer from immobility and chronic diseases such as kidney failure, tumor, cachexia, and a subset of patients with AIDS, specific myostatin inhibitor could also be used to prevent muscle weakness and dystrophy that often manifest over time. However, before advancement can be made, animal studies should first be completed to test the ability of the FST mutant dFSD2 to antagonize myostatin and activin in vivo. Example 2: Myostatin Binding to Follistatin Mutants of Example 1

This study was designed to explore whether any of the follistatin (FST) mutants could differentially antagonize myostatin and activin. FST can bind and neutralize both myostatin and activin with activin being a higher affinity interaction. However, a desirable pharmaceutical intervention could be derived from a mutant FST in which activin binding was substantially decreased compared to myostatin

Methods: Mutant FST clones were prepared from minipreps and sequenced to verify identity. A fixed amount of WT or mutant FST cDNA was transfected, along with the CAGA reporter, into 293 cells. After 24 hours, cells were treated with 5 ng/ml activin or 15 ng/ml myostatin. On the following day, cells were extracted and analyzed for luciferase activity, a measure of activin or myostatin signaling. Since maximal stimulation of activin and myostatin (no FST) were not identical, results are expressed as % of maximum for each ligand. Each experiment included wild type FST as a positive control. Results: We initially screened a panel of 26 FST mutants at a maximal dose (200 ng DNA) compared to wild type FST. We identified 9 mutants which inhibited myostatin better than activin, 1 clone that inhibited activin better than myostatin, and 16 in which myostatin and activin inhibition were equal. An example of typical results are shown in FIGURE 7. Wild type (WT) FST inhibited both activin and myostatin (panels A-C), and deletion of FST domain 3 (dFSD3, Panel A) had no effect on this inhibition, Interestingly, deletion of FST domain 1 (dFSDl, Panel A) reduced inhibition of myostatin more than activin. In contrast, deletion of FST domain 2 (dFSD2, Panel A), swapping the order of the domains, or replacing domain 2 with domain 1 (dFSD2, Panel A; FSD3/1/2, FSD 2/1/3, and FSD 1/1/3, Panel B, respectively) all reduced activin inhibition more than myostatin. In Panel C, clones Yl 1OA and Yl 1OD in FSDl had little effect on either ligand while Yl 85 A had no effect on myostatin inhibition but reduced that of activin. Overall, mutations in FSD2, reduced activin inhibition more than myostatin while mutations in FSDl appeared to alter both ligands, with myostatin being affected more than activin.

We next examined the 9 clones which consistently and significantly inhibited myostatin more than activin at multiple doses to verify differential inhibition (FIGURE 8). Most favored myostatin by about 100-fold. Again, mutations in domain 2, or alterations in the placement of FSD2 affected activin neutralization but had little effect on myostatin. Mutations in domain 1 affected both activin and myostatin about equally.

N-terminal domain mutations also affected both ligands (data not shown). These results demonstrate that the 9 clones identified in the first screen inhibit myostatin more than activin by 10-100 fold, with the most dramatic differences appearing in mutants in which FSD2 is deleted or moved, consistent with the concept that FSD2 is more important for activin binding while FSDl is more critical for myostatin, although it also influences activin action as well.

Discussion: It appears that the binding sites for activin and myostatin within the FST molecule are not entirely overlapping so it is theoretically possible to identify myostatin antagonists with substantially reduced activin antagonist activity. Such a compound would be desirable since it would be less likely to have side effects related to activin inhibition. Using FST without domain 2 might be a good place to start to design such a compound. REFERENCES

The contents of all cited references including literature references, issued patents, published or non-published patent applications cited throughout this application as well as those listed below are hereby expressly incorporated by reference in their entireties. In case of conflict, the present application, including any definitions herein, will control.

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America 100(26): 15824-15826. 10. Sicinski P, Geng Y, Ryder-Cook AS, et al. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 244(4912): 1578-1580.

