AU741350B2

AU741350B2 - Treatment of mammalian myocardium with morphogen locally, or with morphogenically-treated myogenic precursor cells

Info

Publication number: AU741350B2
Application number: AU57119/98A
Authority: AU
Inventors: Charles M. Cohen; Kuber T. Sampath
Original assignee: Curis Inc
Current assignee: Curis Inc
Priority date: 1996-12-20
Filing date: 1997-12-19
Publication date: 2001-11-29
Anticipated expiration: 2017-12-19
Also published as: EP0952845A1; CA2275436A1; WO1998027995A1; JP2001507354A; AU5711998A

Description

WO 98/27995 PCT/US97/23611 TREATMENT OF MAMMALIAN MYOCARDIUM

WITH

MORPHOGEN LOCALLY, OR WITH MORPHOGENICALLY-TREATED MYOGENIC PRECURSOR

CELLS

Field of the Invention The present invention relates generally to methods and preparations for the treatment of mammals, including humans, at risk of, or afflicted with, loss of or damage to myocardium. The methods involve the implantation of mammalian myogenic precursor cells treated with certain morphogens, inducers of those morphogens, agonists of the corresponding morphogen receptors, or with small molecule morphogenic activators.

Background of the Invention Unlike skeletal muscle or smooth muscle, adult mammalian cardiac muscle has extremely limited powers of growth and regeneration. During development, the myocardium arises by endto-end fusion of myogenic precursor cells to form branched myofibers in which individual cardiac myocytes are joined by intercalated disks. The myogenic precursor cells which give rise to the myocardium are derived from the splanchic mesoderm, which is derived from the lateral mesodermal mesenchyme which, in turn, arises from the mesoderm formed after gastrulation. It is generally believed that there are no remaining myogenic precursor cells in adult mammalian myocardium and, therefore, lost or damaged myocardium is typically replaced by fibrotic or scar tissue, rather than new myocardium. See, generally, B.M. Carlson, ed. (1981) Patten's Foundations of Embryology, 4th Edition, McGraw-Hill, New York. As a result, damage or loss of myocardium due, for example, to myocardial infarction, congestive heart failure, physical trauma in an automobile accident), or infection, typically results in a permanent and often progressive loss of functional myocardium.

In contrast, mammalian skeletal muscle has much greater capacity for growth and regeneration, even in adulthood. Like the myocardium, skeletal muscle has its first origins after the induction of the mesoderm. After differentiation of the mesoderm into dorsal, intermediate, and lateral mesoderm, the dorsal mesodermal mesenchyme differentiates to form myotomes which, in turn, differentiate to form the myogenic precursor cells which ultimately form skeletal muscle. Unlike the myogenic precursor cells of the heart, the skeletal muscle precursors fuse side-to-side to form unbranched, multinucleated myofibers. Significantly, some portion of the WO 98/27995 PCT/US97/23611 -2skeletal myogenic precursor cells do not differentiate into myocytes but, rather, attach to the plasmalemmas of the myocytes. These cells may remain, throughout adulthood, as largely undifferentiated, quiescent skeletal muscle "satellite cells." Upon injury of a skeletal muscle, however, these satellite cells are revealed to be myogenic precursor cells, or muscle "stem cells," which proliferate and differentiate into new and functional skeletal muscle. Even after injury, however, a portion of the proliferated satellite cells remain undifferentiated and attach to the newly formed myofibers. Thus, the satellite cells of skeletal muscle provide a constant and renewable source of myogenic precursor cells which allows for skeletal muscle repair and regeneration throughout mammalian life.

The proliferation and differentiation of skeletal muscle satellite cells has been extensively studied in vitro. For example, a simple saline extract of skeletal muscle has been shown to cause satellite cells to proliferate in culture (Bischoff (1989) in Myoblast Transfer Therapy, Griggs and Karpati, eds., pp. 147-158). Similarly, it has been shown that chick embryo extract or the conditioned medium of differentiated myotubes from young mice exhibits a strong mitogenic effect on satellite cells, but that conditioned medium from older murine myotubes has a lesser effect (Mezzogiorno et al. (1993) Mech. Ageing Develop. 70:35-44). In addition, a number of hormones and growth factors have been found to enhance satellite cell proliferation, including FGF, PDGF, ACTH, LIF, and IGF (Bischoff (1989); Mezzogiorno et al. (1993)). Conversely, TGF-3P is widely believed to inhibit satellite cell proliferation, as does contact with the myofiber plasmalemma, but not the basal lamina (Bischoff (1989); but see Hathaway et al. (1991) J. Cell Physiol. 146:435-441).

Curiously, in a rat model of skeletal muscle injury, it was found that there were signs of satellite cell differentiation before there were significant signs of satellite cell proliferation (Rantanen et al. (1995) Lab. Invest. 72:341-347). This suggests the possibility that there are two populations of skeletal muscle satellite cells: "committed satellite cells" which respond to injury by rapidly differentiating to replace the injured tissue, and "stem satellite cells" which respond more slowly by proliferating and, perhaps, renewing the committed satellite cell population. In this scenario, the stem satellite cells may undergo mitosis to produce one daughter cell which remains a stem satellite cell, and another which becomes a committed satellite cell.

WO 98/27995 PCT/US97/23611 -3- In another animal model, autologous mouse skeletal muscle cells were explanted from a healthy muscle, proliferated in vitro, and then implanted into a necrotized skeletal muscle site (Alameddine and Fardeau (1989) in Myoblast Transfer Therapy, Griggs and Karpati, eds., pp.

159-166). In these experiments, it was shown that the transplanted satellite cells were able to populate the necrotized area and differentiate into functional myotubes. Similarly, PCT Publication WO 96/28541 discloses that histocompatible donor mouse myoblasts can be implanted into the weakened muscle of a mouse model of muscular dystrophy and differentiate into myofibers. In addition, it is shown that growth of the myoblasts in bFGF results in significantly more new myofibers at the implant site. Thus, skeletal muscle satellite cells, proliferated in vitro, may be able to serve as a source of myogenic precursor cells for muscle restoration or regeneration therapy.

The ability of skeletal muscle satellite cells to restore or regenerate injured skeletal muscle, has led some researchers to test whether myogenic precursor cells could be used to replace lost or damaged myocardial muscle. For example, mouse fetal cardiomyocytes, which are not terminally differentiated and retain the ability to divide, have been directly injected into the myocardium of a syngeneic adult mouse, and have been shown to form new and apparently functional myocardium (Soonpaa et al. (1994) Science 264:98-101). Significantly, it has been shown that skeletal muscle satellite cells, explanted from adult canine skeletal muscle can be proliferated in vitro and implanted into a site of myocardial cryoinjury, where they appear to differentiate into "cardiaclike" muscle cells, possibly in response to morphogenic signals present in the myocardium (Chiu et al. (1995) Ann. Thorac. Surg. 60:12-18).

Morphogens and Growth Factors A great many proteins have now been identified which appear to act as morphogenetic or growth factors, regulating cell proliferation and/or differentiation. Typically these growth factors exert their effects on specific subsets of cells and/or tissues. Thus, for example, epidermal growth factors, nerve growth factors, fibroblast growth factors, various hormones, and many other proteins inducing or inhibiting cell proliferation or differentiation have been identified and shown to affect some subset of cells or tissues.

One group of morphogenetic proteins, referred to herein as "morphogens," includes members of the family of bone morphogenetic proteins (BMPs) which were initially identified by WO 98/27995 PCT/US97/23611 -4their ability to induce ectopic, endochondral bone morphogenesis. Subsequent characterization of the nucleic acid and amino acid sequences of the BMPs has shown them to be a subgroup of the TGFP superfamily of growth and differentiation factors. Members of the morphogen family have now been shown to include the mammalian osteogenic protein (OP1, also known as BMP7), osteogenic protein2 (OP2), osteogenic protein3 (OP3), BMP2 (also known as BMP2A or CBMP2A), BMP3, BMP4 (also known as BMP2B or CBMP2B), BMP5, BMP6, Vgrl, and GDFI, as well as the Xenopus homologue Vgl and the Drosophila homologues DPP and Members of this family encode secreted polypeptides that share common structural features, and that are similarly processed from a pro-protein to yield a carboxy terminal mature protein of approximately 100-110 amino acids. All members share a conserved pattern of cysteines in this domain and the active form of these proteins is either a disulfide-bonded homodimer of a single family member, or a heterodimer of two different members (see, Massague (1990) Annu.

Rev. Cell Biol. 6:597; Sampath, et al. (1990) J. Biol. Chem. 265:13198).

The members of the morphogen family of proteins are expressed naturally in a variety of tissues during development. BMP-2 BMP-2A), for example, is expressed in embryonic mouse hair follicles, cartilage and bone (Lyons et al. (1989) Genes Develop. 3:1657-1668); BMP3 has been shown to be most highly expressed in human embryonic lung and kidney, highly expressed in intestinal mucosa and skeletal tissues such as the perichondrium and periosteum, expressed in brain, but undetectable in embryonic heart and liver (Vukicevic et al. (1994) L Histochem. Cvtochem. 42:869-875); BMP4 has been shown to be expressed in the developing limbs, heart, facial processes and condensed mesenchyme associated with early whisker follicles in embryonic mice (Jones, et al. (1991) Development 111:531-542); and OPI (BMP7) has been shown immunohistochemically to be present in human embryos in sclerotome, hypertrophied chondrocytes, osteoblasts, periosteum, adrenal cortex, renal convoluted tubules, placenta, smooth, cardiac and skeletal muscles, meninges and neural cells, as well as the basement membranes of the lungs, pancreas and skin (Vukicevic, et al. (1994) Biochem. Biophvs. Res.

Commun. 198:693-700). Some of the morphogens OP2 and BMP2) were not detected in analyses of adult tissues, suggesting only an early developmental role for these morphogens (Ozkaynak, et al. (1992) J. Biol. Chem. 267:25220-25227).

WO 98/27995 PCT/US97/23611 Although, as noted above, several morphogens have been shown to be expressed in embryonic or adult mammalian heart tissue, and various utilities for the morphogens have been proposed and developed, it has never previously been shown or suggested that treatment of myogenic precursor cells with the morphogens, morphogen inducers, agonists of morphogen receptors, or small molecule morphogenic activators is useful in promoting the proliferation and/or differentiation of myogenic precursor cells into new and functional myocardium in a morphogenically permissive environment. Nor has it previously been shown or suggested that morphogenically-treated myogenic precursor cells are useful in the treatment of lost or damaged mammalian myocardium.

Summary of the Invention The present invention is directed to methods of treatment, and pharmaceutical preparations for use in the treatment, of mammalian subjects at risk of, or afflicted with, loss of or damage to myocardium. Such subjects include subjects already afflicted with the loss of myocardial tissue, such as those which have already suffered a myocardial infarction, physical trauma to the heart in an automobile accident, or those already suffering from congestive heart failure, as well as subjects reasonably expected to suffer from myocardial infarction or congestive heart failure. Whether a particular subject is at risk is a determination which may routinely be made by one of ordinary skill in the relevant medical or veterinary art.

In these methods of treatment, myogenic precursor cells are implanted into a mammal at a site at risk of, or afflicted with, loss of or damage to myocardium, and the myogenic precursor cells are morphogenically-treated prior to, simultaneously with, or subject to implantation. Thus, for example, morphogenically-treated mammalian myogenic precursor cells may be implanted into a mammalian heart at the site of a myocardial infarct, or into the damaged or weakened myocardium of a subject with congestive heart failure. The mammalian myogenic precursor cells may be derived from skeletal muscle skeletal muscle satellite cells), from embryonic tissue embryonic mesodermal mesenchyme) or from a myogenic precursor cell line maintained in vitro. Thus, the myogenic precursor cells may be derived from a donor a tissue-type matched donor, sibling, identical twin, or fetus), may be derived from a tissue culture undifferentiated or partly undifferentiated myogenic cells in culture; fetal tissue culture), or may be explanted from the subject and re-implanted after morphogen-induced proliferation and/or WO 98/27995 PCT/US97/23611 -6differentiation. Finally, the morphogenic treatment of the implanted cells may include treatment of the cells with a morphogen, morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator prior to implantation, simultaneously with implantation, or subsequent to implantation.

The present invention is further directed to methods of promoting the proliferation and differentiation of mammalian myogenic precursor cells in vivo or in vitro. Thus, for example, myogenic precursor cells isolated from mammalian skeletal muscle tissue, embryonic myogenic precursor cells, or myogenic precursor cell lines, may be stimulated to proliferate by treatment with a morphogen, an inducer of a morphogen, an agonist of a morphogen receptor, or a small molecule morphogenic activator. Alternatively, or in addition, mammalian myogenic precursor cells may be stimulated to differentiate into myocytes, particularly myocytes which express markers of myocardial tissue, in a morphogenically permissive environment.

The present invention is further directed to therapeutic preparations comprising isolated mammalian myogenic precursor cells and an amount of a morphogen, inducer of a morphogen, agonist ofa morphogen receptor, or small molecule morphogenic activator sufficient to promote proliferation or differentiation of the myogenic precursor cells in a morphogenically permissive environment.

The methods and compositions of the present invention capitalize in part upon the fact that certain proteins of eukaryotic origin, defined herein as morphogens, may be used to treat myogenic precursor cells such that, when these morphogenically-treated myogenic precursor cells are present in a morphogenically permissive environment, they may migrate, proliferate and/or differentiate so as to form new and functional myocardium. In particular, the present invention is based in part upon the fact that treatment of myogenic precursor cells with these morphogens enhances or increases the probability, rate, or efficiency with which these cells migrate, proliferate and/or differentiate into new and functional myocardium in a morphogenically permissive environment. Thus, in accordance with the present invention, morphogenically-treated myogenic precursor cells may be used to restore or regenerate lost or damaged myocardium in a mammal, or to prophylactically treat a mammal at risk of such loss or damage. The present invention is novel in that myocardial tissue is believed to lack a sufficient number of myogenic precursor cells for adequate regeneration or repair of lost or damaged tissue and, therefore, the ability of the WO 98/27995 PCT/US97/23611 -7morphogens to promote the migration, proliferation and/or differentiation of myogenic precursor cells skeletal muscle satellite cells) into functional myocardium is unexpected.

In preferred embodiments, the morphogen is a dimeric protein comprising a pair of folded polypeptides, each having an amino acid sequence that shares a defined relationship with an amino acid sequence of a reference morphogen. Preferred morphogen polypeptides share a defined relationship with a sequence present in morphogenically active human OP-1 (SEQ ID NO: 4).

However, any one or more of the naturally occurring or biosynthetic sequences disclosed herein similarly could be used as a reference sequence. Preferred morphogen.polypeptides share a defined relationship with at least the C-terminal six cysteine domain of human OP-1 (residues 43- 139 of SEQ ID NO: Preferably, morphogen polypeptides share a defined relationship with at least the C-terminal seven cysteine domain of human OP-1 (residues 38-139 of SEQ ID NO: 4).

That is, preferred morphogen polypeptides in a dimeric protein with morphogenic activity each comprise a sequence that corresponds to a reference sequence or is functionally equivalent thereto. Examples of preferred morphogens include mammalian, and particularly human, OP-1, CBMP-2A (BMP-2) and CBMP-2B (BMP-4).

Functionally equivalent sequences include functionally equivalent arrangements of cysteine residues disposed within the reference sequence, including amino acid insertions or deletions which alter the linear arrangement of these cysteines, but do not materially impair their relationship in the folded structure of the dimeric morphogen protein, including their ability to form such intra- or inter-chain disulfide bonds as may be necessary for morphogenic activity.

Functionally equivalent sequences further include those wherein one or more amino acid residues differs from the corresponding residue of a reference morphogen sequence, the C-terminal seven cysteine domain (also referred to herein as the conserved seven cysteine skeleton) of human OP-1, provided that this difference does not destroy morphogenic activity. Accordingly, conservative substitutions of corresponding amino acids in the reference sequence are preferred.

Amino acid residues that are "conservative substitutions" for corresponding residues in a reference sequence are those that are physically or functionally similar to the corresponding reference residues, that have similar size, shape, electric charge, chemical properties including the ability to form covalent or hydrogen bonds, or the like. Particularly preferred conservative substitutions are those fulfilling the criteria defined for an "accepted point mutation" WO 98/27995 PCT/US97/23611 -8in Dayhoff, et al. (1978) Atlas of Protein Sequence and Structure, 5: Suppl. 3, ch. 22 (pp. 354- 352), Natl. Biomed. Res. Found., Washington, D.C. 20007, the teachings of which are incorporated by reference herein.

In certain embodiments, a polypeptide suspected of being functionally equivalent to a reference morphogen polypeptide is aligned therewith using the method of Needleman, et al.

(1970) J. Mol. Biol. 48:443-453, implemented conveniently by computer programs such as the Align program (DNAstar, Inc.). As noted above, internal gaps and amino acid insertions in the candidate sequence are ignored for purposes of calculating the defined relationship, conventionally expressed as a level of amino acid sequence homology or identity, between the candidate and reference sequences. "Amino acid sequence homology" is understood herein to include both amino acid sequence identity and similarity. Homologous sequences share identical and/or similar amino acid residues, where similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding amino acid residues in an aligned reference sequence. Thus, a candidate polypeptide sequence that shares 70% amino acid homology with a reference sequence is one in which any 70% of the aligned residues are either identical to, or are conservative substitutions of, the corresponding residues in a reference sequence.

The present invention alternatively can be practiced with methods and compositions comprising a morphogen inducer in lieu of a morphogen. A "morphogen inducer" is a compound that stimulates the production transcription, translation, and/or secretion) of morphogen by a cell competent to produce and/or secrete a morphogen encoded within the genome of the cell.

Endogenous or administered morphogens can act as endocrine, paracrine or autocrine factors.

Therefore, an inducer of a morphogen may stimulate endogenous morphogen synthesis by the cells in which the morphogenetic responses are induced, by neighboring cells in vivo or in vitro in tissue culture) or by cells of a distant tissue in vivo (in which case the secreted morphogen is transported to the site of morphogenesis, by the individual's bloodstream). In preferred embodiments, the inducer stimulates expression and/or secretion of a morphogen so as to increase amounts thereof available to mammalian myogenic precursor cells in vivo or in vitro.

Thus, to promote the migration, proliferation and/or differentiation of myogenic precursor cells in vivo, an inducer of a morphogen may be administered to induce production of morphogen by the myogenic precursor cells themselves, or by other cells co-cultured with the myogenic precursor WO 98/27995 PCT/US97/23611 -9cells. Similarly, to promote the proliferation and/or differentiation of myogenic precursor cells in vivo, an inducer of a morphogen may administered locally or systemically to induce morphogen production by the myogenic precursor cells themselves, or by neighboring or distant cells in a mammal's body.

In still other embodiments, an agent which acts as an agonist of a morphogen receptor may be administered instead of the morphogen itself. An "agonist" of a receptor is a compound which binds to the receptor, and for which the result of such binding is similar to the result of binding the natural, endogenous ligand of the receptor. That is, the compound must, upon interaction with the receptor, produce the same or substantially similar transmembrane and/or intracellular effects as the endogenous ligand. Thus, an agonist of a morphogen receptor binds to the receptor and such binding has the same or a functionally similar result as morphogen binding induction of morphogenesis). The activity or potency of an agonist can be less than that of the natural ligand, in which case the agonist is said to be a "partial agonist," or it can be equal to or greater than that of the natural ligand, in which case it is said to be a "full agonist." Thus, for example, a small peptide or other molecule which can mimic the activity of a morphogen in binding to and activating the morphogen's receptor may be employed as an equivalent of the morphogen. Preferably the agonist is a full agonist, but partial morphogen receptor agonists may also be advantageously employed. Methods of identifying such agonists are known in the art and include assays for compounds which induce morphogen-mediated responses induction of differentiation of metanephric mesenchyme, induction of endochondral bone formation, and the like). Such an agonist may also be referred to as a morphogen "mimic," "mimetic," or "analog." Alternatively, a small molecule morphogenic activator, as described herein, may be administered instead of the morphogen itself to promote the migration, proliferation, and/or differentiation of myogenic precursor cells by increasing the level of expression of proteins associated with myocardial phenotype. Exemplary methods comprise introducing a small molecule morphogenic activator that regulates some portion or portions of a morphogen-induced regulatory pathway, resulting in an effective increase in expression or activity of myocardiumspecific protein. This may result either from stimulating an increase in the endogenous expression of such protein or from a decrease in the inhibition of normal expression of such protein. For example, a small molecule morphogenic activator may act at the type I or type II morphogen WO 98/27995 PCT/US97/23611 receptor; or at the serine/threonine kinase, or other kinase domains of those receptors. Another target of pathway activation is the Smad proteins, including the monomeric, dimeric (including heteromeric and homomeric complexes) or trimeric forms (including heteromeric and homomeric complexes). Alternately, a small molecule morphogenic activator may lead to activation of a transcription factor (for example, the X-protein shown in Figure 2) that causes phenotype-specific gene expression expression of protein characteristic of myocardium).

Preferably, the morphogens, morphogen inducers, agonists of morphogen receptors, or small molecule morphogenic activators are directly contacted with the myogenic precursor cells in solution either in vitro prior to implantation, in vivo at the time of implantation, or in vivo subsequent to implantation. Alternatively, however, the morphogens, morphogen inducers, agonists of morphogen receptors may be administered by any route which is compatible with the selected agent, and may be formulated with any pharmaceutically acceptable carrier appropriate to the route of administration. Preferred systemic routes of administration are parenteral and, in particular, intravenous and intraperitoneal.

In additional embodiments, the present invention provides pharmaceutical compositions comprising a morphogen, or morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator in combination with one or more of a "muscle extract," conditioned medium from differentiated myotubes grown in culture, bFGF, IGF, PDGF, LIF, ACTH, MSH, or G-CSF. These compositions are useful in promoting the proliferation and/or differentiation of myogenic precursor cells.

Brief Description of the Figures Figure 1. Panels 1-1 through 1-12 of this figure are a tabular alignment of the amino acid sequences of various naturally occurring morphogens with a preferred reference sequence of human OP1, residues 38-139 of SEQ ID NO: 4. Morphogen polypeptides shown in this figure also are identified in the Sequence Listing.

Figure 2. Figure 2 is a schematic representation of a morphogen-activated regulatory pathway for expression of a phenotype-specific gene.

Detailed Description of the Invention I. Definitions WO 98/27995 PCT/US97/23611 -11 In order to more clearly and concisely point out the subject matter of the claimed invention, the following definitions are provided for specific terms used in the following written description and appended claims.

Subjects at risk of, or afflicted with, loss of or damage to mvocardium. As used herein, a subject (preferably a mammal, a human) is said to be at risk of, or afflicted with, loss of or damage to myocardium, if the subject has suffered a loss of functional myocardial tissue which is clinically detectable in terms of reduced or altered cardiac function, or if the subject may reasonably be expected to suffer such a loss. Subjects at risk of, or afflicted with, loss of or damage to myocardium include, but are not limited to, subjects which have already suffered a myocardial infarction, which have suffered a physical trauma to the heart in an automobile accident) which has reduced cardiac function, or which have already been diagnosed with congestive heart failure; as well as subjects which can reasonably be expected to suffer a myocardial infarction or congestive heart failure. Whether a particular subject is at risk is a determination which may routinely be made by one of ordinary skill in the relevant medical or veterinary art.

Mvogenic precursor cells As used herein, the term "myogenic precursor cells" refers to cells capable of myogenesis, or the process of proliferation and differentiation into new and functional muscle when present in a morphogenically permissive environment. Myogenic precursor cells are variously referred to in the literature as "myoblasts," "muscle stem cells" or "satellite cells." Morphogenically permissive environment. As used herein, a "morphogenically permissive environment" is an environment which allows or promotes the differentiation of cells into a specific cell type or types. A "morphogenically permissive environment" is, therefore, sufficiently free of inhibitors of cell differentiation to allow or promote cell differentiation. In addition, a morphogenically permissive environment is one which provides signals through cell-cell contact, cell-extracellular matrix contact, or diffusible factors) which allow or promote a pluripotent cell to follow a particular morphogenic pathway. In particular, with respect to myocardial differentiation, a morphogenically permissive environment includes an environment of intact or damaged myocardial tissue which provides signals to myogenic precursor cells which allow or promote the differentiation of those cells into new and functional myocardium. It is WO 98/27995 PCT/US97/23611 -12 known, for example, that myogenic precursor cells differentiate into myocytes at least partly in response to contact with the plasmalemma of a myofiber. The presence of myofiber plasmalemmas, therefore, may be one element of a morphogenically permissive environment for myogenesis. Similarly, electrical or biochemical stimuli from nerves, as well as a variety of growth factors (see below), appear to be elements of a morphogenically permissive environment for myogenesis. Thus, a morphogenically permissive environment may include one or more of these elements.

II. Description of the Preferred Embodiments A. General The present invention depends, in part, upon the surprising discovery that morphogenically-treated mammalian myogenic precursors cells, when implanted in vivo at a site of lost or damaged mammalian myocardium, undergo a process of proliferation and/or differentiation to produce new and functional mammalian myocardium, thereby restoring or regenerating the lost or damaged tissue in whole or in part. This result is particularly unexpected in light of the fact that mammalian myocardial tissue is believed to lack a sufficient number of myogenic precursor cells for adequate regeneration or repair of lost or damaged tissue and, therefore, mammalian myocardium previously has been believed to be a poor responder for functional restoration or regeneration after tissue loss or damage. In addition, the present invention depends, in part, upon the surprising discovery that non-myocardial cells, such as those obtained from mammalian skeletal muscle or embryonic myogenic precursor cells, may be induced to proliferate and differentiate into myocardium in a morphogenically permissive environment. It is further surprising that the morphogens, morphogen inducers, agonists ofmorphogen receptors, and small molecule morphogenic activators, as described herein, may promote such restoration or regeneration despite the fact that they have no known role in myocardial tissue restoration or regeneration in the adult mammal.

Without being bound to any particular theory of the invention, it is believed that the morphogens, morphogen inducers, agonists of morphogen receptors, or small molecule morphogenic activators may promote the proliferation of myogenic precursor cells and render them more susceptible to differentiation into new and functional myocardium when implanted in a morphogenically permissive environment. Thus, it is believed that the morphogens, morphogen WO 98/27995 PCT/US97/23611 -13 inducers, agonists ofmorphogen receptors, or small molecule morphogenic activators may increase the pluripotentiality of these myogenic precursor cells, such that they may "switch fates" and, rather than differentiating only into smooth or skeletal muscle, they may proliferate and then differentiate into new and functional myocardium.

B. Isolating and Culturing Mammalian Mvogenic Precursor Cells Methods of isolating and culturing mammalian myogenic precursor cells are wellestablished in the art. For example, myogenic precursor cells may be obtained, as further described in the examples below, by dissociation of skeletal muscle and subsequent culturing of the satellite cells. Alternatively, myogenic precursor cells may be obtained from embryonic tissues, where they arise as fetal myoblasts from the myotomes of the somites, after induction of the mesoderm. Myogenic precursor cells may also be obtained from cell lines, such as a pluripotent mesodermal mesenchyme cell line or a partially dedifferentiated laboratory cell line, which may be induced to differentiate into myoblasts after implantation into a morphogenically permissive environment. See, generally, Hathaway, et al. (1991) J. Cell. Physiol. 146:435-441; Mezzogiorno et al. (1993) Mech. Ageing Develop. 70:35-44; Alameddine and Fardeau (1989); Chiu et al. (1995) Ann. Thorac. Surg. 60:12-18.

1. Isolating Myogenic Precursor Cells from Skeletal Muscle In preferred embodiments, the myogenic precursor cells are obtained from skeletal muscle.

The skeletal muscle donor is preferably the subject for myocardial treatment or an identical twin in order to avoid problems of histocompatibility and possible tissue rejection. Alternatively, other family members or histocompatible donors, including transgenic mammals raised for organ transplantation purposes lacking MHC markers or expressing humanized MHC proteins), may be employed as donors of the skeletal muscle tissue. Depending upon the degree of histocompatibility, standard methods ofimmunosuppression may be needed in conjunction with the present invention to prevent rejection of the implanted cells.

Briefly, a sample of skeletal muscle is excised from one or more skeletal muscles of a subject under local or general anesthesia. Any excessive connective tissue and fasciae are dissected away, the muscle is rinsed in sterile solution, and the muscle is dissociated by, for example, mincing with scissors or passage through a meat grinder until substantially homogeneous. The amount of muscle excised will depend, of course, upon the quantity of WO 98/27995 PCT/US97/23611 -14myogenic precursor cells required by the treatment, as well as the degree of myogenic precursor cell proliferation which is to be promoted in vitro. Typically, however, amounts of 1-100 grams, more preferably 10-50 grams, of skeletal muscle tissue are removed. Such quantities may be excised conveniently from one or more of the larger, relatively superficial muscles of the limbs biceps brachii, triceps brachii, brachialis, brachioradialis, rectus femoris, biceps femoris, semitendinosus, gracilis, vastus lateralis, gastrocnemius, tibialis anterior), chest and shoulders pectoralis, deltoid), pelvis and hips gluteus medius, gluteus maximus), back trapezius, latissimus dorsi) or abdomen obliquus abdominis externus, rectus abdominis), but may be obtained from any available skeletal muscle.

Preferably, the dissociated muscle then is incubated with a proteolytic enzyme pronase (Sigma, St. Louis, MO), collagenase (Sigma, St. Louis, MO), hyaluronidase (Sigma, St.

Louis, MO), or trypsin (Difco Laboratories, Inc., Detroit, MI) at 37 0 C for 15 min to 1 hr to remove remaining connective tissue. The mass of digested muscle tissue optionally may be further dissociated by, for example, repeated pipetting or mixing. In addition, the digested mass optionally may be washed, pelleted and resuspended to remove digested connective tissue and enzyme, and any remaining debris may be removed by filtration. The cells are then suspended in a sterile buffer phosphate buffered saline solution) and centrifuged at approximately 500-550 g for approximately 10 minutes to sediment the larger, multinucleated skeletal muscle fibers and myocytes, while leaving the satellite cells in the supernatant. Either before or after centrifugation, serum, such as fetal bovine serum (FBS, GIBCO BRL, Grand Island, NY), may be added to the mixture to halt the enzymatic cleavage process and antibiotics may be added to prevent microbial growth. If desired, satellite cells may be separated from fibroblasts and other remaining cells using a density centrifugation method (see, Yablonka-Reuveni and Nameroff (1987) Histochemistry 87:27-38).

2. Isolating Mvogenic Precursor Cells from Embryos Myogenic precursors cells may be isolated from mammalian embryonic or fetal (together "embryonic") tissues at various stages of development after induction of the mesoderm. Thus, for example, myogenic precursor cells may be obtained from the embryonic mesoderm prior to its further differentiation into dorsal, intermediate, and lateral mesodermal mesenchyme. After this stage of differentiation, any mesodermal cells may be employed but, preferably, cells are employed WO 98/27995 PCT/US97/23611 which arise along the routes of differentiation toward skeletal or cardiac muscle. For example, the dorsal mesodermal mesenchyme differentiates to form the myotomes which, in turn, differentiate to form both the skeletal muscles of the trunk and the limb buds. The mesodermal mesenchyme of the limb buds further differentiates to form the skeletal muscles of the appendages (as well as the appendicular skeleton. Similarly, the lateral mesodermal mesenchyme differentiates, in part, to form the splanchic mesoderm which, in turn, differentiates to form the myocardium and smooth muscles of the viscera (as well as the gonads, circulatory system and other primary elements of the viscera). One of ordinary skill in the art may, therefore, readily choose appropriate embryonic cells for use in the present invention (see, Soonpaa et al.

(1994) Science 264:98-101; also see, generally, B.M. Carlson, ed. (1981) Patten's Foundations of Embryology 4th Edition, McGraw-Hill, New York). Once excised, the embryonic tissue may be treated essentially as described above with respect to skeletal muscle to isolate the myogenic precursor cells.

As with cells obtained from the skeletal muscle of an adult mammal, histocompatibility problems may arise upon implantation of embryonic myogenic precursor cells. Therefore, depending upon the degree of histocompatibility, standard methods of immunosuppression may be needed in conjunction with the present invention to prevent rejection of the implanted cells.

3. Isolating Myogenic Precursor Cells from Established Cell Lines Established cell lines, including myogenic precursor cell lines, myoblast cell lines, or mesenchymal cell lines, may also be employed in the present invention without the need for isolation of the myogenic precursor cells from adult or embryonic tissue. For example, the established murine myoblast cell line C 2

C

1 2 (ATCC CRL 1772) has been implanted into mouse hearts and shown to differentiate into functional myocardium and fuse with native myocardium (Koh et al. (1993) J. Clin. Invest. 92:1548-54). Alternatively, pluripotent mesodermal stem cell lines, including primary dermal fibroblast lines, smooth muscle cell lines, or chondroblast lineages may be caused to differentiate into muscle cells (see, Choi et al. (1990) Proc. Nat. Acad. Sci.

(USA) 87:7988-7992). Finally, it should be noted that a variety of established mammalian myogenic cell lines are commercially available for use in accordance with the present invention including, for example, the human cell line HISM (ATCC CRL 1692), the murine cell lines C2C12 (ATCC CRL 1772), NOR-10 (ATCC CRL 197), and G-8 (ATCC CRL 1456), and the rat WO 98/27995 PCT/US97/23611 -16cell lines A7r5 (ATCC CRL 1444), A10 (ATCC CRL 1476), H9c2 (ATCC CRL 1446), L6 (ATCC CRL 1458) and L8 (ATCC CRL 1769). Following essentially the same protocols as described in the original reports of these cell lines (see the ATCC's Catalogue of Cell Lines Hybridomas, for citations) one of ordinary skill in the art can readily produce comparable cell lines from any mammalian species.

4. Culturing Mvogenic Precursor Cells Myogenic precursor cells may be cultured on solid or in liquid media. Thus, for example, the myogenic precursor cells may be suspended in a flask of liquid medium while maintaining mild or periodic agitation. Alternatively, the cells may be plated on a solid substrate and fed with a liquid medium. Appropriate liquid media are well known in the art and include, but are not limited to, McCoy's, M199, Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (commercially available from, for example, GIBCO BRL, Grand Island, NY, or Sigma Chemical Company, St. Louis, MO), and the like. These media may, of course, be supplemented with additional buffers or nutrient solutions 10% fetal bovine serum, 3% horse serum), or with antimycotics and/or antibiotics 50-5,000 IU/ml penicillin, 50-5,000 .g/ml streptomycin, 5-50 tg/ml gentamicin). Preferably, the liquid media is replaced every 24-48 hrs and the cultures are maintained at a relatively constant temperature of about 37°C under a normal or 5% CO2-enriched humid atmosphere. For culturing on solid substrates, cells are preferably plated at a density of approximately 104-106 cells per 60 mm plate. To promote cell adherence to solid substrates, the plates may optionally be coated with, for example, basement membrane matrigel or laminin (Sigma Chemical Company, St. Louis, MO) although, as described below, adherence and/or confluence may inhibit proliferation.

In order to allow or promote proliferation of the myogenic precursor cells in vitro while inhibiting premature differentiation, a number of steps may be taken. For example, myogenic precursor cell proliferation has been shown to be inhibited by TGF-0 (Allen and Boxhorn (1989) J. Cell Physiol. 138:311-315) and contact with myofiber plasmalemmas, (Bischoff(1989)); and has been shown to be promoted by a saline "muscle extract" (Bischoff(1986) Dev. Biol.

115:140), conditioned medium from differentiated myotubes grown in culture (Mezzogiorno et al.

(1993) Mech. Ageing Develop. 70:35-44), basic fibroblast growth factor (bFGF) (Clegg et al.

(1987) J. Cell. Biol. 105:949-56), insulin-like growth factors (IGF) (Ewton and Florini (1977) WO 98/27995 PCT/US97/23611 -17- Endocrinology 106:577-587; Tollfsen et al. (1989) Proc. Nat. Acad. Sci. (USA) 1543-1547), platelet-derived growth factor (PDGF) (Yablonka-Reuveni et al. (1990) J. Cell Biol. 11:1623- 1629), leukemia-inhibiting factor (LIF) (Austin and Burgess (1991) J. Neuro. Sci. 101:193-197), adrenocorticotrophic hormone (ACTH) (Cossu et al. (1989) Develop. Biol 131:331-336; De Angelis et al. (1992) Dev. Biol. 151:446-458), melanocyte-stimulating factor (MSH) (Cossu et al.

(1989) Develop. Biol. 131:331-336) and granulocyte colony stimulating factor (G-CSF) (Austin and Burgess (1991) J. Neuro. Sci. 101:193-197). Thus, in order to promote proliferation of the myogenic precursors cells in vitro prior to implantation and/or in vivo after implantation, the cells may be grown in the presence of one or more of these factors, or other known mitogens. In addition, as is generally known in the art, proliferation of such cells may be promoted by repeated passaging treatment with dilute trypsin to remove adhered cells from the culture plate and replating at a lower density every 2-3 days), growth in liquid culture, growth in the absence of enhancers of cell adhesion, growth in the presence of inhibitors of cell adhesion, and/or growth at densities below confluence.

There is no absolute requirement that the myogenic precursor cells of the present invention be cultured in vitro prior to implantation. Indeed, if a therapeutically effective number of myogenic precursor cells can conveniently and economically be obtained without culturing, this step may be omitted. On the other hand, when such cells are in scarce supply from fetal tissues) or can be obtained only through invasive measures excision of substantial portions of muscle from a donor or donor/subject), it is preferred that smaller numbers of cells be obtained initially, and then proliferated in vitro. Doubling times will vary depending upon the source of cells, media, and the presence or absence of other growth factors, but doubling times on the order of every 12 hrs have been reported in the literature for muscle satellite cells grown in the presence of muscle abstract (Bischoff, (1989)). Therefore, it is contemplated that culturing times of several days to a week may be employed in the present methods to expand the myogenic precursor cell population prior to implantation.

Myogenic precursor cells may be harvested by brief trypsin treatment to remove any cells adhered to the culture plate or vessel, and centrifugation 10-15 min at 500-1000 The cells may then be resuspended in a physiologically acceptable buffer solution PBS, Ringer's saline) at an appropriate density 103-107 cells/ml).

WO 98/27995 PCT/US97/23611 18- Finally, it should be noted that morphogens, morphogen inducers, agonists of morphogen receptors, and small molecule morphogenic activators may be used to treat the myogenic precursor cells during culturing (if any) to aid in proliferation and/or subsequent differentiation.

Alternatively, the myogenic precursor cells may be treated with a morphogen, morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator either simultaneously with, or subsequent to, implantation. In the case of morphogen inducers, the myogenic precursor cells may be co-cultured with auxiliary cells which respond to these morphogen inducers by producing morphogen. The myogenic precursor cells then may be implanted along with these auxiliary cells, or may be isolated from the co-culture by standard cell separation techniques, which are known in the art, but which will vary with the type of auxiliary cells employed density centrifugation separation, cell type specific cytotoxins).

C. Implantation of Myogenic Precursor Cells at a Myocardial Site Myogenic precursor cells may be implanted at a site of loss of or damage to mammalian myocardium by any of a variety of surgical techniques known in the art. These techniques range from the minimally invasive injection by needle through the thoracic wall) to substantially invasive thoracotomy and incision of the myocardium, followed by implantation, suturing of the implant site and closing of the chest). The technique employed in any given instance will depend upon such factors as the size of the myocardial site to be treated, the accessibility of the site, and the age and stamina of the subject.

Generally, the myogenic precursor cells are implanted in a physiologically acceptable buffer solution. To minimize the volume of solution administered to the treatment site, the cells may be at a relatively high titer within this solution 105-10' cells/ml). The solution may contain growth factors, as described above, to promote further proliferation of the myogenic precursor cells within the implant site, or may be free of such factors so as to promote differentiation into new and functional myocardium in the morphogenically permissive environment of the myocardial implant site. In addition, as noted above, the myogenic precursor cells may be implanted either simultaneously with a morphogen, morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator, or the morphogenic treatment may be subsequent to implantation.

WO 98/27995 PCT/US97/23611 -19- Thus, for example, a solution of myogenic precursor cells and a morphogen, morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator, may be implanted at a site ofmyocardial infarction in essentially the following manner. For example, to treat a myocardial infarct to the anterior wall of the left ventricle, a left thoracotomy is performed on a subject under general anesthesia in an intercostal space the sixth intercostal space) and the site of the infarct is determined by observation. At the discretion of the surgeon, the heart may or may not be stopped and systemic blood flow shunted to a heart-lung machine. Myogenic precursor cells then may be directly injected into one or more sites within the infarct using an intravenous catheter a 16-gauge Teflon catheter from Criticon, Tampa, FL). The initial injection(s) may include a morphogen, morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator, or these may be included in one or more additional injections to the infarct site. Alternatively, a number of non-transmural incisions may be made at the site of the infarct to create "channels" parallel to the direction of the myocardial fibers. The suspension of myogenic precursor cells (with or without morphogen, morphogen inducer or morphogen receptor agonist) then may be introduced within these channels and the channels closed by suturing. Finally, the pericardium is sutured and chest wall are closed by standard surgical techniques (after restarting and returning systemic circulation to the heart from a heartlung machine, if employed).

The treatment of chronically deteriorating mammalian myocardium due to congestive heart failure or chronic myopathy), may be performed similarly except that the implantation sites are chosen to correspond to areas of generalized myocardial deterioration and, therefore, may be more diffuse.

The number of myogenic precursor cells implanted will vary according to the amount of myocardial tissue to be restored or regenerated. The volume of cells to be restored or regenerated may be ascertained by standard techniques of cardiac imaging. Generally, it is expected that on the order of approximately 104-105 myogenic precursor cells will be required to restore or regenerate 1 mg of myocardial tissue (see, Alameddine and Fardeau (1989)).

D. Morphogens. Inducers, Agonists. and Small Molecule Morphogenic Activators Morphogens useful in the present invention include eukaryotic proteins originally identified as osteogenic proteins (see U.S. Patent 5,011,691, incorporated herein by reference), WO 98/27995 PCT/US97/23611 such as the OP1, OP2, OP3, CBMP2A (BMP-2), CBMP-2B (BMP-4) and BMP3 proteins (SEQ ID NOs: 4-9, 15-22, 25-27), as well as amino acid sequence-related proteins such as DPP (SEQ ID NO: 10, from Drosophila), Vgl (SEQ ID NO: 11, from Xenopus), Vgrl (SEQ ID NO: 12, from mouse), GDFI (SEQ ID NOs: 13, 30 and 31, from humans, see Lee (1991), PNAS 88:4250-4254), 60A (SEQ ID NOs: 23 and 24, from Drosophila, see Wharton et al. (1991) PNAS 88:9214-9218), dorsalin-1 (from chick, see Basler et al. (1993) Cell 73:687-702 and GenBank accession number L12032) and GDF5 (from mouse, see Storm et al. (1994) Nature 368:639-643). Additional useful morphogens include biosynthetic morphogen constructs disclosed in U.S. Pat. No. 5,011,691, COPI, 3-5, 7 and 16, as well as others known in the art including dor3, NODAL, UNIVIN, BMP9, BMP10, GDF3, GDF6, GDF7, CDMP2, and SCREW. See also U.S. Pat. No. 4,968,590, incorporated herein by reference.

Naturally occurring proteins identified and/or appreciated herein to be morphogens form a distinct subgroup within the loose evolutionary grouping of sequence-related proteins known as the TGFP superfamily or supergene family. The naturally occurring morphogens share substantial amino acid sequence homology in their C-terminal regions (domains). Typically, the abovementioned naturally occurring morphogens are translated as a precursor, having an N-terminal signal peptide sequence, typically less than about 30 residues, followed by a "pro" domain that is cleaved to yield the mature C-terminal domain. The signal peptide is cleaved rapidly upon translation, at a cleavage site that can be predicted in a given sequence using the method of Von Heijne (1986) Nucleic Acids Research 14:4683-4691. The pro domain typically is about three times larger than the fully processed mature C-terminal domain. Herein, the "pro" form of a morphogen refers to a morphogen comprising a folded pair of polypeptides each comprising the pro and mature domains of a morphogen polypeptide. Typically, the pro form of a morphogen is more soluble than the mature form under physiological conditions. The pro form appears to be the primary form secreted from cultured mammalian host cells.

Table 1, below, summarizes various naturally occurring morphogens identified to date, including their nomenclature as used herein, their Sequence Listing references, and publication sources for the amino acid sequences for the full length proteins not included in the Sequence Listing. Each of the generic terms set forth in Table 1 is intended and should be understood to embrace morphogenically active proteins expressed from nucleic acids encoding the identified WO 98/27995 PCT/US97/23611 -21sequence mentioned below and set forth in the Sequence Listing, or a morphogenically active fragment or precursor thereof, including functional equivalents such as naturally occurring and biosynthetic variants thereof Naturally occurring variants include allelic variant forms isolated from other individuals of a single biological species, and phylogenetic counterpart (species) variant forms (homologues) isolated from phylogenetically distinct biological species. The disclosures of publications mentioned below are incorporated herein by reference.

TABLE 1 "OP I" Refers generically to morphogenically active proteins expressed from nucleic acids encoding OP1 proteins, including at least the human OP1 protein disclosed in SEQ ID NO: 4 and the mouse OPI protein disclosed in SEQ ID NO: In each of human and mouse OP1 proteins, the conserved seven cysteine skeleton is defined by residues 38 to 139. cDNA sequences and amino acid sequences encoded therein and corresponding to the full length proteins are provided in SEQ ID NOs: 15 and 16 (hOP1) and SEQ ID NOs: 17 and 18 (mOP- The mature proteins are defined by residues 293-431 (hOPI) and 292-430 (mOP1). The "pro" regions of the proteins, cleaved to yield the mature, morphogenically active proteins are defined essentially by residues 30-292 (hOP1) and residues 30-291 (mOP1).

"OP2" Refers generically to morphogenically active proteins expressed from nucleic acids encoding the OP2 proteins, including at least the human OP2 protein disclosed in SEQ ID NO: 6 and the mouse OP2 protein disclosed in SEQ ID NO: 7 In each of human and mouse OP2 proteins, the conserved seven cysteine skeleton is defined by residues 38 to 139 of SEQ ID NOs: 6 and 7. cDNA sequences and amino acid sequences encoded therein and corresponding to the full length proteins are provided in SEQ ID NOs: 19 and 20 (hOP2) and SEQ ID NOs: 21 and 22 (mOP2.) The mature proteins are defined essentially by residues 264- 402 (hOP2) and 261-399 (mOP2). The "pro" regions of the proteins, cleaved to yield the mature, morphogenically active proteins are defined essentially by residues 18-263 (hOP2) and residues 18-260 (mOPI).

WO 98/27995 PCT/US97/23611 22 "OP3" Refers generically to morphogenically active proteins expressed from nucleic acids encoding OP3 proteins, including at least the mouse OP3 protein disclosed in SEQ ID NO: 26 The conserved seven cysteine domain is defined by residues 298 to 399 of SEQ ID NO: 26, which shares greater than 79% amino acid identity with the corresponding mOP2 and hOP2 sequences, and greater than 66% identity with the corresponding OPI sequences. A cDNA sequence encoding the abovementioned amino acid sequence is provided in SEQ ID NO: 25. OP3 is unique among the morphogens identified to date in that the residue at position 9 in the conserved seven cysteine domain residue 315 of SEQ ID NO: 26) is a serine, whereas other morphogens typically have a tryptophan at this location.

"CBMP2" Refers generically to morphogenically active proteins expressed from nucleic acids encoding the CBMP2 proteins, including at least the human CBMP2A protein disclosed in SEQ ID NO: 8 (hCBMP2A) and the human CBMP2B protein disclosed in SEQ ID NO: 9 (hCBMP2B). The amino acid sequence for the full length proteins, referred to in the literature as BMP2A and BMP2B, or BMP2 and BMP4, appear in Wozney, et al. (1988) Science 242:1528-1534. The pro domain for BMP2 (BMP2A) likely includes residues 25-248 of the published sequence; the mature protein, residues 249-396. The pro domain for BMP4 (BMP2B) likely includes residues 25-256 of the published sequence; the mature protein, residues 257-408.

"DPP" Refers generically to proteins encoded by the Drosophila DPP gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO: 10. The amino acid sequence for the full length protein appears in Padgett, et al. (1987) Nature 325:81-84. The pro domain likely extends from the signal peptide cleavage site to residue 456 of the published sequence; the mature protein likely is defined by residues 457-588.