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12. Amthor H, Nicholas G, McKinnell I, et al. Follistatin complexes myostatin and antagonizes myostatin-mediated inhibition of myogenesis. Developmental Biology 270: 19-30. 13. Hill J, Davies M, Pearson A, et al. The myostatin propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum. Journal of Biological Chemistry 277(43): 40735-40741. 14. Keutmann H, Schneyer A, Sidis Y. The role of follistatin domains in follistatin biological action. Molecular Endocrinology 18(1): 228-240. 15. Shimasaki S, Koga, M, Esch F, et al. Primary structure of the human follistatin precursor and its genomic organization. Proceedings of the National Academy of Sciences of the United States of America 85: 4218-4222.

16. Tsuchida K, Arai KY, Kuramoto Y, et al. Identification and characterization of a novel follistatin-like protein as a binding protein for the TGF-β family. Journal of Biological Chemistry 275(52): 40788-40796.

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EQUIVALENTS

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The appended claims are not intended to claim all such embodiments and variations, and the full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

Claimed are:

1. An isolated, recombinant mutant follistatin that inhibits myostatin but has substantially reduced activin antagonism activity comprising the following domains in the order N-terminus to C-terminus: FST N-terminal domain (FSTN), FSDl, and any one additional follistatin domain (1, 2, or 3) but no other follistatin domain to the C- terminus of the one additional follistatin domain.

2. An isolated, recombinant mutant follistatin that inhibits myostatin but has substantially reduced activin antagonism activity comprising the following domains in the order N-terminus to C-terminus: FSD3, FSDl and FSD2.

3. An isolated, recombinant mutant follistatin that inhibits myostatin but has substantially reduced activin antagonism activity having at least about 95% identity to SEQ ID NO: 1 and any amino acid except tyrosine (Y) at position 185.

4. The isolated, recombinant mutant follistatin of claim 3, comprising a sequence having at least about 95% identity to SEQ ID NO: 1 and an aspartic acid (D) at position 185.

5. An isolated, recombinant mutant FST 288 that inhibits myostatin but does not alter activin activity polypeptide comprising a sequence having at least about 95% identity to SEQ ID NOs: 1 and any amino acid except leucine (L) at position 191.

6. The isolated, recombinant mutant follistatin of claim 5, comprising a sequence having at least about 95% identity to SEQ ID NO: 1 and an aspartic acid (D) at position 191 rather than a leucine (L).

7. An isolated, recombinant mutant follistatin that inhibits myostatin but has substantially reduced activin antagonism activity comprising a sequence selected from the group consisting of any one of SEQ ED NO: 8, 9, 10, 11 or 12.

8. The isolated, recombinant mutant follistatin of any one of claim 1, 2, 3, 4, 5, 6, or 7 wherein said follistatin is selected from the group consisting of: FST 288, FST 305 and FST 315.

9. The isolated, recombinant polypeptide of any one of claim 1, 2, 3, 4, 5, 6, 7 or 8 wherein said polypeptide is fused to another polypeptide.

10. An isolated nucleic acid encoding the polypeptides of any one of claims 1-9.

11. A vector comprising a nucleic acid of claim 10.

12. The vector of claim 1 1 , wherein said nucleic acid is operably linked to a promoter.

13. A host cell comprising a vector of claim 11 or 12.

14. A pharmaceutical composition comprising a polypeptide of any one of claims 1-9.

15. A pharmaceutical composition comprising a nucleic acid of claim 10.

16. The pharmaceutical composition of claim 15, wherein said nucleic acid is contained in a vector.

17. A method of stimulating muscle growth comprising administering a pharmaceutical composition of claim 14, 15 or 16 to a subject in need thereof.

18. The method of claim 17, further comprising administering a second therapy.

19. The method of claim 18, wherein the second therapy comprises physical therapy.

20. The method of claim 18, wherein the second therapy comprises drug therapy.

21. A method of treating a muscle wasting disorder, comprising administering to a subject in need thereof a pharmaceutical composition of claim 14, 15 or 16.

22. The method of claim 21, further comprising administering a second therapy.

23. The method of claim 22, wherein the second therapy comprises physical therapy.

24. The method of claim 22, wherein the second therapy comprises drug therapy.

25. A kit comprising a polypeptide of any one of claims 1-9.

26. A kit comprising a nucleic acid of claim 10.

27. The kit of claim 25 or 26, further comprising instructions for use.