"Vgl" Refers generically to proteins encoded by the Xenopus Vgl gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO: 11. The amino acid sequence for the full length protein appears in Weeks (1987) Cell 51:861-867. The WO 98/27995 "Vgrl" "GDF1" "60A" PCT/US97/23611 -23prodomain likely extends from the signal peptide cleavage site to residue 246 of the published sequence; the mature protein likely is defined by residues 247-360.

Refers generically to proteins encoded by the murine Vgrl gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO: 12. The amino acid sequence for the full length protein appears in Lyons, et al. (1989) PNAS 86:4554- 4558. The prodomain likely extends from the signal peptide cleavage site to residue 299 of the published sequence; the mature protein likely is defined by residues 300-438.

Refers generically to proteins encoded by the human GDF1 gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO: 13. The cDNA and encoded amino sequence for the full length protein are provided in SEQ ID NOs: 30 and 31. The prodomain likely extends from the signal peptide cleavage site to residue 214; the mature protein likely is defined by residues 215- 372.

Refers generically to morphogenically active proteins expressed from nucleic acid encoding 60A proteins or morphogenically active fragments thereof, including at least the Drosophila 60A protein disclosed in SEQ ID NO: 24. A Drosophila cDNA is disclosed in SEQ ID NO: 23. The prodomain likely extends from the signal peptide cleavage site to residue 324; the mature protein likely is defined by residues 325-455. The active fragment of 60A protein likely is defined by the conserved seven cysteine skeleton of residues 354 to 455 of SEQ ID NO: 24. The protein is considered likely herein to be a phylogenetic counterpart variant of the human and mouse OP1 genes; Sampath, et al. (1993) PNAS 90:6004-6008.

Refers generically to proteins encoded by the human BMP3 gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO: 27. The amino acid sequence for the full length protein appears in Wozney, et al. (1988) Science 242:1528-1534. The pro domain likely extends from the signal peptide cleavage "BMP3" WO 98/27995 PCT/US97/23611 -24site to residue 290 of the published sequence; the mature protein likely is defined by residues 291-472.

Refers generically to proteins encoded by the human BMP5 gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO: 28. The amino acid sequence for the full length protein appears in Celeste, et al. (1991) PNAS 87:9843-9847. The pro domain likely extends from the signal peptide cleavage site to residue 316 of the published sequence; the mature protein likely is defined by residues 317-454.

"BMP6" Refers generically to proteins encoded by the human BMP6 gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO: 29. The amino acid sequence for the full length protein appears in Celeste, et al. (1990) PNAS 87:9843-5847. The pro domain likely extends from the signal peptide cleavage site to residue 374 of the published sequence; the mature protein likely is defined by residues 375-513.

As shown in Figure 1, the OP2 and OP3 proteins have an additional cysteine residue in the conserved C-terminal region see residue 41 of SEQ ID NOs: 6 and in addition to the conserved cysteine skeleton or domain in common with the other known proteins in this family.

The GDFI protein has a four amino acid insert within the conserved skeleton (residues 44-47 of SEQ ID NO: 13) but this insert likely does not interfere with the relationship of the cysteines in the folded structure. Further, the CBMP2 proteins are missing one amino acid residue within the cysteine skeleton. Thus, these morphogen polypeptides illustrate the principles of alignment used herein with respect to the preferred reference morphogen sequence of human OP 1, residues 38- 139 of SEQ ID NO: 4.

In certain preferred embodiments, morphogens useful herein include those in which the amino acid sequences of morphogen polypeptides comprise a sequence sharing at least amino acid sequence homology or "similarity", and preferably 80% homology or similarity with a reference morphogen sequence selected from the foregoing sequences or naturally occurring morphogens. Preferably, the reference morphogen is human OP1, and the reference sequence thereof is the C-terminal seven cysteine domain present in morphogenically active forms of human WO 98/27995 WO 98/27995 PCT/US97/23611 OP1, residues 38-139 of SEQ ID NO: 4. Morphogens useful herein accordingly include alleles, phylogenetic counterparts and other variants of the preferred reference sequence, whether naturally-occurring or biosynthetically produced including "muteins" or "mutant proteins"), as well as novel members of the morphogenic family of proteins including the morphogens set forth and identified above, in connection with Table 1. Certain particularly preferred morphogen polypeptides share at least 60% amino acid identity with the preferred reference sequence of human OP 1, still more preferably at least 65% amino acid identity therewith.

In other preferred embodiments, the family of morphogen polypeptides useful in the present invention, and members thereof, are defined by a generic amino acid sequence. For example, Generic Sequence 7 (SEQ ID NO: 1) and Generic Sequence 8 (SEQ ID NO: 2) disclosed below, accommodate the homologies shared among preferred morphogen protein family members identified to date, including at least OPI, OP2, OP3, CBMP2A, CBMP2B, BMP3, BMPS, BMP6, DPP, Vgl, Vgrl, 60A, and GDF1. The amino acid sequences for these proteins are described herein (see Sequence Listing) and/or in the art, as summarized above. The generic sequences include both the amino acid identity shared by these sequences in the C-terminal domain, defined by the six and seven cysteine skeletons (Generic Sequences 7 and 8, respectively), as well as alternative residues for the variable positions within the sequence. The generic sequences provide an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and contain certain critical amino acids likely to influence the tertiary structure of the folded proteins. In addition, the generic sequences allow for an additional cysteine at position 41 (Generic Sequence 7) or position 46 (Generic Sequence thereby encompassing the morphogenically active sequences of OP2 and OP3.

Generic Sequence 7 (SEQ ID NO: 1) Leu Xaa Xaa Xaa Phe Xaa Xaa 1 Xaa Gly Trp Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa WO 98/27995 PCT/US97/23611 -26- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys Xaa wherein each Xaa independently is selected from a group of one or more specified amino acids defined as follows: "res." means "residue" and Xaa at res. 2 (Tyr or Lys); Xaa at res. 3 Val or Ile); Xaa at res. 4 (Ser, Asp or Glu); Xaa at res. 6 (Arg, Gin, Ser, Lys or Ala); Xaa at res. 7 (Asp or Glu); Xaa at res. 8 (Leu, Val or Ile); Xaa at res. 11 (Gln, Leu, Asp, His, Asn or Ser); Xaa at res. 12 (Asp, Arg, Asn or Glu); Xaa at res. 13 (Trp or Ser); Xaa at res. 14 (Ile or Val); Xaa at res. 15 (Ile or Val); Xaa at res. 16 (Ala or Ser); Xaa at res. 18 (Glu, Gin, Leu, Lys, Pro or Arg); Xaa at res. 19 (Gly or Ser); Xaa at res. 20 (Tyr or Phe); Xaa at res. 21 (Ala, Ser, Asp, Met, His, Gin, Leu or Gly); Xaa at res. 23 (Tyr, Asn or Phe); Xaa at res. 26 (Glu, His, Tyr, Asp, Gin, Ala or Ser); Xaa at res. 28 (Glu, Lys, Asp, Gin or Ala); Xaa at res. (Ala, Ser, Pro, Gin, Ile or Asn); Xaa at res. 31 (Phe, Leu or Tyr); Xaa at res. 33 (Leu, Val or Met); Xaa at res. 34 (Asn, Asp, Ala, Thr or Pro); Xaa at res. 35 (Ser, Asp, Glu, Leu, Ala or Lys); Xaa at res. 36 (Tyr, Cys, His, Ser or Ile); Xaa at res. 37 (Met, Phe, Gly or Leu); Xaa at res. 38 (Asn, Ser or Lys); Xaa at res. 39 (Ala, Ser, Gly or Pro); Xaa at res. 40 (Thr, Leu or Ser); Xaa at res. 44 (Ile, Val or Thr); Xaa at res. 45 (Val, Leu, Met or Ile); Xaa at res. 46 (Gin or Arg); Xaa at res. 47 (Thr, Ala or Ser); Xaa at res. 48 (Leu or Ile); Xaa at res. 49 (Val or Met); Xaa at res. 50 (His, Asn or Arg); Xaa at res. 51 (Phe, Leu, Asn, Ser, Ala or Val); Xaa at res. 52 (Ile, Met, Asn, Ala, Val, Gly or Leu); Xaa at res. 53 (Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res. 54 (Pro, Ser or Val); Xaa at res. 55 (Glu, Asp, Asn, Gly, Val, Pro or Lys); Xaa at res. 56 (Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, Ile or His); Xaa at res. 57 (Val, Ala or lie); Xaa at res. 58 (Pro or Asp); Xaa at res. 59 (Lys, Leu or Glu); Xaa at res. 60 (Pro, Val or Ala); Xaa at res. 63 (Ala or Val); Xaa at res. 65 (Thr, Ala or Glu); Xaa at res. 66 (Gln, Lys, Arg or Glu); Xaa at res. 67 (Leu, Met or Val); Xaa at res. 68 WO 98/27995 PCT/US97/23611 -27- (Asn, Ser, Asp or Gly); Xaa at res. 69 (Ala, Pro or Ser); Xaa at res. 70 (Ile, Thr, Val or Leu); Xaa at res. 71 (Ser, Ala or Pro); Xaa at res. 72 (Val, Leu, Met or Ile); Xaa at res. 74 (Tyr or Phe); Xaa at res. 75 (Phe, Tyr, Leu or His); Xaa at res. 76 (Asp, Asn or Leu); Xaa at res. 77 (Asp, Glu, Asn, Arg or Ser); Xaa at res. 78 (Ser, Gin, Asn, Tyr or Asp); Xaa at res. 79 (Ser, Asn, Asp, Glu or Lys); Xaa at res. 80 (Asn, Thr or Lys); Xaa at res. 82 (Ile, Val or Asn); Xaa at res. 84 (Lys or Arg); Xaa at res. 85 (Lys, Asn, Gin, His, Arg or Val); Xaa at res. 86 (Tyr, Glu or His); Xaa at res. 87 (Arg, Gin, Glu or Pro); Xaa at res. 88 (Asn, Glu, Trp or Asp); Xaa at res. 90 (Val, Thr, Ala or lie); Xaa at res. 92 (Arg, Lys, Val, Asp, Gin or Glu); Xaa at res. 93 (Ala, Gly, Glu or Ser); Xaa at res. 95 (Gly or Ala) and Xaa at res. 97 (His or Arg).

Generic Sequence 8 (SEQ ID NO: 2) includes all of Generic Sequence 7 and in addition includes the following sequence (SEQ ID NO: 14) at its N-terminus: Cys Xaa Xaa Xaa Xaa 1 Accordingly, beginning with residue 7, each "Xaa" in Generic Sequence 8 is a specified amino acid defined as for Generic Sequence 7, with the distinction that each residue number described for Generic Sequence 7 is shifted by five in Generic Sequence 8. Thus, "Xaa at res. 2 =(Tyr or Lys)" in Generic Sequence 7 refers to Xaa at res. 7 in Generic Sequence 8. In Generic Sequence 8, Xaa at res. 2 (Lys, Arg, Ala or Gin); Xaa at res. 3 (Lys, Arg or Met); Xaa at res. 4 (His, Arg or Gin); and Xaa at res. 5 (Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr).

As noted above, certain currently preferred morphogen polypeptide sequences useful in this invention have greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the conserved six or seven cysteine skeleton ofhOPI residues 43-139 or 38-139 of SEQ ID NO: These particularly preferred sequences include allelic and phylogenetic counterpart variants of the OP1 and OP2 proteins, including the Drosophila protein (SEQ ID NO: 24). Accordingly, in certain particularly preferred embodiments, useful morphogens include active proteins comprising pairs of polypeptide chains within the generic WO 98/27995 PCT/US97/23611 -28amino acid sequence herein referred to as "OPX" (SEQ ID NO: which corresponds to the seven cysteine skeleton and accommodates the homologies between several identified variants of OP1 and OP2. As described therein, each Xaa at a given position independently is selected from the residues occurring at the corresponding position in the C-terminal sequence of mouse or human OP 1 or OP2 (see SEQ ID NOs: 4-7 and/or SEQ ID NOs: 15-22).

In still other preferred embodiments, useful morphogen polypeptides have amino acid sequences comprising a sequence encoded by a nucleic acid that hybridizes, under stringent hybridization conditions, to DNA or RNA encoding reference morphogen sequences,

C-

terminal sequences defining the conserved seven cysteine domains ofOPi or OP2, e.g., nucleotides 1036-1341 and nucleotides 1390-1695 of SEQ ID NO: 15 and 19, respectively. As used herein, stringent hybridization conditions are defined as hybridization according to known techniques in 40% formamide, 5 X SSPE, 5 X Denhardt's Solution, and 0.1% SDS at 37 0

C

overnight, and washing in 0.1 X SSPE, 0.1% SDS at 50 0

C.

As noted above, morphogens useful in the present invention generally are dimeric proteins comprising a folded pair of the above polypeptides. Morphogens are inactive when reduced, but are active as oxidized homodimers and when oxidized in combination with other morphogens of this invention to produce heterodimers. Thus, members of a folded pair of morphogen polypeptides in a morphogenically active protein can be selected independently from any of the specific morphogen polypeptides mentioned above.

The morphogens useful in the methods, compositions and devices of this invention include proteins comprising any of the polypeptide chains described above, whether isolated from naturally-occurring sources, or produced by recombinant DNA or other synthetic techniques, and includes allelic and phylogenetic counterpart variants of these proteins, as well as biosynthetic variants (muteins) thereof, and various truncated and fusion constructs. Deletion or addition mutants also are envisioned to be active, including those which may alter the conserved Cterminal six or seven cysteine domain, provided that the alteration does not functionally disrupt the relationship of these cysteines in the folded, biologically active, structure. Accordingly, such active forms are considered the equivalent of the specifically described constructs disclosed herein. The proteins may include forms having varying glycosylation patterns, varying N-termini, a family of related proteins having regions of amino acid sequence homology, and active truncated WO 98/27995 PCT/US97/23611 -29or mutated forms of native or biosynthetic proteins, produced by expression of recombinant

DNA

in host cells.

The morphogenic proteins can be expressed from intact or truncated cDNA or from synthetic DNAs in prokaryotic or eukaryotic host cells, and purified, cleaved, refolded, and dimerized to form morphogenically active compositions. Currently preferred host cells include E.

coli or mammalian cells, such as CHO, COS or BSC cells. A detailed description of the morphogens useful in the methods, compositions and devices of this invention is disclosed in published application W092/15323, the disclosure of which is incorporated by reference herein.

Thus, in view of this disclosure, skilled genetic engineers can isolate genes from cDNA or genomic libraries of various different biological species, which encode appropriate amino acid sequences, or construct DNAs from oligonucleotides, and then can express them in various types of host cells, including both prokaryotes and eukaryotes, to produce large quantities of active proteins capable of stimulating the morphogenesis of, and/or inhibiting damage or loss of, mammalian myocardial tissue.

As noted above, a protein is morphogenic herein generally if it induces the developmental cascade of cellular and molecular events that culminate in the formation of new, organ-specific tissue. Preferably, a morphogen comprises a pair of polypeptides having a sequence that corresponds to or is functionally equivalent to at least the conserved C-terminal six or seven cysteine skeleton of human OP1, included in SEQ ID NO: 4. The morphogens generally are competent to induce a cascade of events including all of the following, in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells. Details of how the morphogens useful in this invention first were identified, as well as a description on how to make, use and test them for morphogenic activity are disclosed in published application W092/15323. As disclosed therein, the morphogens can be purified from naturally-sourced material or recombinantly produced from prokaryotic or eukaryotic host cells, using the genetic sequences disclosed therein.

Alternatively, novel morphogenic sequences can be identified following the procedures disclosed therein.

WO 98/27995 PCT/US97/23611 Exemplary useful morphogens include naturally derived proteins comprising a pair of polypeptides, the amino acid sequences of which comprise sequences selected from those disclosed in the Sequence Listing and Figure 1. Other useful sequences include those of the naturally derived morphogens dorsalin-1, SCREW, NODAL, UNIVIN and GDF5, discussed herein in connection with Table 1, as well as biosynthetic constructs disclosed in U.S. Pat.

5,011,691, the disclosure of which is incorporated herein by reference COP COP3, COP4, COP7, and COP16).

Accordingly, certain preferred morphogens useful in the methods and compositions of this invention can be described as morphogenically active proteins having amino acid sequences sharing 70% or, preferably, 80% homology with a reference morphogen sequence described above, residues 38-139 ofSEQ ID NO: 4, where "homology" is as defined herein above.

Alternatively, in other preferred embodiments, morphogens useful in the methods and compositions disclosed herein fall within the family of polypeptides described by Generic Sequence 7, SEQ ID NO: 1, more preferably by Generic Sequence 8, SEQ ID NO: 2.

Figure 1 herein sets forth an alignment of the amino acid sequences of the active regions of exemplary naturally occurring proteins that have been identified or appreciated herein as morphogens, including human OP1 (hOP1, SEQ ID NOs: 4 and 15-16), mouse OP1 (mOP1, SEQ ID NOs: 5 and 17-18), human and mouse OP2 (SEQ ID NOs: 6, 7, and 19-22), mouse OP3 (SEQ ID NOs: 25-26), CBMP2A (SEQ ID NO: CBMP2B (SEQ ID NO: BMP3 (SEQ ID NO: 27), DPP (from Drosophila, SEQ ID NO: 10), Vgl, (from Xenopus, SEQ ID NO: 11), Vgrl (from mouse, SEQ ID NO: 12), GDF1 (from mouse and/or human, SEQ ID NOs: 13, 30 and 31), protein (from Drosophila, SEQ ID NOs: 23 and 24), BMP5 (SEQ ID NO: 28) and BMP6 (SEQ ID NO: 29). The sequences are aligned essentially following the method of Needleman, et al. (1970) J. Mol. Biol., 48:443-453, calculated using the Align Program (DNAstar, Inc.). In Figure 1, three dots indicates that the amino acid in that position is the same as the corresponding amino acid in hOP 1. Three dashes indicates that no amino acid is present in that position, and are included for purposes of illustrating homologies. For example, amino acid residue 60 is "missing" in both CBMP2A and CBMP2B. Of course, both of these amino acid sequences in this region comprise Asn-Ser (residues 58, 59), with CBMP2A then comprising Lys and Ile, whereas CBMP- 2B comprises Ser and Ile. Figure 1 also illustrates the handling of insertions in the morphogen WO 98/27995 PCT/US97/23611 -31amino acid sequence: between residues 56 and 57 of BMP3 is an inserted Val residue; between residues 43 and 44 of GDF1 is inserted the amino acid sequence, Gly-Gly-Pro-Pro. Such deviations from the reference morphogen sequence are ignored for purposes of calculating the defined relationship between, GDFI and hOP1. As is apparent from the amino acid sequence comparisons set forth in Figure 1, significant amino acid changes can be made from the reference sequence while retaining morphogenic activity. For example, while the GDF1 protein sequence depicted in Figure 1 shares only about 50% amino acid identity with the hOP1 sequence described therein, the GDFI sequence shares greater than 70% amino acid sequence homology with the hOP 1 sequence, where "homology" is as defined above.

In other embodiments, as an alternative to the administration of a morphogenic protein, an effective amount of an agent competent to stimulate or induce increased endogenous morphogen expression in a mammal may be administered by any of the routes described herein. Such an inducer of a morphogen may be provided to a mammal, by local or systemic administration to the mammal or by direct administration to implanted myogenic precursor cells, or may be provided to auxiliary cells co-cultured with myogenic precursor cells. Methods for identifying and testing inducers (stimulating agents) competent to modulate the level of production of morphogens by a given tissue or cell type are described in detail in published applications W093/05172 and W093/05751, the teachings of which are incorporated herein by reference.

Briefly, candidate compounds can be identified and tested by incubation in vitro with a test tissue or cells thereof, or a cultured cell line derived therefrom, for a time sufficient to allow the compound to affect the production, the expression and/or secretion, of a morphogen produced by the cells of that tissue. Suitable tissue, or cultured cells of a suitable tissue, preferably can be selected from renal epithelium, ovarian tissue, fibroblasts, and osteoblasts.

In other embodiments, an agent which acts as an agonist of a morphogen receptor may be administered instead of the morphogen itself. Such an agent may also be referred to an a morphogen "mimic," "mimetic," or "analog." Thus, for example, a small peptide or other molecule which can mimic the activity of a morphogen in binding to and activating the morphogen's receptor may be employed as an equivalent of the morphogen. Preferably the agonist is a full agonist, but partial morphogen receptor agonists may also be advantageously employed. Methods of identifying such agonists are known in the art and include assays for WO 98/27995 PCT/US97/23611 -32compounds which induce morphogen-mediated responses induction of differentiation of metanephric mesenchyme, induction ofendochondral bone formation). For example, methods of identifying morphogen inducers or agonists of morphogen receptors may be found in U.S. Ser.

No. 08/478,097 filed June 7, 1995 and U.S. Ser. No. 08/507,598 filed July 26, 1995, the disclosures of which are incorporated herein by reference.

In yet other embodiments, a small molecule morphogenic activator may be used for promoting the migration, proliferation, and/or differentiation of myogenic precursor cells by increasing the level of expression of proteins associated with myocardial phenotype. Exemplary methods comprise introducing a small molecule morphogenic activator that regulates some portion or portions ofa morphogen-induced regulatory pathway, resulting in an effective increase in expression or activity ofmyocardium-specific protein. This may result either from stimulating an increase in the endogenous expression of such protein or from a decrease in the inhibition of normal expression of such protein. For example, a small molecule morphogenic activator may act at the type I or type II morphogen receptor; or at the serine/threonine kinase, or other kinase domains of those receptors. Another target of pathway activation is the Smad proteins, including the monomeric, dimeric (including heteromeric and homomeric complexes) or trimeric forms (including heteromeric and homomeric complexes). The Smads have been characterized, and are known in the art. See, Baker, et al., Curr. Op. Genet. Develop., 7: 467-473 (1997), incorporated by reference herein.

Alternately, a small molecule morphogenic activator may lead to activation of a transcription factor (for example, the X-protein shown in Figure 2) that causes phenotype-specific gene expression expression of protein characteristic ofmyocardium). A small molecule morphogenic activator may act to facilitate, mimic, or, if desired, prevent any one or several of the following: type I and/or type II receptor binding, phosphorylation of the type I receptor, phosphorylation of the Smad molecules, Smad complex formation, Smad translocation into the nucleus, nuclear accumulation of the Smad complex, or transcription modulation of the Smad complex. Furthermore, a small molecule morphogenic activator may act on Smads or Smad complexes to alter tertiary structure, thereby to facilitate or inhibit interaction of the Smad or Smad complex with a receptor kinase domain, other Smads, DNA binding proteins, or DNA itself.

WO 98/27995 PCT/US97/23611 -33 In a particularly-preferred embodiment, a small molecule morphogenic activator is contacted with myogenic precursor cells in vivo or in vitro, or is administered to a patient, wherein the small molecule morphogenic activator facilitates formation of Smad complexes, particularly complexes comprising molecules of Smad1, Smad2, Smad3, Smad4, Smad5 and/or Smad8 in order to induce myogenic precursor cells to migrate, proliferate and/or differentiate into cells expressing markers of a myocardial tissue phenotype. Also in a preferred embodiment, methods comprise administering a small molecule morphogenic activator composition that activates a serine/threonine kinase domain associated with a morphogen type I or type II receptor, thereby to activate the pathway involved in morphogen-induced gene expression. In another embodiment, methods of the invention comprise activating Smad4 association with Smadi, thereby to induce morphogen-responsive phenotype. Methods of the invention may also facilitate Smad interaction with specific nucleic acids, such as promoters of myocardial tissue phenotypespecific gene expression expression of genes for a phenotypic protein; a protein associated with preservation, restoration, or enhancement of phenotype, including a protein which is critical for production of non-protein phenotypic markers, such as characteristic lipids or carbohydrates; a protein associated with performance of a phenotypic function or morphology; or a morphogen).

Such interaction may be, for example, in association with a transcription control factor that is capable of binding to a regulatory portion of a gene and, simultaneously, to one or more regulatory proteins such as a Smad complex (See Figure 2).

An exemplary morphogen-activated pathway is shown in Figure 2. Morphogens are ligands for the type I and type II receptors. Following phosphorylation of the type I receptor by the type-II receptor, the type I receptor specifically phosphorylates Smadl homodimers. The type I receptor also specifically phosphorylates Smad5 homodimers. The homodimers then separate to form, in association with a phosphorylated Smad4 molecule, a phosphorylated heteromeric complex comprising at least a Smadl and a Smad4. A phosphorylated Smadl/Smad5/Smad4 heterotrimer may alternatively be formed. The heteromeric complex then translocates into the nucleus, and accumulates therein. In the nucleus, the Smad complex binds operative DNA, either alone or in association with a specific DNA binding protein (the X-protein in Figure to initiate DNA transcription. The "X-protein" acts as a DNA-binding protein, binding the Smad heteromeric complex to the DNA. The Smad Smad2, Smad3 and Smad5 proteins consist of WO 98/27995 WO 98/27995 PCT/US97/23611 -34conserved amino- and carboxy-terminal domains linked by a region that is more divergent among the Smads. The carboxy-terminal domain has an effector function. The amino-terminal domain interacts physically with the carboxy-terminal domain, inhibiting its effector activity, and contributes to DNA binding. Receptor-mediated phosphorylation of the serine residues at the end of the carboxy-terminal domain relieves the carboxy-terminal domain from the inhibitory action of the amino-terminal domain. Phosphorylated Smad molecules form a heteromeric complex with at least'one other specific Smad family molecule. The resulting Smad complex then translocates into and accumulates in the cell nucleus. There, the heteromeric Smad complexes regulate transcriptional responses either alone or by specific interaction with a DNA-binding protein, such as forkhead activin signal transducer-1 (FAST I).

Other intracellular pathways are induced by morphogens, and may be affected in the manner described herein by use of a small molecule morphogenic activator.

In a preferred embodiment, a small molecule morphogenic activator for use in the invention is a compound that affects one or more intracellular pathways that normally are under morphogen regulation. Such small molecule morphogenic activators preferably have the ability to enter the cell and target one or more intracellular pathway components in order to stimulate or inhibit their activity. For example, a small molecule morphogenic activator that promotes Smad complex formation between Smadl, Smad4, and Smad5 will stimulate pathways leading to expression of genes encoding phenotype-specific proteins.

One way in which to identify a candidate small molecule morphogenic activator is to assay for the ability of the candidate to modulate the effective systemic or local concentration of a morphogen. This may be done, for example, by incubating the candidate in a cell culture that produces the morphogen, and assaying the culture for a parameter indicative of a change in the production level of the morphogen according the methods of U.S.S.N. 08/451,953 and/or U.S.

5,650,276, the teachings of each of which are incorporated by reference herein. Alternatively, candidate compounds are screened for their ability to induce phenotype-specific protein production in a cell culture in which morphogen activity is not present. Examples of compositions which may be screened for their effect on the production of morphogens or other phenotype-specific proteins include but are not limited to chemicals, biological response modifiers lymphokines, cytokines, hormones, or vitamins), plant extracts, microbial broths and WO 98/27995 PCT/US97/23611 extracts medium conditioned by eukaryotic cells, body fluids, or tissue extracts. Useful candidate compositions then may be tested for in vivo efficacy in a suitable animal model. These compositions then may be used in vivo to up-regulate morphogen-activated regulatory pathways ofphenotype-specific protein expression.

A simple method of determining if a small molecule composition has effected a change in the level of a phenotype-specific protein in cultured cells is provided in co-owned, co-pending patent application, U.S.S.N. 08/451,953, the disclosure of which is incorporated by reference herein. The level of a target phenotype-specific protein in a cell resulting from exposure to a small molecule is measured. Alternatively, a change in the activity or amount of an intracellular pathway component is measured in response to application of a candidate small molecule.

Candidates having the desired affect on protein production or pathway regulation are selected for use in methods of the invention. If, for example, a composition up-regulates the production of OP-1 by a kidney cell line, it would then be desirable to test systemic administration of this compound in an animal model to determine if it up-regulates the production of OP-I in vivo.

The level of morphogen in the body may be a result of a wide range of physical conditions, e.g., tissue degeneration such as occurs in diseases including arthritis, emphysema, osteoporosis, kidney diseases, lung diseases, cardiomyopathy, and cirrhosis of the liver. The decrease in level of morphogens in the body may also occur as a result of the normal process of aging. The same strategy is used for compositions affecting intracellular pathway components. A composition selected by these screening methods is then used as a treatment or prophylactic.

An appropriate test cell is any cell comprising DNA defining a morphogen-responsive transcription activating element operatively associated with a reporter gene encoding a detectable phenotype-specific gene product. Such DNA can occur naturally in a test cell or can be a transfected DNA. The induced intracellular effect typically is characteristic of morphogenic biological activity, such as Smad activation, or activation of a cascade of biochemical events, such as described above, or involving, for example, cyclic nucleotides, diacylglycerol, and/or and other indicators of intracellular signal transduction such as activation or suppression of gene expression, including induction of mRNA resulting from gene transcription and/or induction of protein synthesis resulting from translation of mRNA transcripts indicative of tissue morphogenesis.

Exemplary morphogen-responsive cells are preferably of mammalian origin and include, but are WO 98/27995 WO 98/27995 PCT/US97/23611 -36not limited to, osteogenic progenitor cells; calvaria-derived cells; osteoblasts; osteoclasts; osteosarcoma cells and cells of hepatic or neural origin. Any such morphogen responsive cell can be a suitable test cell for assessing whether a candidate substance is a small molecule morphogenic activator.

A preferred identification method is carried out by exposing a test cell to at least one candidate substance, and detecting whether such exposure induces expression of the detectable phenotype-specific gene product that is in operative association with the morphogen-responsive transcription activating element. Expression of this gene product indicates that the candidate substance induces a morphogen-mediated biological effect. Skilled artisans can, in light of guidance provided herein, construct a test cell with a responsive element from a morphogenresponsive cell and a reporter gene of choice, using recombinant vectors and transfection techniques well-known in the art. There are numerous well-known reporter genes useful herein.

These include, for example, chloramphenicol acetyltransferase (CAT), luciferase, human growth hormone (hGH), beta-galactosidase, and assay systems and reagents which are available through commercial sources. As will be appreciated by skilled artisans, the listed reporter genes represent only a few of the possible reporter genes that can be used herein. Examples of such reporter genes can be found in Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley Sons, New York, (1989). Broadly, any gene that encodes a detectable product, any product having detectable enzymatic activity or against which a specific antibody can be raised, can be used as a reporter gene in the present identification method.

A currently preferred reporter gene system is the firefly luciferase reporter system. Gould, et al., Anal. Biochem., 7:404-408 (1988), incorporated herein by reference. The luciferase assay is fast and sensitive. In this assay system, a lysate of the test cell is prepared and combined with ATP and the substrate luciferin. The encoded enzyme luciferase catalyzes a rapid, ATPdependent oxidation of the substrate to generate a light-emitting product. The total light output is measured and is proportional to the amount of luciferase present over a wide range of enzyme concentrations. CAT is another frequently used reporter gene system; a major advantage of this system is that it has been an extensively validated and is widely accepted as a measure of promoter activity. Gorman et al., Mol. Cell. Biol., 2:1044-1051 (1982), incorporated by reference herein. In this system, test cells are transfected with CAT expression vectors and WO 98/27995 WO 98/27995 PCT/US97/23611 -37incubated with the candidate substance within 2-3 days of the initial transfection. Thereafter, cell extracts are prepared. The extracts are incubated with acetyl CoA and radioactive chloramphenicol. Following the incubation, acetylated chloramphenicol is separated from nonacetylated form by thin layer chromatography. In this assay, the degree of acetylation reflects the CAT gene activity with the particular promoter.

Another suitable reporter gene system is based on immunologic detection ofhGH. This system is also quick and easy to use. Selden, et al., Mol. Cell. Biol., 6:3173-3179 (1986), incorporated by reference herein. The hGH system is advantageous in that the expressed hGH polypeptide is assayed in the media, rather than in a cell extract. Thus, this system does not require the destruction of the test cells. It will be appreciated that the principle of this reporter gene system is not limited to hGH but rather adapted for use with any polypeptide for which an antibody of acceptable specificity is available or can be prepared.

A small molecule morphogenic activator composition may up-regulate a morphogenactivated pathway by acting at any one or more point. For example, small molecule morphogenic activator potentiation of the pathway may be initiated at the receptor level. Depending on the pathway, the transmembrane receptors may be type I and/or type II, or may be comprise variations on either type I or type II receptors. For example, OP-I is capable of activating regulatory pathways comprising at least two variations of both type I and type II receptors (ActR- 1 and BMPR-1B, and ActRII and BMPR-II, respectively). A small molecule morphogenic activator may stimulate the pathway by acting as a ligand and binding to any of the receptors, thereby inducing phosphorylation of type I receptors and/or Smad molecules. Similarly, a small molecule morphogenic activator may activate the regulatory pathway at the level of the serine/threonine kinase domain of the receptors, thereby stimulating phosphorylation of type I receptors and/or Smad molecules.

As a further alternative, a small molecule morphogenic activator may activate the regulatory pathway at the level of Smad complex formation. A small molecule morphogenic activator may stimulate the formation of Smad family homodimers, heterodimers, or other homomeric or heteromeric complexes. Furthermore, a small molecule morphogenic activator may activate the pathway by interacting with a Smad molecule or Smad complex, thereby altering its tertiary structure.

WO 98/27995 PCT/US97/23611 -38- Alternatively, or in addition, a small molecule morphogenic activator may activate the regulatory pathway by facilitating translocation of a Smad molecule or Smad complex or accumulation of the Smad molecule or Smad complex within the nucleus of the cell. By acting as a DNA binding protein or a transcriptional activator, a small molecule morphogenic activator may activate the regulatory pathway by increasing transcriptional activity caused by the Smad molecule or Smad complex.

Furthermore, a small molecule morphogenic activator can act to stimulate the regulatory pathway by interfering with an inhibitor of the pathway. For example, Smad6 and Smad7, which are structurally different than Smadi, Smad2, Smad3 and Smad5, act as inhibitors of certain types of desirable phenotype-specific protein expression by activating TGF-P to induce scar tissue formation). Smad6 forms a stable association with type I receptors and interferes with the phosphorylation of other Smad proteins, including Smad2 and Smad 1, and their subsequent heteromerization with Smad4. Smad7 also forms a stable association with activated type I receptors and blocks access and phosphorylation of certain Smad molecules, thereby preventing formation of certain Smad heteromeric complexes. Smad7 also inhibits nuclear accumulation of Smad heteromeric complexes. A small molecule morphogenic activator may interfere with the inhibitory activity of these Smad proteins by, for example, tightly binding to either one or both proteins and rendering either protein incapable of stable association with type I receptors, or by competitively binding and stimulating the morphogen-activated transmembrane receptors.

Alternatively, a small molecule morphogenic activator may activate the inhibitory effects of these Smads in order to inhibit an undesirable effect TGF3 activity).

E. Subjects for Treatment As a general matter, the methods of the present invention may be utilized for any mammalian subject at risk of, or afflicted with, loss of or damage to myocardium. Mammalian subjects which may be treated according to the methods of the invention include, but are not limited to, human subjects or patients. In addition, however, the invention may be employed in the treatment of domesticated mammals which are maintained as human companions dogs, cats, horses), which have significant commercial value dairy cows, beef cattle, sporting animals), which have significant scientific value captive or free specimens of endangered species), or which otherwise have value. In addition, as a general matter, the subjects for WO 98/27995 PCT/US97/23611 -39treatment with the methods of the present invention need not present indications for morphogen treatment other than those associated with loss of or damage to myocardium. That is, the subjects for treatment generally are expected to be otherwise free of indications for morphogen treatment. In some number of cases, however, the subjects may present with other symptoms osteoporosis, chronic renal failure) for which morphogen treatment also would be indicated.

In such cases, the morphogen treatment should be adjusted accordingly to avoid excessive dosing.

One of ordinary skill in the medical or veterinary arts is trained to recognize subjects at risk of, or afflicted with, loss of or damage to myocardium. In particular, clinical and non-clinical indications, as well as accumulated experience, relating to the presently disclosed and other methods of treatment, are expected to inform the skilled practitioner in deciding whether a given individual is a subject at risk of, or afflicted with, loss of or damage to myocardium and whether any particular treatment is best suited to the subject's needs, including treatment according to the present invention.

As a general matter, a mammalian subject may be regarded as a subject at risk of, or afflicted with, loss of or damage to myocardium if that subject has already been diagnosed as at risk of, or afflicted with, loss of or damage to myocardium. Such subjects include, but are not limited to, those which have already suffered a myocardial infarction, which have suffered a physical trauma to the heart, or which have been diagnosed with congestive heart failure.

E. Formulations and Methods of Treatment The morphogens, morphogen inducers, agonists of morphogen receptors, or small molecule morphogenic activators of the present invention may be provided to myogenic precursor cells by any suitable means, preferably directly in vitro or locally after implantation, as by addition to culture medium, injection or topical administration to a tissue locus) or systemically parenterally or orally). Preferably, the morphogen, morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator comprises part of an aqueous, physiologically acceptable solution so that in addition to delivery of the desired agent to the target cells, the solution does not otherwise adversely affect the cells' or subject's electrolyte and/or volume balance. The aqueous medium for the agent thus may comprise normal physiologic saline 9.85% NaCI, 0.15M, pH Such an aqueous solution containing the agent can be made, for example, by dissolving or dispersing the agent in 50% ethanol containing acetonitrile in WO 98/27995 PCT/US97/23611 0.1% trifluoroacetic acid (TFA) or 0.1% HC1, or equivalent solvents. One volume of the resultant solution then is added, for example, to ten volumes of phosphate buffered saline (PBS), which further may include 0.1-0.2% human serum albumin (HSA). The resultant solution preferably is vortexed extensively.

For systemic administration, the morphogens, morphogen inducers, agonists of morphogen receptors, or small molecule morphogenic activators of the present invention may be administered by any route which is compatible with the particular morphogen, inducer, or agonist employed. Where the agent is to be provided parenterally, such as by intravenous, subcutaneous, intramuscular, intraorbital, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, buccal, rectal, vaginal, intranasal or by aerosol administration, the agent preferably comprises part of an aqueous solution. In addition, administration may be by periodic injections of a bolus of the morphogen, inducer, agonist, or small molecule morphogenic activator, or may be made more continuous by intravenous or intraperitoneal administration from a reservoir which is external an i.v. bag) or internal a bioerodable implant, or a colony of implanted, morphogen-producing cells).

If desired, a given morphogen or other agent may be made more soluble by association with a suitable molecule. For example, association of the mature morphogen dimer with the pro domain results in the pro form of the morphogen which typically is more soluble or dispersible in physiological solutions than the corresponding mature form. In fact, endogenous morphogens are thought to be transported secreted and circulated) in the mammalian body in this form. This soluble form of the protein can be obtained from culture medium of morphogen-secreting mammalian cells, cells transfected with nucleic acid encoding and competent to express the morphogen. Alternatively, a soluble species can be formulated by complexing the mature dimer (or an active fragment thereof) with a morphogen pro domain or a solubility-enhancing fragment thereof (described more fully below). Another molecule capable of enhancing solubility and particularly useful for oral administrations, is casein. For example, addition of 0.2% casein increases solubility of the mature active form of OP1 by 80%. Other components found in milk and/or various serum proteins also may be useful.

Useful solutions for parenteral administration may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences WO 98/27995 PCT/US97/23611 -41- (Gennaro, Mack Pub., 1990. Formulations of the therapeutic agents of the invention may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Formulations for direct administration, in particular, may include glycerol and other compositions of high viscosity to help maintain the agent at the desired locus. Biocompatible, preferably bioresorbable, polymers, including, for example, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, lactide, and glycolide polymers and lactide/glycolide copolymers, may be useful excipients to control the release of the agent in vivo. Other potentially useful parenteral delivery systems for these agents include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or cutric acid for vaginal administration. Suppositories for rectal administration also may be prepared by mixing the morphogen, inducer, agonist, or small molecule morphogenic activator with a non-irritating excipient such as cocoa butter or other compositions which are solid at room temperature and liquid at body temperatures.

Formulations for local or topical administration to a tissue or skin surface may be prepared by dispersing the morphogen, inducer, agonist or small molecule morphogenic activator with an acceptable carrier such as a lotion, cream, ointment or soap. Particularly useful are carriers capable of forming a film or layer over the skin or tissue to localize application and inhibit removal. For local or topical administration to internal tissue surfaces, the agent may be dispersed in a liquid tissue adhesive or other substance known to enhance adsorption to a tissue surface.

For example, hydroxypropylcellulose or fibrinogen/thrombin solutions may be used to advantage.

Alternatively, tissue-coating solutions, such as pectin-containing formulations may be used.

Alternatively, the agents described herein may be administered orally. Oral administration of proteins as therapeutics generally is not practiced as most proteins are readily degraded by digestive enzymes and acids in the mammalian digestive system before they can be absorbed into the bloodstream. However, the morphogens described herein typically are acid stable and WO 98/27995 PCT/US97/23611 -42protease-resistant (see, for example, U.S. Pat. No. 4,968,590). In addition, at least one morphogen, OP1, has been identified in mammary gland extract, colostrum and 57-day milk.

Moreover, the OP1 purified from mammary gland extract is morphogenically active and also is detected in the bloodstream. Maternal administration, via ingested milk, may be a natural delivery route ofTGFP superfamily proteins. Letterio et al. (1994), Science 264:1936-1938, report that TGFp is present in murine milk, and that radiolabeled TGFP is absorbed by gastrointestinal mucosa of suckling juveniles. Labeled, ingested TGF3 appears rapidly in intact form in the juveniles' body tissues, including lung, heart and liver. Finally, soluble form morphogen, e.g., mature morphogen associated with the pro domain, is morphogenically active. These findings, as well as those disclosed in the examples below, indicate that oral and parenteral administration are viable means for administering TGFp superfamily proteins, including the morphogens, to an individual. In addition, while the mature forms of certain morphogens described herein typically are sparingly soluble, the morphogen form found in milk (and mammary gland extract and colostrum) is readily soluble, probably by association of the mature, morphogenically active form with part or all of the pro domain of the intact sequence and/or by association with one or more milk components. Accordingly, the compounds provided herein also may be associated with molecules capable of enhancing their solubility in vitro or in vivo.

The compounds provided herein also may be associated with molecules capable of targeting the morphogen, inducer, agonist or small molecule morphogenic activator to the desired tissue. For example, an antibody, antibody fragment, or other binding protein that interacts specifically with a surface molecule on cells of the desired tissue, may be used. Useful targeting molecules may be designed, for example, using the single chain binding site technology disclosed, for example, in U.S. Pat. No. 5,091,513. Targeting molecules can be covalently or noncovalently associated with the morphogen, inducer, agonist, or small molecule morphogenic activator.

As will be appreciated by one of ordinary skill in the art, the formulated compositions contain therapeutically effective amounts of the morphogen, morphogen inducers, agonists of morphogen receptors, or small molecule morphogenic activators. That is, they contain amounts which provide appropriate concentrations of the agent to the mammalian myogenic precursor cells for a time sufficient to stimulate morphogenesis of new and functional myocardium, and/or to WO 98/27995 PCT/US97/23611 -43prevent, inhibit or delay further significant loss of myocardium or decline of myocardial function.

As will be appreciated by those skilled in the art, the concentration of the compounds described in a therapeutic composition of the present invention will vary depending upon a number of factors, including the biological efficacy of the selected agent, the chemical characteristics hydrophobicity) of the compounds employed, the formulation of the compound excipients, the administration route, and the treatment envisioned, including whether the active ingredient will be administered directly to cells in vitro, directly into a tissue site, or systemically. The preferred dosage to be administered also is likely to depend on such variables such as the condition of the diseased or damaged tissues, and the overall health status of the particular subject.

As a general matter, for systemic administration, daily or weekly dosages of 0.00001- 1000 mg ofa morphogen are sufficient, with 0.0001-100 mg being preferable, and 0.001 to 10 mg being even more preferable. Alternatively, a daily or weekly dosage of 0.01-1000 tg/kg body weight, more preferably 0.1-100 pg/kg body weight, may be advantageously employed. Dosages are preferably administered continuously, but daily, multi-weekly, weekly or monthly dosages may also be employed. In addition, in order to facilitate frequent infusions, implantation of a semipermanent stent intravenous, intraperitoneal, intracisternal or intracapsular) may be advisable. It should be noted that no obvious morphogen induced pathological lesions arise when mature morphogen OP1, 20 mg) is administered daily to normal growing rats for 21 consecutive days. Moreover, 10 mg systemic injections of morphogen OP1) injected daily for 10 days into normal newborn mice does not produce any gross abnormalities.

The morphogens, inducers, agonists or small molecule morphogenic activators of the invention may, of course, be administered alone or in combination with other molecules known to be beneficial in the treatment of the conditions described herein. Thus, in other embodiments the present invention provides pharmaceutical compositions in which a morphogen, morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator is combined with other agents which promote or enhance the proliferation and differentiation of myogenic precursor cells into new and functional myocardium. Thus, the present invention provides pharmaceutical compositions comprising a morphogen, or morphogen inducer, or agonist of a morphogen receptor, or small molecule morphogenic activator, in combination with one or more of a "muscle extract," conditioned medium from differentiated myotubes grown in culture, bFGF, WO 98/27995 WO 98/27995 PCT/US97/23611 -44- IGF, PDGF, LIF, ACTH, MSH, or G-CSF. In each such composition, the ratios or the morphogenic and mitogenic agents may be adjusted based upon their activities, as disclosed in the literature or as determined through simple experimentation, to provide a therapeutically effective dosage of each compound in a single unit dosage. The morphogenic and mitogenic agents in such a composition each preferably comprise at least about and more preferably more than 5% or of the dry weight of the composition. The compositions may, however, include other pharmaceutical carriers and active agents, as described above and, generally, in Remington's Pharmaceutical Sciences (Gennaro, Mack Pub., 1990, and, therefore, the morphogenic and mitogenic agents may each comprise a small fraction of the final weight of the pharmaceutical composition.

Practice of the invention, including additional preferred aspects and embodiments thereof, will be still more fully understood from the following examples, which are presented herein for illustration only and should not be construed as limiting the invention in any way.

Examples Preparation of Soluble Morphogen Complexes A currently preferred form of the morphogen useful herein, having improved solubility in aqueous solutions, is a dimeric morphogenic protein comprising at least the C-terminal seven cysteine domain characteristic of the morphogen family, complexed with a peptide comprising a pro region of a member of the morphogen family, or a solubility-enhancing fragment thereof, or an allelic, species or other sequence variant thereof. Preferably, the dimeric morphogenic protein is complexed with two pro region peptides. Also, the dimeric morphogenic protein preferably is noncovalently complexed with the pro region peptides. The pro region peptides preferably comprise at least the N-terminal eighteen amino acids that define the pro domain of a given naturally occurring morphogen, or an allelic or phylogenetic counterpart variant thereof In other preferred embodiments, peptides defining substantially the full length pro domain are used.

Other soluble forms of morphogens include dimers of the uncleaved pro forms of these proteins, as well as "hemi-dimers" wherein one subunit of the dimer is an uncleaved pro form of WO 98/27995 PCT/US97/23611 the protein, and the other subunit comprises the mature form of the protein, including truncated forms thereof, preferably noncovalently associated with a cleaved pro domain peptide.

As described above and in published application W094/03600, the teachings of which are incorporated herein by reference, useful pro domains include the full length pro regions, as well as various truncated forms hereof, particularly truncated forms cleaved at proteolytic Arg-Xaa-Xaa- Arg cleavage sites within the pro domain polypeptide. For example, in OP1, possible pro sequences include sequences defined by residues 30-292 (full length form); 48-292; and 158-292.

Soluble OPI complex stability is best enhanced when the pro region comprises the full length form rather than a truncated form, such as the residues 48-292 truncated form, in that residues 30-47 show sequence homology to the N-terminal portions of other morphogens, and currently are believed to have particular utility in enhancing complex stability for all morphogens.

Accordingly, currently preferred pro domains include peptides comprising at least the N-terminal fragment, amino acid residues 30-47 of a naturally occurring morphogen pro domain, or a biosynthetic variant thereof that retains the solubility and/or stability enhancing properties of the naturally-occurring peptide.

As will be appreciated by those having ordinary skill in the art, useful sequences encoding the pro region can be obtained from genetic sequences encoding known morphogens.

Alternatively, chimeric pro regions can be constructed from the sequences of one or more known morphogens. Still another option is to create a synthetic sequence variant of one or more known pro region sequences.

In another preferred aspect, useful pro region peptides include polypeptide chains comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a DNA or RNA sequence encoding at least the N-terminal eighteen amino acids of the pro region sequence for OPI or OP2, nucleotides 136-192 and 152-211 of SEQ ID NOs: 15 and 19, respectively.

A. Isolation from conditioned media or body fluid Morphogens are expressed from mammalian cells as soluble complexes. Typically, however the complex is disassociated during purification, generally by exposure to denaturants often added to the purification solutions, such as detergents, alcohols, organic solvents, chaotropic agents and compounds added to reduce the pH of the solution. Provided below is a WO 98/27995 PCT/US97/23611 -46currently preferred protocol for purifying the soluble proteins from conditioned media (or, optionally, a body fluid such as serum, cerebrospinal or peritoneal fluid), under non-denaturing conditions. The method is rapid, reproducible and yields isolated soluble morphogen complexes in substantially pure form.

Soluble morphogen complexes can be isolated from conditioned media using a simple, three step chromatographic protocol performed in the absence of denaturants. The protocol involves running the media (or body fluid) over an affinity column, followed by ion exchange and gel filtration chromatographies. The affinity column described below is a Zn-IMAC column. The present protocol has general applicability to the purification of a variety of morphogens, all of which are anticipated to be isolatable using only minor modifications of the protocol described below. An alternative protocol also envisioned to have utility includes an immunoaffinity column, created using standard procedures and, for example, using antibody specific for a given morphogen pro domain (complexed, for example, to a protein A-conjugated Sepharose column).

Protocols for developing immunoaffinity columns are well described in the art (see, for example, Guide to Protein Prification, M. Deutscher, ed., Academic Press, San Diego, 1990, particularly sections VII and XI thereof).

In this study, OP1 was expressed in mammalian (CHO, Chinese hamster ovary) cells as described in the art (see, for example, international application US90/05903 (W091/05802). The CHO cell conditioned media containing 0.5% FBS was initially purified using Immobilized Metal- Ion Affinity Chromatography (IMAC). The soluble OPI complex from conditioned media binds very selectively to the Zn-IMAC resin and a high concentration ofimidazole (50 mM imidazole, pH 8.0) is required for the effective elution of the bound complex. The Zn-IMAC step separates the soluble OP1 from the bulk of the contaminating serum proteins that elute in the flowthrough and 35 mM imidazole wash fractions. The Zn-IMAC purified soluble OP1 is next applied to an S- Sepharose cation-exchange column equilibrated in 20 mM NaPO 4 (pH 7.0) with 50 mM NaCI.

This S-Sepharose step serves to further purify and concentrate the soluble OP1 complex in preparation for the following gel filtration step. The protein was applied to a Sephacryl S-200HR column equilibrated in TBS. Using substantially the same protocol, soluble morphogens also can be isolated from one or more body fluids, including serum, cerebrospinal fluid or peritoneal fluid.

WO 98/27995 PCT/US97/23611 -47- IMAC was performed using Chelating-Sepharose (Pharmacia) that had been charged with three column volumes of 0.2 M ZnSO 4 The conditioned media was titrated to pH 7.0 and applied directly to the Zn-IMAC resin equilibrated in 20 mM HEPES (pH 7.0) with 500 mM NaC1. The Zn-IMAC resin was loaded with 80 mL of starting conditioned media per mL of resin.

After loading, the column was washed with equilibration buffer and most of the contaminating proteins were eluted with 35 mM imidazole (pH 7.0) in equilibration buffer. The soluble OPI complex then is eluted with 50 mM imidazole (pH 8.0) in 20 mM HEPES and 500 mM NaCI.

The 50 mM imidazole eluate containing the soluble OP1 complex was diluted with nine volumes of 20 mM NaPO 4 (pH 7.0) and applied to an S-Sepharose (Pharmacia) column equilibrated in 20 mM NaPO 4 (pH 7.0) with 50 mM NaC1. The S-Sepharose resin was loaded with an equivalent of 800 mL of starting conditioned media per mL of resin. After loading, the S- Sepharose column was washed with equilibration buffer and eluted with 100 mM NaCI followed by 300 mM and 500 mM NaCI in 20 mM NaPO 4 (pH The 300 mM NaCI pool was further purified using gel filtration chromatography. Fifty mis of the 300 mM NaCI eluate was applied to a 5.0 X 90 cm Sephacryl S-200HR (Pharmacia) equilibrated in Tris buffered saline (TBS), 50 mM Tris, 150 mM NaCI (pH The column was eluted at a flow rate of 5 mL/minute collecting mL fractions. The apparent molecular mass of the soluble OP1 was determined by comparison to protein molecular weight standards (alcohol dehydrogenase (ADH, 150 kDa), bovine serum albumin (BSA, 68 kDa), carbonic anhydrase (CA, 30 kDa) and cytochrome C (cytC, 12.5 kDa).

The purity of the S-200 column fractions was determined by separation on standard polyacrylamide SDS gels stained with Coomassie blue. The identity of the mature OP 1 and the pro-domain was determined by N-terminal sequence analysis after separation of the mature OP1 from the pro-domain using standard reverse phase C18 HPLC.

The soluble OP1 complex elutes with an apparent molecular weight of 110 kDa. This agrees well with the predicted composition of the soluble OP 1 complex with one mature OP 1 dimer (35-36 kDa) associated with two pro-domains (39 kDa each). Purity of the final complex can be verified by running the appropriate fraction in a reduced 15% polyacrylamide gel.

The complex components can be verified by running the complex-containing fraction from the S-200 or S-200HR columns over a reverse phase C18 HPLC column and eluting in an acetonitrile gradient (in 0.1% TFA), using standard procedures. The complex is dissociated by WO 98/27995 PCT/US97/23611 -48this step, and the pro domain and mature species elute as separate species. These separate species then can be subjected to N-terminal sequencing using standard procedures (see, for example, Guide to Protein Purification, M. Deutscher, ed., Academic Press, San Diego, 1990, particularly pp. 602-613), and the identity of the isolated 36 kDa, 39 kDa proteins confirmed as mature morphogen and isolated, cleaved pro domain, respectively. N-terminal sequencing of the isolated pro domain from mammalian cell produced OPI revealed two forms of the pro region, the intact form (beginning at residue 30 of SEQ ID NO: 16) and a truncated form, (beginning at residue 48 of SEQ ID NO: 16.) N-terminal sequencing of the polypeptide subunit of the isolated mature species reveals a range of N-termini for the mature sequence, beginning at residues 293, 300, 313, 315, 316, and 318, of SEQ ID NO: 16, all of which are active, as demonstrated by the standard bone morphogenesis assay set forth in published application WO92/15323 as incorporated herein by reference.

B. In Vitro Soluble Morphogen Complex Formation As an alternative to purifying soluble complexes from culture media or a body fluid, soluble complexes can be formulated from purified pro domains and mature dimeric species.

Successful complex formation apparently requires association of the components under denaturing conditions sufficient to relax the folded structure of these molecules, without affecting disulfide bonds. Preferably, the denaturing conditions mimic the environment of an intracellular vesicle sufficiently such that the cleaved pro domain has an opportunity to associate with the mature dimeric species under relaxed folding conditions. The concentration of denaturant in the solution then is decreased in a controlled, preferably step-wise manner, so as to allow proper refolding of the dimer and pro regions while maintaining the association of the pro domain with the dimer. Useful denaturants include 4-6M urea or guanidine hydrochloride (GuHCI), in buffered solutions of pH 4-10, preferably pH 6-8. The soluble complex then is formed by controlled dialysis or dilution into a solution having a final denaturant concentration of less than 0.1-2M urea or GuHCl, preferably 1-2 M urea of GuHCl, which then preferably can be diluted into a physiological buffer. Protein purification/renaturing procedures and considerations are well described in the art, and details for developing a suitable renaturing protocol readily can be determined by one having ordinary skill in the art. One useful text on the subject is Guide to WO 98/27995 PCT/US97/23611 -49- Protein Purification, M. Deutscher, ed., Academic Press, San Diego, 1990, particularly section V.

Complex formation also may be aided by addition of one or more chaperone proteins.

C. Stability of Soluble Morphogen Complexes The stability of the highly purified soluble morphogen complex in a physiological buffer, Tris-buffered saline (TBS) and phosphate-buffered saline (PBS), can be enhanced by any of a number of means. The currently preferred method is by means of a pro region that comprises at least the first 18 amino acids of the pro sequence residues 30-47 of SEQ ID NO: 16 for OPand preferably is the full length pro region. Residues 30-47 show sequence homology to the N-terminal portion of other morphogens and are believed to have particular utility in enhancing complex stability for all morphogens. Other useful means for enhancing the stability of soluble morphogen complexes include three classes of additives. These additives include basic amino acids L-arginine, lysine and betaine); nonionic detergents Tween 80 or Nonldet P- 120); and carrier proteins serum albumin and casein). Useful concentrations of these additives include 1-100 mM, preferably 10-70 mM, including 50 mM, basic amino acid;, 0.01preferably 0.05-0.2%, including 0.1% nonionic detergent;, and 0.01-1.0%, preferably 0.05-0.2%, including 0.1% carrier protein.

Equivalents The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.

WO 98/27995 PCT/US97/23611 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: COHEN, CHARLES

M.

(ii) TITLE OF INVENTION: TREATMENT OF MAMMALIAN MYOCARDIUM

WITH

MORPHOGENICALLY-TREATED MYOGENIC PRECURSOR

CELLS

(iii) NUMBER OF SEQUENCES: 31 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: TESTA, HURWITZ THIBEAULT,

LLP

STREET: 125 HIGH STREET CITY: BOSTON STATE: MA COUNTRY: USA ZIP: 02110 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM:

PC-DOS/MS-DOS

SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION

DATA:

APPLICATION

NUMBER:

FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT

INFORMATION:

NAME: TWOMEY, MICHAEL J REGISTRATION NUMBER: 38,349 REFERENCE/DOCKET NUMBER: CRP-123 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 617/248-7000 TELEFAX: 617/248-7100 INFORMATION FOR SEQ ID NO:1: SEQUENCE

CHARACTERISTICS:

LENGTH: 97 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..97 OTHER INFORMATION: /label= Generic-Seq-7 /note= "wherein each Xaa is independently selected from a group of one or more specified amino acids as defined in the specification." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: WO 98/27995 PCT/US97/23611 -51- Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa 40 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro 55 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 70 75 Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys 90 Xaa INFORMATION FOR SEQ ID NO:2: SEQUENCE

CHARACTERISTICS:

LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /label= Generic-Seq-8 /note= "wherin each Xaa is independently selected from a group of one or more specified amino acids as defined in the specification." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly 25 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala 40 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 55 Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 70 75 Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val 90 Xaa Xaa Cys Xaa Cys Xaa 100 WO 98/27995 PCT/US97/23611 -52- INFORMATION FOR SEQ ID NO:3: SEQUENCE

CHARACTERISTICS:

LENGTH: 102 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /label= OPX /note= "WHEREIN EACH XAA IS INDEPENDENTLY

SELECTED

FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS AS DEFINED IN THE SPECIFICATION" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xaa Asp 10 Leu Gly Trp Xaa Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa 25 Ala Tyr Tyr Cys Glu Gly Asn His Ala Glu Cys Xaa Phe Pro Leu Xaa Xaa Met Asn Ala Thr Ile Xaa Gin Xaa Leu Val Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr 70 Xaa Leu Xaa Ala Xaa 75 Ser Val Leu Tyr Xaa Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xaa 90 Arg Asn Met Val Val Xaa Ala Cys Gly Cys His 100 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens TISSUE TYPE: HIPPOCAMPUS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 WO 98/27995 PCT/US97/23611 -53- OTHER INFORMATION: /label= hOPI-MATURE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro Lys 1 5 10 Asn Gin Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 25 Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 40 Asp Leu Gly Trp Gin Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 55 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 70 75 Ala Thr Asn His Ala Ile Val Gin Thr Leu Val His Phe Ile Asn Pro 90 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: MURIDAE TISSUE TYPE: EMBRYO (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATION: /label= MOP1-MATURE (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Thr Gly Gly Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro Lys 1 5 10 Asn Gin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn Ser Ser Ser .25 Asp Gln Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 40 WO 98/27995 PCT/US97/23611 -54- Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro G1l 55 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asr 70 75 Ala Thr Asn His Ala Ile Val Gin Thr Leu Val His 90 Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gir 100 105 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile 115 120 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 INFORMATION FOR SEQ ID NO:6: SEQUENCE

CHARACTERISTICS:

LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS TISSUE TYPE: HIPPOCAMPUS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATION: /label= HOP2-MATURE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Ala Val Arg Pro Leu Arg Arg Arg Gin Pro Lys Lys 1 5 10 Pro Gin Ala Asn Arg Leu Pro Gly Ile Phe Asp Asp 25 His Gly Arg Gin Val Cys Arg Arg His Glu Leu Tyr 40 Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pro Gin 55 Tyr Tyr Cys Glu Gly Glu Cys Ser Phe Pro Leu Asp 70 75 Ala Thr Asn His Ala Ile Leu Gin Ser Leu Val His 90 Asn Ala Val Pro Lys Ala Cys Cys Ala Pro Thr Lys 100 105 Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile 115 120 1 Gly Tyr Ala Ala n Ser Tyr Met Asn SPhe Ile Asn Pro SLeu Asn Ala Ile 110 Leu Lys Lys Tyr 125 Ser Val Val Gly Ser Leu Leu Leu 125 Glu Gly Phe Ser Met Lys Ala Lys Leu Ser Gin Ala Asn Pro Thr His WO 98/27995 PCT/US97/23611 Arg Asn Met Val Val Lys Ala Cys Gly Cys His 130 135 INFORMATION FOR SEQ ID NO:7: SEQUENCE

CHARACTERISTICS:

LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL

SOURCE:

ORGANISM:

MURIDAE

TISSUE TYPE: EMBRYO (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATION: /label= MOP2-MATURE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn Glu Leu 1 5 10 Pro His Pro Asn Lys Leu Pro Gly Ile Phe Asp Asp Gly His Gly Ser 25 Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Arg 40 Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pro Gin Gly Tyr Ser Ala 55 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys Met Asn 70 75 Ala Thr Asn His Ala Ile Leu Gin Ser Leu Val His Leu Met Lys Pro 90 Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr 100 105 110 Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg Lys His 115 120 125 Arg Asn Met Val Val Lys Ala Cys Gly Cys His 130 135 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 101 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 98/27995 PCT/US97/23611 -56- (vi) ORIGINAL

SOURCE:

ORGANISM: bovinae (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..101 OTHER INFORMATION: /label= CBMP-2A-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Cys Lys Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn 1 5 10 Asp Trp Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly 25 Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala 40 Ile Val Gin Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala 55 Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp 70 75 Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gin Asp Met Val Val Glu 90 Gly Cys Gly Cys Arg 100 INFORMATION FOR SEQ ID NO:9: SEQUENCE

CHARACTERISTICS:

LENGTH: 101 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS TISSUE TYPE: hippocampus (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..101 OTHER INFORMATION: /label= CBMP-2B-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn 1 5 10 Asp Trp Ile Val Ala Pro Pro Gly Tyr Gin Ala Phe Tyr Cys His Gly 25 Asp Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala 40 WO 98/27995 WO 987995 PCT/US97/23611 -57- Ile Val Gin Thr Leu Val Asn Ser Val Asn Ser Ser Ile Pro Lys Ala 55 Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp 70 75 Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr Gin Glu Met Val Val Glu 90 Gly Cys Gly Cys Arg 100 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: DROSOPHILA

MELANOGASTER

(ix) FEATURE: NAME/KEY: Protein LOCATION: 1..101 OTHER INFORMATION: /label= DPP-FX (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asp 1 5 10 Asp Trp Ile Val Ala Pro Leu Gly Tyr Asp Ala Tyr Tyr Cys His Gly 25 Lys Cys Pro Phe Pro Leu Ala Asp His Phe Asn Ser Thr Asn His Ala 40 Val Val Gin Thr Leu Val Asn Asn Asn Asn Pro Gly Lys Val Pro Lys 55 Ala Cys Cys Val Pro Thr Gin Leu Asp Ser Val Ala Met Leu Tyr Leu 70 75 Asn Asp Gin Ser Thr Val Val Leu Lys Asn Tyr Gin Glu Met Thr Val 90 Val Gly Cys Gly Cys Arg 100 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 98/27995 PCT/US97/23611 -58- (vi) ORIGINAL SOURCE: ORGANISM: XENOPUS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /label=

VGL-FX

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Cys Lys Lys Arg His Leu Tyr Val Glu Phe Lys Asp Val Gly Trp Gin 1 5 10 Asn Trp Val Ile Ala Pro Gin Gly Tyr Met Ala Asn Tyr Cys Tyr Gly 25 Glu Cys Pro Tyr Pro Leu Thr Glu Ile Leu Asn Gly Ser Asn His Ala 40 Ile Leu Gin Thr Leu Val His Ser Ile Glu Pro Glu Asp Ile Pro Leu 55 Pro Cys Cys Val Pro Thr Lys Met Ser Pro Ile Ser Met Leu Phe Tyr 70 75 Asp Asn Asn Asp Asn Val Val Leu Arg His Tyr Glu Asn Met Ala Val 90 Asp Glu Cys Gly Cys Arg 100 INFORMATION FOR SEQ ID NO:12: SEQUENCE

CHARACTERISTICS:

LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL

SOURCE:

ORGANISM:

MURIDAE

(ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /label= VGR-1-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Cys Lys Lys His Glu Leu Tyr Val Ser Phe Gin Asp Val Gly Trp Gin 1 5 10 Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly 25 Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 40 WO 98/27995 PCT/US97/23611 -59- Ile Val Gin Thr Leu Val His Val Met Asn Pro Glu Tyr Val Pro Lys 55 Pro Cys Cys Ala Pro Thr Lys Val Asn Ala Ile Ser Val Leu Tyr Phe 70 75 Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val 90 Arg Ala Cys Gly Cys His 100 INFORMATION FOR SEQ ID NO:13: SEQUENCE

CHARACTERISTICS:

LENGTH: 106 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(vi) ORIGINAL

SOURCE:

ORGANISM: Homo sapiens TISSUE TYPE: brain (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..106 OTHER INFORMATION: /note= "GDF-1 (fx)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp His 1 5 10 Arg Trp Val Ile Ala Pro Arg Gly Phe Leu Ala Asn Tyr Cys Gin Gly 25 Gin Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala 40 Leu Asn His Ala Val Leu Arg Ala Leu Met His Ala Ala Ala Pro Gly 55 Ala Ala Asp Leu Pro Cys Cys Val Pro Ala Arg Leu Ser Pro Ile Ser 70 75 Val Leu Phe Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gin Tyr Glu 90 Asp Met Val Val Asp Glu Cys Gly Cys Arg 100 105 INFORMATION FOR SEQ ID NO:14: SEQUENCE

CHARACTERISTICS:

LENGTH: 5 amino acids WO 98/27995 PCT/US97/23611 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Cys Xaa Xaa Xaa Xaa 1 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 1822 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS TISSUE TYPE: HIPPOCAMPUS (ix) FEATURE: NAME/KEY: CDS LOCATION: 49..1341 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /function= "OSTEOGENIC

PROTEIN"

/product= "OPl" /evidence=

EXPERIMENTAL

/standardname= "OPl" (xi) SEQUENCE DESCRIPTION: SEQ ID GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA

GCGCGTAGAG

CCGGCGCG ATG CAC GTG Met His Val CGC TCA CTG CGA GCT GCG GCG CCG CAC AGC TTC GTG GCG CTC TGG GCA Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala 1() CTG TTC CTG CTG Leu Phe Leu Leu

CGC

Arg TCC GCC CTG GCC Ser Ala Leu Ala TTC AGC CTG GAC AAC Phe Ser Leu Asp Asn GAG GTG CAC TCG AGC TTC ATC CAC CGG Glu Val His Ser Ser Phe Ile His Arg CTC CGC AGC CAG Leu Arg Ser Gin GAG CGG Glu Arg CGG GAG ATG Arg Glu Met

CAG

Gin CGC GAG ATC CTC TCC ATT TTG GGC TTG Arg Glu Ile Leu Ser Ile Leu Gly Leu CCC CAC CGC Pro His Arg WO 98/27995 PCT/US97/2361 1 61 CCG CGC CGG CAC CTC GAG GGC AAG GAG AAC TCG GCA CCC ATO TTG ATG A rLg Pro~ h£seu Gln Gly Lys His Asn Ser Ala Pro Met Phe Met

CTG

Leu

GGC

Gly 100

CCC

Pro

ATG

Met

GAC

His

CCA

Pro

TAG

Tyr 180

CAG

Gin

GAC

Asp

ATC

Ile

GGC

Gly

AAG

Lys 260

TTC

Phe

CGG

Arg.

GAC

Asp

CAG

Gin

CT

Pro

GTC

Val1

GCA

Pro

GAA

Giu 165

ATC

Ile

GTG

Val1

AGC

Ser

ACA

Thr

CTG

Leu 245

TTG

Leu

ATG

M'et

TCC

Ser

GTG

Leu

GGC

Giy

GTG

Leu

ATG

Met

CGG

Arg 150

GGG

Gly

CGG

Arg

CTC

Leu

CGT

Arg

GCC

Ala 230

GAG

Gin

GG

Ala

GTG

Val

ACG

Thr

TAC

TTC

Phe

GCC

Al a

AGC

Ser 135

TAG

Tyr

GAA

Glu

GAA

Glu

GAG

Gln

ACG

Thr 215

ACG

Thr

CTG

Leu

~GGC

Gly

GT

Alia

GGG

Mly 295

AAG

Asn

TGG

Ser

AGG

Ser 120

TTG

Phe

GAG

His

GGT

Ala

GG

Arg

GAG

Glu 200

GTG

Leu

AGG

Ser

TG

Ser

GTG

Leu

TTG

Phe 280

AGG

Ser

GCC

Ala

TAG

Tyr 105

GTG

Leu

GTG

Val

GAT

His

GTC

Val1

TTG

Phe 185

CAG

His

TG

rrp

~AG

As n

GTG

NTT

Ilie 265

E'TG

The -,ys

ATG

Met 90

GGG

Pro

GAA

Gin

AAG

As n

GGA

Arg

AG

Thr 170

GAG

Asp

TTG

Leu

GGG

Ala

GAG

His

GAG

Glu 250

GGG

Gly.

AAG

Lys

GAG

Gin

GG

Ala

TAC

Tyr

GAT

Asp

GTG

Leu

GAG

Glu 155

GGA

Ala

AAT

As n

GGC

G ly

TG

Ser

TGG

Trp 235

AG

Thr

GG

Arg

GGG

Ala

CGC

Arg

GTG

*Val

AAG

Lys

AGG

Ser

GTG

Val1 140

TTG

Phe

GGG

Al a

GAG

Glu

AGG

Arg

GAG

Glu 220

GTG

Val

CTG

Leu

GAG

His

AG

Thr

AGG

Ser 300

GAG

Glu

GGG

Al a

GAT

His 125

GAA

Glu

GG

Arg

GAA

Glu

AG

Thr

GAA

Glu 205

GAG

Glu

GTG

Val1

GAT

ksp

G

Mly

GAG

Mlu 285

CAG

Mln

GAG

Glu

GTG

Val1 110

TTG

Phe

GAT

His

TTT

Phe

TTG

Phe

TTG

Phe 190

TCG

Ser

GC

Gly

AAT

Asn

GGG

Gly

GGG

Pro 270

GTG

Val

AAG

Asn

GGC

Oiy

TTC

Phe

GTG

Leu

GAG

Asp

GAT

Asp

GG

Arg 175

GG

Arg

GAT

Asp

TGG

Trp

CCG

Pro

CAG

Mln

CAG

Mln

CAG

-is

CGG

D.rg

GGG

Gly

AGT

Ser

ACG

Thr

AAG

Lys

GTT

Leu 160

ATG

Ile

ATG

Ile

GTG

Leu

GTG

Leu

GG

Arg 240

AGG

Ser

AAG

Asn

TTG

Phe

TGG

Ser

GGC

Gl

AGCC

Thr

GAC

Asp

GAA

Glu 145

TGG

Ser

TAG

Tyr

AGG

Ser

TTC

Phe

GTG

Val1 225

GAG

His

ATG

Ile

AAG

Lys

CGG

Arg Lys 305

GGG

Pro

GAG

*Gin

GGG

Ala 130

*TTC

Phe

AAG

Lys

AAG

Lys

GTT

Val1

GTG

Leu 210

TTT

Phe

AAG

Asn

AAG

Asn

GAG

Gln

AGG

Ser 290

AG

Thr

GGC

Gly

GGG

Gly 115

GAG

Asp

TTG

Phe

ATG

Ile

GAG

Asp

TAT

Tyr 195

CTC

Leu

GAG

Asp

CTG

Leu GGc Pro CGc Pro 275

%TG

Ile GGc Pro 345 393 441 489 537 585 633 681 729 777 825 873 921 969 AAG AAG GAG Lys Asn Gin 310 GAA GGG GTG CGG ATO GGG AAG GTG GGA GAG Glu Ala Leu Arg Met Ala Asn Val Ala Glu 315 320 AAG AGG AGG Asn Ser Ser 1017 WO 98/27995 PCT/US97/2361 1 62 AGC GAC GAG AGG CAG GCC TGT AAG AAG CAC Ser Asp 325 CGA GAC Arg Asp Gln Arg Gin Ala Lys Lys His TGG ATC ATG Trp Ile Ile CTG GGG TGG Leu Gly Trp 340

CAG

Gin 345

GGG

Gly

GAG

Giu

GCG

Al a 350

CCT

Pro GAA GGC TAC Glu Gly Tyr CTG TAT GTC AGC Leu Tyr Val Ser

TTC

Phe

GCG

Ala 355 GGC TAC TAG TGT Ala Tyr Tyr Cys GAG TGT GCG Glu Cys Ala

TTC

Phe 365

ACG

Thr CTG AAC TCG Leu Asn Ser TAC ATG Tyr Met 370 AAC GGC ACG Asn Ala Thr GCG GAA ACG Pro Glu Thr 390 ATC TCC GTC Ile Ser Val GCG ATC GTG Ala Ile Val CTG GTC CAC Leu Val His CCC AAG CCC Pro Lys Pro

TGC

Cys 395

GAC

Asp GCG CCC ACG Ala Pro Thr

CAG

Gin 400

ATC

Ile TTC ATG AAC Phe Ile Asn 385 CTG AAT GC Leu Asn Ala CTG AAG AAA Leu Lys Lys CTC TAG TTG Leu Tyr Phe 405 TAG AGA Tyr Arg

GAT

Asp 410

CGG

Arg AGC TGG AAG Ser Ser Asn 1065 1113 1161 1209 1257 1305 1351 1411 1471 1531 1591 1651 1711 1771 1822 AAG ATG GTG Asn Met Val GCC TGT GOC Ala Gys Gly TGC GAG Cys His 430

TAGGTGGTGG

420

GAGAATTGAG

GAAGCAGGAG

TGTGAGAGTA

ATGGAATGAA

GGATAAAGAA

GGTTTGGAGA.

GGGGTGGGAA

GTGTAATAAA

AGGGTTTGGG

AGGAAGTGGG

TTAGGAAAGA

GAAGATGGTA

AAATGGCGG

GGTAATTATG

GGGGTGGGGA

TGTGAGAATA

GGGAAGTTTT

TTTTGTGAGA.

TGAGGAGGAT

GAAGGTGTGG

GGGAGGTGAT

AGCGGGTAGC

GATTGGTGTG

AAAGGAATGA

TGTGGATGGT

GGTTGGGGTG

ATGGGTTTTG

AGGGAAAAGG

TGGGTGGGAA

AGCGAGGGGA

TGTGGGAAAG

ATGAAAAAAA

GGATTGGTG

GGTATCGGGA

ATCAGTTTTT

TAGGAGGAAA

GTCTGAGGGA

GGGAGGGGTG

GAAAATTGAG

AAAAZAAA

GGTTGGGGAG

AGTTTAAAGG

GAGTGGGAGC

AAAAAAGAAC

TGGAGGGAGT

GGAGGAAGGG

GGGGAAGTTG

A

INFORMATION FOR SEQ ID NO:i6: Wi SEQUENGE GHARAGTERISTIGS: LENGTH: 431 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLEGULE TYPE: protein (xi) SEQUENGE DESGRIPTION: SEQ ID NO:16: Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 1 5 10 Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 25 WO 98/27995 PCT/US97/2361 1 63 Leu Asp Asn Giu Val His Ser Ser Phe Ile His Arg 40 Leu Arg Her Gin Pro Met Gly Thr Asp Giu 2145 Ser Tyr Ser Phe Vai 225 His Lys C Arg Lys '2 305 Asn S Vai S Git His Phe Pro Gin Ala 130 Phe Lys Lys Vali Leu 210 Phe s n ksn 1n er 'hr er er j Arc Arc Met Gi Giy Asp Phe Ile Asp 195 Leu Asp Leu Pro Pro 275 Ile Pro Her Phe 3Arg I Pro Leu Gly 100 Pro PMet His Pro Tyr 180 Gin Asp Ile Gly Lys 260 Phe Arg Lys2 Ser2 Arg 340 Giu Met Gin Arc Arg Asp Gin Pro Vali Pro Glu 165 Ile Val1 Her Thr Leu 245 Leu vlet Ser %sn ksp 325 ksp Prc 70 Leu Gly Leu Met Arg 150 Gly Arg Leu Arg Ala 230 Gin Al a Val1 I'hr Glm 310 Glm Lieu 55 His Tyr Phe Al a Her 135 Tyr Giu Giu Gin Thr 215 Thr Leu Gly Ala Gly 295 Glu2 Arg C Gly I2 Let.

Asr Ser Her 120 Phe His Ala Arg Glu 200 Leu Ser Ser Lieu ?he 280 :,er kl a ?rp jGlu Ala -Tyr 105 Leu Val His Val1 Phe 185 His Trp Asn Val Ile 265 Phe Lys Leu2 Ala Gin I Ile Leu Ser Ile Leu Gly Leu Giy Met 90 Pro Gin Asn Arg Thr 170 Asp Leu Al a Hils Glu 250 31 y Lays krg 330 ~sp Lys His Asn Ser Ala Pro 75 Ala Tyr Asp Leu Glu Ala As n Gly Her Trp 235 Thr Arg Ala Arg Met 315 Lys Trp ValI Lys Her Val1 140 Phe Ala Glu Arg Giu 220 Val1 Leu His I'hr Ser 300 A~la Lys Ile Gil *Ala *His 125 Giu Arg Glu Thr Glu 205 Glu Val1 Asp Gly Giu 285 Gin Asn His Ile Phe 365 IGiu Val 110 Phe His Phe Phe Phe 190 Ser Gly As n Gly Pro 270 Val Asn Val2 Glu Ala 350 Gi~ PhE Leu Asp Asp Arg 175 Arg Asp Trp Pro Glm 255 Gln His %rg kl a eu 335 ?ro iGly Ser Thr Lys Leu 160 Ile Ile Leu Leu Arg 240 Her As n Phe Her Glu 320 Tyr Glu Gly Tyr Ala Ala Tyr Tyr Cys Giu Gly Glu Cys Ala Pro Leu Asn WO 98/27995 PCT/US97/23611 -64- Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gin Thr Leu Val His 370 375 380 Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin 385 390 395 400 Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile 405 410 415 Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425 430 INFORMATION FOR SEQ ID NO:17: SEQUENCE

CHARACTERISTICS:

LENGTH: 1873 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(vi) ORIGINAL SOURCE: ORGANISM:

MURIDAE

TISSUE TYPE: EMBRYO (ix) FEATURE: NAME/KEY: CDS LOCATION: 104..1393 OTHER INFORMATION: /function= "OSTEOGENIC

PROTEIN"

/product= "MOP1" /note= "MOP1 (CDNA)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG

CCACCTGGG(

CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGGC GCG ATG CAC GTG CGC

G

TCG

Ser

CTG

Leu

GTG

Val

GAG

Glu

CGC

CTG

Leu

TTC

Phe

CAC

His

ATG

Met

CCG

GCT

Ala

CTG

Leu

AGC

Ser

CGG

Arg

CTC

GCG

Ala

CGC

Arg

TTC

Phe

GAG

Glu

CAG

GCG

Ala 10

TCC

Ser

ATC

Ile

ATC

Ile

GGA

CCA

Pro

GCC

Ala

CAC

His

CTG

Leu

AAG

CAC

His

CTG

Leu

CGG

Arg

TCC

Ser

CAT

AGC

Ser

GCC

Ala

CGC

Arg 45

ATC

Ile

AAT

TTC

Phe

GAT

Asp 30

CTC

Leu

TTA

Leu

TCG

Met His Val Arg 1 GCG CTC TGG GCG CCT Ala Leu Trp Ala Pro AGC CTG GAC AAC GAG Ser Leu Asp Asn Glu AGC CAG GAG CGG CGG Ser Gin Glu Arg Arg TTG CCC CAT CGC CCG Leu Pro His Arg Pro CCC ATG TTC ATG TTG WO 98/2 7995 PCT/US97/2361 1 65 Arg

GAC

Asp

GCC

G ly

TTA

Leu

ATG

Met Pro

CTG

Leu

TTC

Phe

GCC

Ala

AGC

Ser His

TAC

Tyr

TCC

Ser

AGC

Ser

TTC

Phe 135 Leu Gin AAC GCC Asn Ala TAC CCC Tyr Pro 105 CTG CAG Leu Gin 120 CTC AAC Val Asn Gly

ATG

Met 90

TAC

Tyr

GAC

Asp

CTA

Lys 75

GCG

Ala

AAG

Lys

AGC

Ser

GTG

His

GTG

Val1

GCC

Ala

CAT

His

GAA

Asn Ser GAG GAG Clu Giu GTC TTC Val Phe 110 TTC CTC Phe Leu 125 CAT CAC His Asp Ala

AGC

Ser 95

AGT

Ser

ACT

Thr

AAA

Lys Pro 80

GGC

Gly

ACC

Thr

CAC

Asp

GAA

Glu Met

CCG

Pro

CAG

Gin

GCC

Ala

TTC

Phe

CAC

Asp

GCC

C ly

GAC

Asp 130

TTC

Met

GGA

Giy

CCC

Pro 115

ATG

Met

CAC

Leu

CAC

Gin 100

CCT

Pro

GTC

Val1

CCT

403 451 499 547 Leu Va140l 145Ph CGA TAC CAC CAT CCC GAG TTC CCC TTT CAT CTT TCC AAC ATC CCC GAG Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys Ile Pro Glu 150 1rr

GC

Gly 165

CCC

Arg

CTC

Leu

CC

Arg

CC

Ala

CAG

Gin 245

GCA

Ala

GTG

Val

ACG

Thr

CMA

Clu

GAG

Clu

CAG

Gin

ACC

Thr

ACC

Thr 230

CTC

Leu

GC

G

1 y

GCC

'GG

31y

CGC

Arg

CGA

Arg

GAG

Ciu

ATC

Ile 215

AC

Ser

TCT

Ser

CTG

Leu

TTC

Phe

GCC

Gly 295

GTG

Vali

TTT

Phe

CAC

His 200

TGC

Trp

AAC

Asn

GTG

Val1

ATT

Ile

TTC

Phe 280

AAG

Lys ACC CCA CCC GAA Thr

GAC

Asp 185

TCA

Ser

CCT

Ala

CAC

His

GAG

Ciu

CCA

Cly 265

AAC

Lys

CAG

Gin Al a 170

AAC

Asn

GGC

Cly

TCT

Ser

TGC

Trp

ACC

Thr 250

CCC

Arg

GCC

Al a

CC

Arg Ala

GAG

Glu

AG

Arg

GAG

Ciu

GTC

Val1 235

CTC

Leu

CAT

His

ACC

Thr

AGC

Ser Giu

ACC

Thr

GAG

Ciu

GAG

Clu 220

CTC

Val1

CAT

Asp

GA

C ly

GAA

G1u

CAG,

3mn

TTC

Phe

TTC

Phe

TCG

Ser 205

GCC

Gly

AAC

As n

CCC

Cly

CCC

Pro

GTC

Val1 285

AAT

Asn

AGG

Arc

CAG

Gln 190

CAC

Asp

TCC

Trp

CCT

Pro

CAG

Gin

CAG

Gin 270

CAT

His

CC

Arg

ATC

Ile 175

ATC

Ile

CTC

Leu

TTC

Leu

CGC

Arg

AC

Ser 255

AAC

Asn

CTC

Leu

TCC

Ser

TAT

Tyr

ACA

Thr

TTC

Phe

CTC

Val1

CAC

His 240

ATC

Ile

AAC

Lys

CCT

Arg

AAC

Lys

AAC

Lys

CTC

Val1

TTC

Leu

TTT

Phe 225

AAC

Asn

AAC

As n

CAA

Gin

ACT

Ser

ACG

Thr 305

CAC

Asp

TAT

TPyr

CTG

Leu 210

CAT

Asp

CTC

Leu

CCC

Pro

CCC

Pro

ATC

Ilie 290

CCA

Pro

TAC

Tyr

CAG

Gin 195

CAC

Asp

ATC

Ilie

GCC

Gly

AAC

Lys

TTC

Phe 275

CCC

Arg

AAC

Lys

ATC

Ile 180

CTC

Val1

AC

Ser

ACA

Thr

TTA

Leu

TTC

Leu 260

ATG

Me t

TCC

Ser

AC

ks n 643 691 739 787 835 883 931 979 1027 1075 CAA GAC Gin Ciu 310 CCC CTC AGC ATG Ala Leu Arg Met ccc ACT CTG CCA Ser Val Ala CAA AAC Clu Asn 320 ACC ACC ACT GAC Ser Ser Ser Asp WO 98/27995 PCT/US97/23611 -66-

CAG

Gin 325

CTT

Leu AGG CAG GCC Arg Gin Ala TGC AAG AAA CAT GAG CTG Cys Lys Lys His Giu Leu

TAC

Tyr 335 GTC AGC TTC CGA Val Ser Phe Arg S(GC TGG Gly Trp TAC TGT GAG Tyr Cys Glu ACC AAC CAC Thr Asn His 375 ACA GTA CCC Thr Val Pro CAG GAC Gin Asp 345 GGA GAG Gly Glu 360 GCC ATC Ala Ile ATC ATT GCA Ile Ile Ala

CCT

Pro 350

CTG

Leu GAA GGC TAT GCT Glu Gly Tyr Ala TGC GCC TTC Cys Ala Phe AAC TCC TAC Asn Ser Tyr

ATG

Met 370

AAC

Asn GCC TAC Ala Tyr 355 AAC GCC Asn Ala CCA GAC Pro Asp GTC CAG Val Gin

ACA

Thr 380

GCG

Ala GTT CAC TTC Val His Phe AAG CCC TGC Lys Pro Cys CCC ACC CAG Pro Thr Gin 390 GTC CTC Val Leu 405

CTC

Leu 400 TAC TTC GAC Tyr Phe Asp GCC ATC TCT Ala Ile Ser AAG TAC AGA Lys Tyr Arg 420 GAC AGC TCT AAT GTC Asp Ser Ser Asn Val 410 ATC CTG AAG Ile Leu Lys 415 1123 1171 1219 1267 1315 1363 1413 1473 1533 1593 1653 1713 1773 1833 1873 AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCTTCC

TGAGACCCTG

Asn Met Val Val Arg Ala Cys Gly Cys His 425 430 ACCTTTGCGG GGCCACACCT TTCCAAATCT TCGATGTCTC ACCATCTAAG

TCTCTCJ

CCCACCTTGG CGAGGAGAAC AGACCAACCT CTCCTGAGCC TTCCCTCACC

TCCCAA(

AAGCATGTAA GGGTTCCAGA AACCTGAGCG TGCAGCAGCT GATGAGCGCC

CTTTCCT

GGCACGTGAC GGACAAGATC CTACCAGCTA CCACAGCAAA CGCCTAAGAG

CAGGAA-

GTCTGCCAGG AAAGTGTCCA GTGTCCACAT GGCCCCTGGC GCTCTGAGTC

TTTGAGG

AATCGCAAGC CTCGTTCAGC TGCAGCAGAA GGAAGGGCTT AGCCAGGGTG

GGCGCTG

TCTGTGTTGA AGGGAAACCA AGCAGAAGCC ACTGTAATGA TATGTCACAA

TAAAACC

GAATGAAAAA AAAAAAAAAA AAAAAA7ApJA

AAAAGAATTC

&CTG

CGG

'TCT

~AAT

AGT

GCG

CAT

INFORMATION FOR SEQ ID NO:18: SEQUENCE

CHARACTERISTICS:

LENGTH: 430 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 1 5 10 Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 25 Leu Asp Asn Giu Val His Ser Ser Phe lie His Arg Arg Leu Arg Ser WO 98/27995 PCT/US97/23611 -67- Glu His Arg Arg Arg Pro Glu Arg Met Pro Gin 55 His Arg Leu Glu Gin Leu Lys Ser 60 His Ile Asn Leu Ser Gly Ala Leu Pro Gly Thr Met Phe Met Leu Asp Leu Tyr Asn Aia Met Q n Pro Asp Giy Gin Giy Phe 100 Gin Giy Pro Pro Leu Ala Tyr Leu Ala Val Giu Glu Ser Lys Ala Vai Phe Ser 110 Ala Phe 145 Lys Lys Va1 Leu Phe 225 Asn Asn Gin Ser Thr 305 Ser Ser Tyr Asr 13( Phe Ile Asp Tyr Leu 210 Asp Leu Pro Pro Ile 290 Pro Ser Phe Ala 115 Met His Pro Tyr Gin 195 Asp Ile Gly Lys Phe 275 Arg Lys Ser Arg Ala Ser His Phe Leu Thr Asp Val Prc Glu Ile 180 Va1 Ser Thr Leu Leu 260 Met Ser Asn Asp Nsp 340 Met Arg Gly 165 Arg Leu Arg Ala Gin 245 Ala Val Thr Gin Gin 325 Leu 120 Ser Phe Val 135 Tyr His His 150 Glu Arg Val Glu Arg Phe Gin Glu His 200 Thr Ile Trp 215 Thr Ser Asn 230 Leu Ser Val Gly Leu Ile Ala Phe Phe 280 Gly Gly Lys 295 Glu Ala Leu 310 Arg Gin Ala Gly Trp Gin Cys Glu Gly Asn Lei Arg Thr Asp 185 Ser Ala His Glu Gly 265 Lys Gin Arg Cys Asp 345 31u Glu Ala 170 Asn Gly Ser Trp Thr 250 Arg Ala Arg Met Lys 330 Trp Cys Vai Phe 155 Ala Glu Arg Glu Va1 235 Leu His Thr Ser Ala 315 Lys Ile Ala I Gb.

140 Arc Glu Thr Glu Glu 220 Va1 Asp Gly Glu Gin 300 Ser His lie ?he 125 1 His Phe Phe Phe Ser 205 Gly Asn Gly Pro Val 285 Asn Val Glu Ala Pro Asp Asp Arg Gin 190 Asp Trp Pro Gin Gin 270 His Arg Ala Leu Pro 350 eu Lys Leu Ile 175 Ile Leu Leu Arg Ser 255 Asn Leu Ser Glu Tyr 335 Glu Asn Glu Ser 160 Tyr Thr Phe Va1 His 240 Ile Lys Arg Lys Asn 320 Val ,ly 3er Pyr Tyr 355 365 Tyr Met Asn Aia Thr Asn His Ala Ile Val Gin Thr Leu Val His Phe WO 98/27995 PCT/US97/23611 -68- Ile 385 Pro Asp Thr 375 Pro Lys Pro Cys Cy 39 Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Sei 405 410 Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys 420 425 INFORMATION FOR SEQ ID NO:19: SEQUENCE

CHARACTERISTICS:

LENGTH: 1723 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens TISSUE TYPE: HIPPOCAMPUS (ix) FEATURE: NAME/KEY: CDS LOCATION: 490..1696 OTHER INFORMATION: /function= /product= "hOP2-PP" /note= "hOP2 (cDNA)" 380 s Ala Pro Thr Gin Leu 5 400 Ser Asn Val Ile Leu 415 Gly Cys His 430 "OSTEOGENIC PROTEIN" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGCGCCGGCA

GAGCAGGAGT

GGGCTGGAGG

GCTCCCTATG

CCACACCGCA

CCAAGCGGTG

GCGGCCACAG

CCGGACTGGC

CCGCAGAGTA

GCCCCGGCCT

GACAGGTGTC

GCGCGGCGGG

CGCCCCGCCC

CGCCGCCCGC

AGGCCCTGGG

TCGGCCGCGG

CGGCCTGCC ATG ACC GCG Met Thr Ala GGCTGGAGGA GCTGTGGTTG GAGCAGGAGG

TGGCACGGCA

AGTGGCGGAG ACGGCCCAGG AGGCGCTGGA

GCAACAGCTC

GCTGCAGGAG CTCGCCCATC GCCCCTGCGC

TGCTCGGACC

GGGTACGGCG GCGACAGAGG CATTGGCCGA

GAGTCCCAGT

CGAGGCGGTG GCGTCCCGGT CCTCTCCGTC CAGGAGCCAG GCTCCAGGGA CCGCGCCTGA GGCCGGCTGC CCGCCCGTCC CGCCCGCCGA GCCCAGCCTC CTTGCCGTCG

GGGCGTCCCC

AGCCGATGCG CGCCCGCTGA GCGCCCCAGC TGAGCGCCCC CTC CCC GGC CCG CTC TGG CTC CTG GGC CTG Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu 120 180 240 300 360 420 480 528 576 GCG CTA-TGC GCG CTG GGC GGG GGC GGC CCC GGC CTG CGA CCC CCG CCC Ala Leu Cys Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pro Pro Pro 20 GGC TGT CCC CAG CGA CGT CTG GGC GCG CGC GAG CGC CGG GAC GTG CAG Gly Cys Pro Gin Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Val Gin 35 40 WO 98/27995 PCT/US97/23611 -69-

CGC

Arg

GCG

Ala

CTG

Leu

CCC

Pro

AAC

Asn 110

AAG

Lys

GAG

Glu

CCA

Pro

GAC

Asp

GCG

Ala

ATG

Met

GAG

Glu ATC CTG Sle Leu *CCC GCC Pro Ala CTG TAC Leu Tyr CGAG CG Glu Arg GTG GAG Val Glu TTC CGC Phe Arg

GCG

Ala

CC

Ala

CAC

His

CGC

Arg

CGA

Arg

TTT

GTG

Val

*TCC

Ser 0CC Ala

CTG

Leu

GAC

Asp 115

GAC

Asp

CTC

Leu

CG

Arg

ATG

Met

GGC

Gly 100

CGT

Arg

CTG

Leu

GGG

Gly

CTO

Leu

GCC

Ala 85

CGC

Arg 0CC Ala

ACC

Thr

CTG

Leu

CCC

Pro 70

GGC

Gly

GCC

Ala

CTG

Leu

CAG

Gln CCT 000 Pro Gly 55 GCG TCC Ala Ser GAC GAC Asp Asp GAC CTG Asp Leu 0CC CAC Gly His 120 ATC CCG Ile. Pro 135 CGG CCC *Arg Pro GCG CCG Ala Pro GAC GAG Asp Olu GTC ATG Val Met 105 CAG GAG Gin Glu OCT 000 Ala Gly

COG

Arg

CTC

Leu

GAC

Asp

AGC

Ser

CCC

Pro

GAG

3 lu

CAC

4is 155 3AG CCC COC FPro Arg TTC ATO Phe Met GGC OCG Oly Ala TTC OTT Phe Val CAT TOG His Trp 125 OCO GTC Ala Val 672 720 768 816 864 912 960 1008 130

ACA

Thr

AAC

Asn

AAC

As n

OGA

Gly 190

TG

Trp

ACT

Thr

CAA

Gin 0CC Ala

AGO

Arg 270

GCI

Ala

AGO

Arg

AGO

Arg 175

GAC

Asp

TTO

Leu

GAG

Glu

COO

1Arg 4,GT Ser 255

%GG

krg

OCO

*Ala

ACC

Thr 160

GAO

Olu

GAO

Olu

CTG

Leu

GAC

Asp 0CC Ala 240

CCG

Pro

CAG

Gin

GAC

Gh 145

CTC

Leui

TCTI

Ser

C

Oly

AAO

Lys 300

G

1 y 225

CCA

Pro

%GT

3er

~CG

~ro TTC COO ATT TAC AAO iPhe

CAC

His

GAC

Asp

TOO

Trp

COT

Arg 210

CAC

His

CGC

Arg

CCC

Pro

AAG

Lys Arg Ile OTC AGC Val Ser TTO TTC Leu Phe 180 CTO OTO Leu Val 195 CAC AAO His Lys AOC GTG Ser Val TCC CAA Ser Oln ATC COC Ile Arg 260 AAA ACC Lys Ser Tyr

ATO

Met 165

TTT

Phe

CTG

Leu

GAC

Asp

OAT

Asp

CAG

Gin 245 Lys 150

TTC

Phe

TTG

Leu

OAT

Asp

CTG

Leu

CCT

Pro 230

CCT

Pro Val CCC AOC

ATC

Pro Ser c

CTG

Leu

'AG

Oln Val Val Gin 170 U

OAT

Asp

OTC

Val1

OGA

Oly 215

GOC

Gly

TTC

Phe

CTT

Leu

ACA

Thr 200

CTC

Leu

CTG

Leu

GTO

Val1

GCA

Ala

CCG

Pro

CAG

Gin 185

GCA

Al a

COC

Arg 0CC Al a

OTC

Val1

GTO

Val1 265

CAG

Gin

ACG

Thr 0CC Ala

CTC

Leu

OGC

Oly

ACT

Thr 250

AGO

Arg 0CC Ala

CTC

Leu

AGT

Ser

TAT

Tyr

CTO

Leu 235

TTC

Phe

CCA

Pro

AAC

COA

Arg

GAC

Asp

GTG

Val1 220

CTO

Leu

TTC

Phe

CTO

Leu

CGA

Arg

OCT

Ala

TOC

Cys 205

GAO

O iu

GT

Oly

AGO

Arg

AGO

Arg

CTC

Leu 1056 1104 1152 1200 1248 1296 1344 1392 Thr Pro Arg AAC GAO CTO Asn Oiu Leu CCA 000 Pro Gly ATC TTT OAT GAC GTC CAC 0CC TCC CAC GOC COO CAG 290 As~p Val His Gly Ser 295 His Oly Arg Gin OTC TOC Val Cys 300 WO 98/27995 PCT/US97/23611 CGT CGG CAC GAG CTC TAC GTC AGC TTC CAG Arg Arg His Glu Leu Tyr Val Ser Phe Gin 305 310 TGG GTC ATC GCT CCC CAA GGC TA TCG GCC Trp Val Ile Ala Pro Gin Gly Tyr Ser Ala 320 325 TGC TCC TTC CCA CTG GAC TCC TGC ATG AAT Cys Ser Phe Pro Leu Asp Ser Cys Met Asn 335 340 CTG CAG TCC CTG GTG CAC CTG ATG AAG CCA Leu Gin Ser Leu Val His Leu Met Lys Pro 350 355 TGC TGT GCA CCC ACC AAG CTG AGC GCC ACC Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr 370 375 AGC AGC A A G AAC AAT GTG A TG G AAA CAC Ser Ser Asn Asn Val Ile Leu Arg Lys His 385 390 GCC TGC GGC TGC CAC T GAGTCAGCCC

GCCCAGC

Ala Cys Gly Cys His 400 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 402 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID I Met Thr Ala Leu Pro Gly Pro Leu Trp Leu I 1 5 10 Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg P 25 Gin Arg Arg Leu Gly Ala Arg Glu Arg Arg A 40 Leu Ala Val Leu Gly Leu Pro Gly Arg Pro A 55 Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro L 70 Tyr His Ala Met Ala Gly Asp Asp Asp Glu A 90 Arg Arg Leu Gly Arg Ala Asp Leu Val Met S 100 105 Glu Arg Asp Arg Ala Leu Gly His Gin Glu P GAC CTC GGC Asp Leu Gly TAT TAC TGT Tyr Tyr Cys 330 GCC ACC AAC Ala Thr Asn 345 AAC GCA GTC Asn Ala Val 360 TCT GTG CTC Ser Val Leu CGC AAC ATG Arg Asn Met CCT ACTGCAG jeu Gly Leu A >ro Pro Pro G Lsp Val Gin A irg Pro Arg A eu Phe Met L 75 ,sp Gly Ala P: er Phe Val A] 1i ro His TrD L

TGG

Trp 315

GAG

Glu

CAC

His

CCC

Pro

TAC

Tyr

GTG

Val 395 CTG GAC Leu Asp GGG GAG Gly Glu GCC ATC Ala Ile AAG GCG Lys Ala 365 TAT GAC Tyr Asp 380 GTC AAG Val Lys 1440 1488 1536 1584 1632 1680 1723 ~la ly rg la eu ro sn ys Leu Cys Glu Pro Asp Ala Met Glu Cys Pro Ile Pro Leu Glu Val Phe r WO 98/27995 PCT/US97/2361 1 71 Arg Glu 145 Leu Phe 130 Phe His 115 Asp Arg Val1 Leu Ile Ser Thr Tyr Met 165 Gin Lys 150 Phe Ile 135 Val1 Gin 120 Pro Pro Val1 Ala Ser Val1 Gly Ile Gl Hi 15 G1 .u Ala 140 s Leu 5 u Gin 125 Val1 Leu Ser Thr Ala Asn Arg Asn Arg Ala Thr 160 Glu 170 Ser Asp Leu Phe 180 Phe Leu Asp Leu Gin Thr Leu Arg Ala Gly Asp Glu Gly Trp Leu Val Leu Asp Val 195 Thr Ala Ala 200 Ser Asp Val1 220 Leu Glu Lys Gly 225 Pro Ser Pro Phe Giu 305 Ala Pro Leu Pro Asn 385 Cys (2) Arg 210 His Arg Pro Lys Asp 290 Leu Pro Leu Val Thr 370 His Ser S er Ile Lys 275 Asp Tyr ksp Lys Val1 Gin Arg 260 Ser Val1 Val1 Gly Ser 340 ASP Leu Gly Leu 215 Asp Pro Gly Leu 230 Gin Pro Phe Val 245 Thr Pro Arg Ala Asn Glu Leu Pro 280 His Gly Ser His 295 Ser Phe Gin Asp 310 Tyr Ser Ala Tyr 325 Cys Met Asn Ala Arg Leu Ala Gly Val Thr 250 Val Arg 265 Gin Ala Gly Arg Leu Gly Tyr Cys 330 Thr Asn 345 Tyr Leu 235 Leu Giy Gin Arg Ala Phe Phe Arg Pro Leu Arg Asn Arg Leu 285 Gin Val Cys 300 Trp Leu Asp 315 Glu Gly Giu His Ala Ile Pro Lys Ala 365 Al a Arg 270 Pro Arg Trp Cys Leu 350 Cys Ser 255 Arg Gly Arg Vali Ser 335 Gin Cys Pro Gin Ile His Ile 320 Phe Ser Ala iis 355 Leu Met Lys Pro Asn Ala Vai 360 Leu Ser Ala Thr Ser Val Leu 375 Tyr Tyr Asp Ser Ser Asn Val Ile Leu Arg Lys His Arg Asn Met 390 His INFORMATION FOR SEQ ID NO:2i: SEQUENCE

CHARACTERISTICS:

LENGTH: 1926 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear Val1 395 Val Lys Ala Cys WO 98/27995 PCT/US97/2361 1 72 (vi) ORIGINAL

SOURCE:

ORGANISM:

MURIDAE

TISSUE TYPE: EMBRYO (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 93. .1289 OTHER INFORMATION: /function= "OSTEOGENIC

PROTEIN"

/product= "-mOP2-Pp", /note= "ImOP2 cDNA", (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2i: GCCACOCACA GOTGCGCCGT CTOGTCCTCC CCGTCTOCCG TCAGCCCAOC

CCOACCAGCT

ACCAGTGGAT GCGCGCCCGC TGAAACTCCG AG ATG OCT ATG COT CCC GOG CCA Met Ala Met Arg Pro Oly Pro CTC TOG CTA TTO GOC CTT Leu Trp Leu Leu Oly Leu

CCG

Pro

CC

Arg

COO

Arg

GCG

Ala

GAC

Asp

AGC

Ser

CCA

Pro 120

GAG

Oiu

CAC

His

COT

Arg

COC

Arg

CCC

Pro

CCC

Pro

GOC

Oly

TTC

Phe 105

CAC

His

OCT

Ala

CCG

Pro

CCC

Pro

GAC

Asp

COA

Arg

CTC

Leu 000 Cly

OTC

Val1

TOO

Trp

OTC

Val

CTC

Leu

CCG

Pro

ATG

Met

CCC

Pro

TTC

Phe

CCA

Pro

AAC

Asn

AAG

Lys

ACA

Thr

AAC

Asn 155

CAC

His

CAG

Gin

COT

Arg

ATO

Met

CCA

Pro

ATG

Met

GAA

Olu

OCT

Ala 140

ACA

Thr

ACC

Thr

COT

Arg 45

OCA

Al a

TTO

Leu

CAG

01n

OTO

Val1

TTC

Phe 125

OCT

Al a

ACC

Thr

OCT

Al a

TOT

Cys 30

GAA

Olu

CAA

Gin

GAC

Asp

OCT

Ala

OAA

Olu 110

CAC

His

GAG

Olu

CTC

Leu

CTG

Leu

CCC

Pro

ATC

Ile

CCC

Pro

CTA

Leu

CAC

His 95

COC

Arg

TTT

Phe

TTC

Phe

CAC

His

TOC

Cys

CAG

Gin

CTO

Leu 0CC Ala

TAC

Tyr 80

TTA

Leu

CAC

Asp

GAC

Asp

COO

Arg

ATC

Ile 160 0CC Al a

COT

Arg

GCO

Ala

OCT

Ala 65

CAC

His

GCC

Gly

COT.

Arg

CTA

Leu

ATC

Ile 145

ACC

Ser

CTG

Leu

COC

Arg

OTG

Val1 50 0CC Al a 0CC Aila

COT

Arg

ACC

Thr

ACC

Thr 130

TAC

T'yr

A.TG

ie t

OCA

Cly

CTO

Leu

CTC

Leu

CG

Arg

ATO

Met 0CC Al a

CTG

iLeu 115

CAC

Gin

AAA

Lys

TTC

Phe 0C Oiy 20

GA

Oly Oly

CAC

Gin

ACC

Thr

GAC

Asp 100 0C Oly

ATC

Ile

OAA

Giu

GAA

Glu

GCC

Oly

GCG

Ala

CTA

Leu

CCA

Pro

OAT

Asp

CTO

Leu

TAC

Tyr

CCT

Pro

CCC

Pro

OTG

Val

CAC

His

COC

Arg

CCG

Pro

C

Ala

GAC

Asp

OTC

Val

CAG

Gin

OCT

Al a

AGC

Ser 150

GTC

Val1

OCT

Oly

GAC

Giu

OGA

Gly

TCC

Ser

GAC

Asp

ATO

Met

GAO

Giu

GG

Cly 135

ACC

Thr

CAA

Gin 161 209 257 305 353 401 449 497 545 593 GAG CAC TCC AAC AGO GAO TCT GAC TTO TTC TTT TTO OAT CTT CAC ACO Giu His Ser Asn Arg Oiu Ser Asp Leu Phe Phe Leu Asp Leu Gin Thr WO 98/27995 PCTIUS97/23611 -73 CTC CGA Leu Arg 185 TCT GGG GAC GAG Ser Gly Asp Glu TGG CTG GTG CTG Trp Leu Val Leu ATC ACA GCA GCC Ile Thr Ala Ala GAC CGA TGG CTG Asp Arg Trp Leu

CTG

Leu 205 AAC CAT CAC AAG Asr His His Lys

GAC

Asp 210 CTG GGA CTC CGC Leu Gly Leu Arg 689 737 785 TAT GTG GAA ACC Tyr Val Giu Thr

GCG

Ala 220 GAT GGG CAC AGC Asp Gly His Ser GAT CCT GOC CTG Asp Pro Gly Leu GCT GGT Ala Gly 230 CTG CTT GGA Leu Leu Gly TTC TTC AGG Phe Phe Arg 250

CGA

Arg 235 CAA GCA CCA CGC Gin Ala Pro Arg

TCC

Ser 240 AGA GAG CCT TTC Arg Gin Pro Phe ATG GTA ACC Met Val Thr 245 GCA GCG AGA Ala Ala Arg GCC AGC CAG AGT Ala Ser Gin Ser

CCT

Pro 255 GTG CGG GCC CCT Val Arg Ala Pro CCA CTG Pro Leu 265 AAG AGG AGG CAG Lys Arg Arg Gin

CCA

Pro 270 AAG AAA ACG AAC Lys Lys Thr Asn

GAG

Glu 275 CTT CCG CAC CCC Leu Pro His Pro

AAC

Asn 280 AAA CTC CCA GGG Lys Leu Pro Gly TTT GAT GAT GGC Phe Asp Asp Gly

CAC

His 290 GGT TCC CGC GGC Gly Ser Arg Gly

AGA

Arg 295 GAG GTT TGC CGC Glu Val Cys Arg

AGG

Arg 300 CAT GAG CTC TAC His Glu Leu Tyr

GTC

Val 305 AGC TTC CdT GAC Ser Phe Arg Asp CTT GGC Leu Gly 310 TGG CTG GAC Trp Leu Asp GAG GGG GAG Glu Gly Glu 330 GTC ATC GCC CCC Val Ile Ala Pro GGC TAC TCT GCC Gly Tyr Ser Ala TAT TAC TGT Tyr Tyr Cys 325 TGT GCT TTC CCA CTG GAC TCC TGT ATG AAC GCC ACC AAC Cys Ala Phe Pro Leu Asp Ser Cys Met Asn Ala Thr Asn CAT GCC His Ala 345 ATC TTG CAG TCT Ile Leu Gin Ser

CTG

Leu 350 GTG CAC CTG ATG Val His Leu Met

AAG

Lys 355 CCA GAT GTT GTC Pro Asp Val Val 929 977 1025 1073 1121 1169 1217 1265 1319 1379 1439 1499

CCC

Pro 360 AAG GCA TGC TGT Lys Ala Cys Cys CCC ACC AAA CTG Pro Thr Lys Leu GCC ACC TCT GTG Ala Thr Ser Val

CTG

Leu 375 TAC TAT GAC AGC Tyr Tyr Asp Ser

AGC

Ser 380 AAC AAT GTC ATC CTG CGT AAA CAC CGT Asn Asn Val Ile Leu Arg Lys His Arg 385 AAC ATG Asn Met 390 GTG GTC AAG Val Val Lys

GCC

Ala 395 TGT GGC TOC CAC Cys Gly Cys His TGAGGCCCCG CCCAGCATCC

TGCTTCTACT

ACCTTACCAT CTGGCCGGGC CCCTCTCCAG AGGCAGAAAC CCTTCTATGT

TATCATAGCT

CAGACAGGGG CAATGGGAGG CCCTTCACTT CCCCTGGCCA CTTCCTGCTA

AAATTCTGGT

CTTTCCCAGT TCCTCTGTCC TTCATGGGGT TTCGGGGCTA TCACCCCGCC

CTCTCCATCC

WO 98/27995 PCTIUS97/23611 74

TCCTACCCCA

CTGGGGTCAG

AATGGCAJXAT

CTCTGCACCA

GATCAATGCA

CCAGGTATAG

CTGTGAGTTC

GGAATTC

AGCATAGACT

CACTGAAGGC

TCTGGATGGT

TTCATTGTGG

TCGCTGTACT

CGGTGCATGT

AAGGCCACAT

GAATGCACAC

CCACATGAGG

CTAAGAAGGC

CAGTTGGGAC

CCTTGAAATC

CATTAATCCC

AGAAAGAGC C

AGCATCCCAG

AAGACTGATC

CCTGGAATTC

ATTTTTAGGT

AGAGCTAGCT

AGCGCTAAAG

TGTCTCGGGA

AGCTATGCTA

CTTGGCCATC

TAAACTAGAT

ATAACAGACA

TGTTAGAAAA

AGACAGAGAC

GCAGGAAAA

ACTGAGAGGT

CTCAGCCCAC

GATCTGGGCT

CATACACTTA

AGAATCAGAG

AGGAGAATCT

AAAAAAAAAC

1559 1619 1679 1739 1799 1859 1919 1926 INFORMATION FOR SEQ ID NO:22: SEQUENCE

CHARACTERISTICS:

LENGTH: 399 amino acids TYPE: amino acid TOPOLOGY: linear Met 1 Ala (ii) MOLECULE (xi) SEQUENCE Ala Met Arg Pro 5 Leu Gly Gly Gly TYPE: protein DESCRIPTION: SEQ ID NO:22: Gly Pro Leu Trp Leu Leu G1~ His Gly Pro Arg Pro Pro Hi 25 y Leu Ala Leu Cys sThr Arg Arg Leu Ala Val Leu Gly Ala Arg Glu Arg Arg 40 Asp Met Gin Arg Cys Pro Gin Glu Ile Leu Gin Pro Ala Gly Leu Pro Ala Ala Arg Pro Arg Pro Arg Pro Leu Phe Met Ala Arg Gin Pro His Ala 70 Ser Ala Leu Asp Leu Ala Met Thr Asp Asp Asp Gly Pro Gin Ala Gly Arg Ala Arg Thr Leu 115 Leu Thr Gin Asp Leu 100 Gly Tyr Ile Pro Val Met Ser Gin Giu Pro 120 Ala Gly Giu Asn Met Val His Leu Arg Asp Phe Asp Trp Lys Giu Ala Val Thr 130 Ile Tyr Giu Phe Arg Lys Giu Pro His Pro Leu As n 155 As n Thr Leu His Met Phe Glu Val1 165 Gin Glu His Ser Arg Glu Ser Phe Phe Leu Asp Leu Gin Thr Leu Arg Ser Gly Asp Glu Gly Trp Leu WO 98/27995 WO 987995 PCT/US97/23611 180 185 190 Val Leu Asp Ile Thr Ala Ala Ser Asp Arg Trp Leu Leu Asn His His 195 200 205 Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Ala Asp Gly His Ser 210 215 220 Met Asp Pro Gly Leu Ala Gly Leu Leu Gly Arg Gin Ala Pro Arg Ser 225 230 235 240 Arg Gln Pro Phe Met Val Thr Phe Phe Arg Ala Ser Gin Ser Pro Val 245 250 255 Arg Ala Pro Arg Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys 260 265 270 Thr Asn Glu Leu Pro His Pro Asn Lys Leu Pro Gly Ile Phe Asp Asp 275 280 285 Gly His Gly Ser Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr 290 295 300 Val Ser Phe Arg Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pro Gin 305 310 315 320 Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp 325 330 335 Ser Cys Met Asn Ala Thr Asn His Ala Ile Leu Gin Ser Leu Val His 340 345 350 Leu Met Lys Pro Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys 355 360 365 Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile 370 375 380 Leu Arg Lys His Arg Asn Met Val Val Lys Ala Cys Gly Cys His 385 390 395 INFORMATION FOR SEQ ID NO:23: SEQUENCE

CHARACTERISTICS:

LENGTH: 1368 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1368 OTHER INFORMATION: /label= (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: ATG TCG GGA CTG CGA AAC ACC TCG GAG GCC GTT GCA GTG CTC GCC TCC 48 Met Ser Gly Leu Arg Asn Thr Ser Glu Ala Val Ala Val Leu Ala Ser 1 5 10 WO 98/27995 PCTIUS97/23611 -76- CTG GGA Leu Cly GCC CTT Ala Val

CTC

Leu

GAG

Glu

GGA

Gly

GCC

Ala

ATG

Met

ACC

Thr

GTT

Va1

CAG

Gin CTC ATC Leu Met 25 GGG ATT Gly Ile 40

TTC

Phe

TAC

Tyr

GTG

Val

ATA

Ile GCG ACC Ala Thr GAC AAC Asp Asn

CCG

Pro

AAG

Lys

CCC

Pro

GAC

Asp

CAC

Gin

TCG

Ser

CTG

Leu

CTG

Leu

GAT

Asp

CAC

Asp

CTC

Leu 145

AAC

Asn

CGC

Arg I ATG C Met I ACC C Thr 1 2 ACG C Thr L 225 GGG G Cly A

ACC

Thr

TAC

ATC ATC CAC ACA GTG CTG ACC GAG GAC CAC AAG CTC Ile met GAG ATC Leu Ser Glu Asi CTG GGC ATC GC Tyr Glu Ile Leu

AGC

Ser

CAC

Asp

GAG

Glu

CTC

Leu 130

GAC

ksp

AG

:ys

'TG

~eu ;cc la ;cc la 110

TG

leu

AC

sp

AGC

Ser

OTC

Va1

GAC

Asp 115

GAG

Glu

AAG

Lys

CGC

Arg

TGC

Trp

GAG

Clu 195

AAC

Asn

GGC

Gly

TAC

Tyr CAC CAC T His

TAC

Tyr 100

CAC

Asp

GAG

Glu

CGG

Arg

CAC

His

TTC

Phe 180

CTC

Leu

AGG

Arg

CAC

3ml 3TC Val Gin

CAC

His

CAC

Asp

GAT

Asp

GCC

Ala

CAC

His 165

CAC

Asp

CGC

Arg

GAG

Glu

CAC

His

GGC

Cly 245

L

C

A

T

Gi

G:

A2

AE

C']

Ve

A]

Ii

TT

Ph

AC

Th 23

TG

Tr Uiu Phe Leu 70 'TG TCG CTG eu Ser Leu CC ATC ACC rg Ile Thr AC GAA CGC yr Glu Arg 120 AG GGC GAG lu Gly Glu 135 [C CAC GAG le Asp Glu 50 1T GTC GAC ,n Vai Asp rC TCC AAC 1i Ser Asn 2C TAT CAG .e Tyr Gin 200 'C ACC ATC Le Thr Ile 215 C ATO GAG ir Met Glu 0 C TTC GAG p Leu Glu Glj

AGG

Arg

GCG

Ala 105

GGC

Cly

CAC

Gin

AGC

Ser

CAA

Glu

GTC

Va1 185

AAC

Asn

ACG

Thr

CCC

Pro

CTC

Leu Ile

AAC

Lys 90

GAG

Glu

CAT

His

CAC

Gin

GAC

Asp

CTC

Leu 170

CCC

Pro

GCC

Ala

CTA

Val CTG 2 Leu AAC Asn 250 Al 7E

TCC

Sex

GAO

Glu

CGG

Arg

AAG

Lys

ATC

Ile 155

CGT

Arg

AAC

Asn

AAC

ksn

TAC

Pyr

FCC

3er 135

TG

Ial p Asp 60

GAA

1 Glu

OCT

Ala

CGT

Cly

TCC

Ser

AAC

Asn 140

ATC

Ile

CAC

His

GAC

Asp GAG C Glu GCC j Ala I 220 TCG C Ser ACC C Thr C

CAC

Asp

ACG

Thr

TTC

GTC

Va1

CAC

His

CTG

Pro Lys Phe Leu

CTC

Leu

AGG

Arg 125

TTC

Phe

ATG

M2et

GAG

Jlu

AC

%sn 3GC f7y 105 kTT Ele

TG

Tal

;AC

flu

AGC

Ser 110

AGO

Arg

ATC

Ile

ACC

Thr

CAC

His

TAC

Tyr 190

AAC

Lys

GC

Gly

AAC

Asn

CGC

Cly

CAT

Asp

AGC

Ser

ACC

Thr

TTC

Phe

GGC

Cly 175

CTC

Leu

TGG

Trp

ACC

Thr

ACC

Thr

CTC

Leu 255

CAC

Gin

GCC

Ala

CAC

Asp

CTG

Leu 160

CGT

Arg

GTC

Va1

CTG

Leu

GGC

Gly

ACC

Thr 240

CAC

His 192 240 288 336 384 432 480 528 576 624 672 720 768 GAO TGG CTG CTC AAC TCG AAG GAC AAT CAT GOC ATC TAO ATT GCA CCA Glu Trp Leu Val Lys Ser Lys Asp Asn His Gly Ile Tyr Ile Cly Ala WO 98/27995 PCT/US97/23611 -77- CAC GCT GTC His Ala Val 275 AAC CGA CCC GAC Asn Arg Pro Asp

CGC

Arg 280 GAG GTG AAG CTG Glu Val Lys Leu GAC ATT GGA Asp Ile Gly CTG ATC Leu Ile 290 CAC CGC AAG GTG His Arg Lys Val

GAC

Asp 295 GAC GAG TTC CAG Asp Glu Phe Gin

CCC

Pro 300 TTC ATG ATC GGC Phe Met Ile Gly TTC TTC CGC GGA CCG Phe Phe Arg Gly Pro 305 CAC AGG AGC AAG CGA His Arg Ser Lys Arg 325 CTG ATC AAG GCG Leu Ile Lys Ala GCC CAC AGC AGC Ala His Ser Ser AGC GCC AGC CAT Ser Ala Ser His

CCA

Pro 330 CGC AAG CGC AAG Arg Lys Arg Lys AAG TCG Lys Ser 335 GTG TCG CCC Val Ser Pro AGC TGC CAG Ser Cys Gin 355 AAC GTG CCG CTG Asn Val Pro Leu GAA CCG ATG GAG Glu Pro Met Glu AGC ACG CGC Ser Thr Arg 350 CTG GGC TGG Leu Gly Trp ATG CAG ACC CTG Met Gin Thr Leu

TAC

Tyr 360 ATA GAC TTC AAG Ile Asp Phe Lys CAT GAC His Asp 370 TGG ATC ATC GCA Trp Ile Ile Ala

CCA

Pro 375 GAG GGC TAT GGC Glu Gly Tyr Gly

GCC

Ala 380 TTC TAC TGC AGC Phe Tyr Cys Ser 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1368

GGC

Gly 385 GAG TGC AAT TTC Glu Cys Asn Phe CTC AAT GCG CAC Leu Asn Ala His AAC GCC ACG AAC CAT Asn Ala Thr Asn His GCG ATC GTC CAG Ala Ile Val Gin

ACC

Thr 405 CTG GTC CAC CTG Leu Val His Leu

CTG

Leu 410 GAG CCC AAG AAG Glu Pro Lys Lys GTG CCC Val Pro 415 AAG CCC TGC Lys Pro Cys CAC CTG AAC His Leu Asn 435 GCT CCG ACC AGG Ala Pro Thr Arg GGA GCA CTA Gly Ala Leu CCC GTT CTG TAC Pro Val Leu Tyr 430 AGA AAC ATG ATT Arg Asn Met Ile 445 GAC GAG AAT GTG Asp Glu Asn Val CTG AAA AAG TAT Leu Lys Lys Tyr GTG AAA Val Lys 450 TCC TGC GGG TGC Ser Cys Gly Cys CAT TGA His 455 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 455 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Met Ser Gly Leu Arg Asn Thr Ser Glu Ala Val Ala Val Leu Ala Ser WO 98/27995 PCT/US97/2361 1 -78- Gly Leu Gly Met Val Leu Leu Met Phe 25 Val Ala Thr Thr Pro Pro Ala Val Glu Ala Thr Gin Ser Gin Ser Leu Leu Asp Asp Leu 145 As n Arg Met Thr Thr 225 Gly Glu His Leu Phe 305 His1 Thi Tyr Sex Asp Glu Leu 130 Asp Lys Leu Al a Ala 210 Leu A~sp Trp kl a Ile 290 ?he krg Sle *Giu Ser Val Asp 115 Giu Lys Arg Trp Glu 195 Asn Gly Tyr Leu Val2 275 His Arg C Ser I Gly 40 Ile Tyr Ile Asp Mel His Asp Glu Arg His Phe 180 Leu Arg Gin Ja 1 la 1 260 Isn krg ly t His -Leu Gin *His Asp Asp Ala His 165 Asp Arg Glu His Gly 245 Lys Arg Lys Pro Arg 325 Asn Gly Lys Asp Lys Leu Asp Val Arg Val Leu Ser Giu Asp Gil 70 Leu Arg Tyr Glu Ile 150 Asn Val Ile Phe Thr 230 rrp Ser Pro la 1 'iu 310 3er IPhe Ser Ile Giu Gly 135 Asp Val1 Ser Tyr Thr 215 Met Leu Lys Asp Asp 295 Leu Ala Asp Le.

Thr Arg 120 Giu Glu Asp As n Gin 200 Ile Glu Giu Asp Arg 280 ksp Ilie Ser IGl Arg *Ala 105 *Gly Gin Ser Giu Val1 185 Asn Thr Pro Leu Asn 265 Giu Glu Lys His Ile Ala 75 Lys Ser 90 Giu Glu His Arg Gin Lys Asp Ile 155 Leu Arg 170 Pro Asn Ala Asn Vai Tyr Leu Ser 235 Asn Val 250 His Gly Val Lys Phe Gin Ala Thr2 315 Pro Arg I Giu Arg Pro Thr His Ala~ G ly Ser As n 140 Ile His Asp Giu Ala 220 Ser I'hr Ilie i eu Pro 300 kla iy5 Pro *Leu Arg 125 Phe Met Giu Asn Gly 205 Ile Val Glu Tyr Asp 285 Phe His Arg Lys Ser 110 Arg Ile Thr His Tyr 190 Lys Gly Asn Gly Ile 270 Asp "let Ser ys Phe Asp Ser Thr Phe Gly 175 Leu Trp Thr Thr Leu 255 Gly Ile Ile Ser Lys Leu Gin Ala Asp Leu 160 Arg Val1 Leu Gly Thr 240 His Al a Gly Gly His 320 Ser Val Ser Pro Asn Asn Val Pro Leu Leu Giu Pro Met Glu Ser Thr Arg WO 98/27995 WO987995 PCT/US97/23611 -79- 340 345 350 Ser Cys Gin Met Gin Thr Leu Tyr Ile Asp Phe Lys Asp Leu Gly Trp 355 360 365 His Asp Trp Ile Ile Ala Pro Glu Gly Tyr Gly Ala Phe Tyr Cys Ser 370 375 380 Gly Glu Cys Asn Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 385 390 395 400 Ala Ile Val Gin Thr Leu Val His Leu Leu Glu Pro Lys Lys Val Pro 405 410 415 Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Ala Leu Pro Val Leu Tyr 420 425 430 His Leu Asn Asp Glu Asn Val Asn Leu Lys Lys Tyr Arg Asn Met Ile 435 440 445 Val Lys Ser Cys Gly Cys His 450 455 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1674 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: CDS LOCATION: 69..1268 OTHER INFORMATION: /note= "mOP3-PP" (xi) SEQUENCE DESCRIPTION: SEQ ID GGATCCGCGG CGCTGTCCCA TCCTTGTCGT CGAGGCGTCG CTGGATGCGA GTCCGCTAAA CGTCCGAG ATG GCT GCG CGT CCG GGA CTC CTA TGG CTA CTG GGC CTG GCT 110 Met Ala Ala Arg Pro Gly Leu Leu Trp Leu Leu Gly Leu Ala 1 5 CTG TGC GTG TTG GGC GGC GGT CAC CTC TCG CAT CCC CCG CAC GTC TTT 158 Leu Cys Val Leu Gly Gly Gly His Leu Ser His Pro Pro His Val Phe 20 25 CCC CAG CGT CGA CTA GGA GTA CGC GAG CCC CGC GAC ATG CAG CGC GAG 206 Pro Gin Arg Arg Leu Gly Val Arg Glu Pro Arg Asp Met Gin Arg Glu 40 ATT CGG GAG GTG CTG GGG CTA GCC GGG CGG CCC CGA TCC CGA GCA CCG 254 Ile Arg Glu Val Leu Gly Leu Ala Gly Arg Pro Arg Ser Arg Ala Pro 55 GTC GGG GCT GCC CAG CAG CCA GCG TCT GCG CCC CTC TTT ATG TTG GAC 302 Val Gly Ala Ala Gln Gin Pro Ala Ser Ala Pro Leu Phe Met Leu Asp WO 98/27995 PCT/US97/23611 80

CTG

Leu

CAC

His

CGC

Arg

TTT

Phe

TTC

Phe

CAC

His

GAC

Asp 175

TGG

Trp

CAT

His

CAC

His

CGC

Arg

CCT

Pro 255

AAT

Asn

GAT

Asp I CTC Leu

TAC

Tyr

TTG

Leu

GAC

Asp

GAC

Asp

CG

Arg

ATC

Ile 160

TTG

Leu

CTG

Leu

CAC

His

A\GC

Ser

TCC

Ser 240 3TG Jal 'AA2

'AT

~sp( EAT C Eyr

CGI

Arg

GAC

Asp

COT

Arg

CTA

Leu

ATC

Ile 145

AGC

Ser

TTC

Phe

CTG

Val1

AAG

Liys

ATA

Ile 225 k.GA krg

CGG

%rg

%TC

Ile 3GC

TC

al1 GCC ATG ACG GAT GAC ACT GGC~ GGT GG IAla

CGI

Arg

ACC

Thr

ACC

Thr 130

TAC

Tyr

ATG

Met

TTT

Phe

CTG

Leu

GAC

Asp 210

GAT

Asp

CAG

Gin

CC

Ala

AAC

Asn

CAC

His 290

AGC

Ser Met C1 Ala

CTG

Leu 115

CAG

Gin

AAA

Lys

TTC

Phe

TTC

Leu

GAC

Asp 195

CTA

Leu

CCT

Pro

CCT

Pro

CCT

Pro

CAG

Gin 275

GT

Oly

TTC

Phe Thr

CAC

Asp 100

GCC

IGly

ATC

Ile

CAA

Giu

GAA

Giu

OAT

Asp 180

ATC.

Ile

OGA

Oly

GC

Gly

TTC

Phe

COA

Arg 260

CTO

Leu

TCT

Ser

COTC

Arg Asj

CTC

Le.

TAC

Tyx

CC']

Pro

CCC

Pro

GTG

Val1 165

CTT

Leu

ACA

Thr

CTC

Leu

E!TA

Leu k.TG 4e t 245

NCA

rhr C0 Pro

'AC

iis

AC

D

5 Asp Ser Oly Oly G ily

ACC

Thr

CCC

Pro

CCT

Pro 2ATT

CAG

Gin

OCT

Ala

ACT

Ser 150

GTC

Val1

CAG

Gin

OCA

Ala

CC

Arg

OCT

Ala 230

OTT

Val

OCA

Ala

CAC

His

GCC

Gly

CTT

ATG

Met

GAG

Giu

CG

Oly 135

ACC

Thr

CAA

Gin

ACO

Thr

CC

Ala

CTC

Leu 215

OCT

Gly

GOT

Gly

AGA

A.rg

TCC

Ser 8.GA %\rg 295

GGC

31 ly

IAGC

*Ser

CCA

Pro 120

GAG

Giu

CAC

His

GAG

Glu

CTC

Leu

ACT

Ser 200

TAT

Tyr

CTG

Leu

TTC

Phe

CCA

Pro

AAC

Asn 280

OAA

Glu

TG

Trp I

TTT

Phe 105

CAC

His

OCT

Ala

CCC

Pro

CAC

His

CGA

Arg 185

GAC

Asp

OTO

Val1

CTT

Leu

TTC

Phe CT0 eu 265

%AA

3TT Ja 1

"TG

~eu

OTC

Val1

TOO

Trp,

GTC

Val1

CTC

Leu

TCC

Ser 170

TCT

Ser

CGA

Arg

GAA.

Glu

GCA

Gly

AGO

Arg 250

AAO

Lys

CAC

His

TGC

Cys

GAC

Asp Asn Ile Val Glu

AAO

Lys

ACA

Thr

AAC

As n 155

AAC

Asn 000 Gly

TG

Trp

ACC

Thr

CGA

Arg 235

GCC

Al a

PAOG

Lys

CTA

Leu

C

krg

FCT

'er

OAA

Oiu

OCT

Ala 140

ACA

Thr

AGO

Arg

GAC

Asp

CTO

Leu

GAG

Olu 220

CAA

Gin

AAC

Asn

AAG

Lys

OGA

Gly

AGO

Arg 300

GTC

ValI

TTC

Phe 125

OCT

Aila

ACC

Thr

GAO

Clu

GAO

Oiu

CTO

Leu 205

GAT

Asp

GCA

Ala

CAG

Gln CA0 .ln

%TC

Ile 285

'AT

-[is kTT lie

CAC

His

GAG

Olu

CTC

Leu

TCT

Ser

GOC

Gly 190

AAC

As n 0CC Oly

CCA

Pro

ACT

Ser

CTA

Leu 270

CTT

Leu

GAG

Oiu

CC

Ala 446 494 542 590 638 686 734 782 830 878 926 974 1022 310 CCC CAC GOC TAC TCC 0CC TAT TAC TOT OCT GOG GAO TOC ATC TAC CCA 1070 WO 98/27995 PCTIUS97/23611 -81- Pro Gin 320 CTG AAC Leu Asn Gly Tyr Ser Ala Tyr 325

TCC

Ser Tyr Cys Ala Gly Giu Cys Ile Tyr Pro TCC TGT ATG Ser Cys Met 335

GTA

Va1

AAC

Asn 340

CCA

Pro ACC AAC CAC Thr Asn His ACT ATG CAG GCC Thr Met Gin Ala

CTG

Leu 350 CAT CTG ATG His Leu Met GAT ATC ATC Asp Ile Ile

CCC

Pro 360 GTG TGC TGT Vai Cys Cys GTG CCT Vai Pro 365 ACT GAG CTG Thr Giu Leu GTC ATC CTG Val Ile Leu 385 AGT GCC Ser Ala 370 CGC AGG Arg Arg ATT TCT CTG Ile Ser Leu GAG CGC AAC Glu Arg Asn CTC TAC Leu Tyr 375 ATG GTA Met Val TAT GAT AGA Tyr Asp Arg AAC AAT AAT Asn Asn Asn 380 GTC CAG GCC TGT GG Val Gin Ala Cys Gb 395 C TGC {r Cys CAC TGAGTCCCTG CCCAACAGCC TGCTGCCATC

CCATCTATCT

His 400 CTCTTCCAAG GCAGGAAACC AACAAAGAGG GAAGGCAGTG

CTTI

TTCACAGTCT TGGCCCTCTC TGTTCTTTTT GCCAAGGCTG

AGAI

ACCCTGGTGA CCTCAGTAGC CCGATCTCTC ATCTCCCCAA

ACTC

GCATCTATGT CCTTTGGGAT TGGGCACAGA AGTCCAATTT

ACCP

CTACTGGCCC AGCCTGGACT TGAACCTGGA ACACAGGGTA

GAGC

CCATCAGAAG ATTTAGGTGT GTGCAGACAT GACCACACTC r"'

AGTCAGGCCT

1118 1166 1214 1262 1315 1375 1435 1495 1555 1615 1674

.CAACTC

~GATGGT

:CCCAAT

ACTTAT

.TCAGGC

TAGCAC

CATGTCCACA

CCTAGTTATA

GCAGCCAGGG

TCATGAGTCA

TCTTCAGTAT

TCCATAGCC

INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 399 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE (xi) SEQUENCE Met Ala Ala Arg Pro 1 5 Val Leu Gly Gly Gly Arg Arg Leu Gly Val Glu Val Leu Gly Leu TYPE: protein DESCRIPTION: SEQ ID Gly Leu Leu Trp Leu 10 NO: 26: Leu Gly Leu Ala Leu Cys His Leu Ser His Pro Pro His 25 Arg Glu Pro Arg Asp Met Gin 40 Ala Gly Arg Pro Arg Ser Arg 55 Ala Ser Ala Pro Leu Phe Met 70 75 Va1 Arg Ala Phe Pro Gin Glu Ile Arg Pro Val Gly Ala Ala Gin Gin Pro Leu Asp Leu Arg Ala Met Thr Asp Asp Ser Gly Gly Gly Thr Pro Gin Pro His Leu WO 98/27995 PCT/US97/23611 82 Asp Arg Ala Asp Leu Ile Met Ser Phe Val Asn Ile Vai Glu Arg Asp 100 105 110 Arg Thr Leu 115 Gly Tyr Gir Leu Ile 145 Ser Phe Val1 Lys Ile 225 Arg Arg Ile Gly Val1 305 Gly Ser Leu Leu Leu 385 Th2 13( Tyi Met Phe Leu Asp 210 Asp Gin Ala Asn His 290 Ser Tyr C:ys Ser 370 Dirg Gin Ile Pro Ala *Lys *Phe *Leu Asp 195 Leu Pro Pro Pro Gin 275 Gly Phe Ser Met Lys 355 Ala Arg C Gil Glu Asp 180 Ile Giy Gly Phe Arg 260 Leu Ser Arg 41 a ksn 340 Pro Ilie 1 u IPrc Val 165 Leu Thr Leu Leu Met 245 Thr Pro His Asp Tyr 325 Ser Asp Ser Arg Ser 150 Val Gin Ala Arg Ala 230 Val1 Ala.

His Gly Leu 310 Tyr Thr Ile Leu Asn r 390 Gil Gly 135 Thr Gin Thr Ala Leu 215 Gly Gly Arg Ser Arg 295 ?Aiy Ile jeu 375 e t 1Pro His 120 Glu Ala His Pro Giu His Leu Arg 185 Ser Asp 200 Tyr Val Leu Leu Phe Phe Pro Leu 265 Asn Lys 280 Giu Val Trp Leu2 Ala Gly His Ala 345 Pro Lys\ 360 Tyr Tyr I Val Val G Tr Va Let Sez 170 Sex Arc Giu Gly Arg 250 Lys His :ys ),sp .flu 330 'hr Pal tsp in p 1- Lys Glu Phe His Phe Asp 125 Thr Ala Ala Giu Phe Arg IAsn 155 Asn Gly Trp Thr Arg 235 Ala Lys Leu Arg Ser 315 Cys Met Cys Arg 1 Ala C 395 140 Thr Arg Asp Leu Giu 220 Gin As n Lys 3 iy Arg 300 lal1 Ile ,ln 'ys s n 180 ~ys Thr Giu Giu Leu 205 Asp Ala Gin Gin Ile 285 His Ile Tyr Val 365 Asn I Gly

C

Leu Ser Gly 190 As n Gly Pro Ser Leu 270 Leu Glu klia Pro jeu 350 ?ro s n ~ys *His Asp 175 Trp His His Arg Pro 255 As n Asp.

Leu Pro Leu 335 Val Thr Val His Ile 160 Leu Leu His Ser Ser 240 Val1 Gin Asp Tyr Gln 320 4Csn 3lu Ile INFORMVATION FOR SEQ ID NO:27: SEQUENCE

CHARACTERISTICS:

WO 98/27995 PCT/US97/23611 -83- LENGTH: 104 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..104 OTHER INFORMATION: /note= "BMP3" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp Ile Gly Trp Ser 1 5 10 Glu Trp Ile Ile Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ser Gly 25 Ala Cys Gln Phe Pro Met Pro Lys Ser Leu Lys Pro Ser Asn His Ala 40 Thr Ile Gln Ser Ile Val Ala Arg Ala Val Gly Val Val Pro Gly Ile 55 Pro Glu Pro Cys Cys Val Pro Glu Lys Met Ser Ser Leu Ser Ile Leu 70 75 Phe Phe Asp Glu Asn Lys Asn Val Val Leu Lys Val Tyr Pro Asn Met 90 Thr Val Glu Ser Cys Ala Cys Arg 100 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /note= (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gin 1 5 10 Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala Phe Tyr Cys Asp Gly 25 WO 98/27995 PCT/US97/23611 -84- Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 40 Ile Val Gin Thr Leu Val His Leu Met Phe Pro Asp His Val Pro Lys 55 Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe 70 75 Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val 90 Arg Ser Cys Gly Cys His 100 INFORMATION FOR SEQ ID NO:29: SEQUENCE

CHARACTERISTICS:

LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /note= "BMP6" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Cys Arg Lys His Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Gin 1 5 10 Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly 25 Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 40 Ile Val Gin Thr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro Lys 55 Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe 70 75 Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Tyr Arg Trp Met Val Val 90 Arg Ala Cys Gly Cys His 100 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1247 base pairs TYPE: nucleic acid WO 98/27995 PCT/US97/23611 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS TISSUE TYPE: BRAIN (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 84..1199 OTHER INFORMATION: /product= "GDF-1" /note= "GDF-1 CDNA" (xi) SEQUENCE DESCRIPTION: SEQ ID GGGGACACCG GCCCCGCCCT CAGCCCACTG GTCCCGGGCC

GCCGCGGACC

CTGCGCACTC

TCTGGTCATC GCCTGGGAGG AAG ATG CCA CCG CCG CAG CAA GGT CCC TGC Met Pro Pro Pro Gin Gin Gly Pro Cys

GGC

Gly CAC CAC CTC His His Leu CTC CTC CTC CTG GCC CTG CTG CTG CCC Leu Leu Leu Leu Ala Leu Leu Leu Pro TCG CTG CCC Ser Leu Pro CTG ACC CGC GCC Leu Thr Arg Ala

CCC

Pro GTG CCC CCA GGC Val Pro Pro Gly GCC GCC GCC CTG CTC CAG Ala Ala Ala Leu Leu Gin GCT CTA GGA Ala Leu Gly GTT CCC CCG Val Pro Pro

CTG

Leu CGC GAT GAG CCC Arg Asp Glu Pro

CAG

Gin 50 GGT GCC CCC AGG Gly Ala Pro Arg CTC CGG CCG Leu Arg Pro CCC CAG GAG Pro Gin Glu GTC ATG TGG CGC Val Met Trp Arg TTT CGA CGC CGG Phe Arg Arg Arg ACC AGG Thr Arg TCT GGC TCG CGG Ser Gly Ser Arg ACG TCC CCA GGG Thr Ser Pro Gly

GTC

Val ACC CTG CAA CCG Thr Leu Gin Pro 158 206 254 302 350 398 446 494 542

TGC

Cys CAC GTG GAG GAG His Val Glu Glu

CTG

Leu 95 GGG GTC GCC GGA Gly Val Ala Gly ATC GTG CGC CAC Ile Val Arg His

ATC

Ile 105 CCG GAC CGC GGT Pro Asp Arg Gly

GCG

Ala 110 CCC ACC CGG GCC TCG GAG CCT GTC TCG Pro Thr Arg Ala Ser Glu Pro Val Ser GCC GCG Ala Ala 120 GGG CAT TGC Gly His Cys CCC GCT GAG Pro Ala Glu 140 GAG TGG ACA GTC Glu Trp Thr Val TTC GAC CTG TCG Phe Asp Leu Ser GCT GTG GAA Ala Val Glu 135 TTC GCG GCG Phe Ala Ala CGC CCG AGC CGG Arg Pro Ser Arg CGC CTG GAG CTG Arg Leu Glu Leu GCG GCG Ala Ala 155 GCG GCA GCC CCG GAG GGC GGC TGG GAG Ala Ala Ala Pro Glu Gly Gly Trp Glu

CTG

Leu 165 AGC GTG GCG CAA Ser Val Ala Gin WO 98/27995 PCT/US97/23611 86 GCG GGG GAG GGC GCG GGC GCG GAG CCC Ala Gly Gin Gly Ala Gly Ala Asp Pro 170 175 GGG CCG Gly Pro 180 GTG CTG GTC CGC CAG Val Leu Leu Arg Gln

TTG

Leu

GCT

Ala

GCG

Al a

TCG

Ser

CGG

Arg 250

GCT

Al a

CAC

His

GGT

G ly

GCG

Al a

GGA

Gly 330

TC

Ser

GAG

GTG

Val1

TGG

Trp

CTA

Leu

CTG

Leu 235

CCG

Pro

TGT

Cys

CGC

Arg

CAG

Gin

CTG

Leu 315

GCC

Al a

GTG

Val1

GAC

CCC

Pro

GGT

Al a

CGC

Arg 220

CTG

Leu

CGG

Arg

CGC

Arg

TGG

Trp

TGG

Cys 300

AAC

Asn

CC

Ala

GTC

Leu

ATG

GGG

Ala

CC

Arg 205 Ccc Pro

GTG

Leu

CGG

Arg

GCG

Ala

GTC

Val1 285

GCG

Ala

CAC

His

GAC

Asp

TTC

Phe

GTG

CTG

Leu 190

AAC

Asn

CGG

Arg

GTG

Val1

GAC

As~p

CGG

Arg 270

ATC

Ile

GTG

Leu

GGT

Al a

GTG

Leu

TTT

Phe 350

GTG

GGG

Gly

GCG

Ala

GCC

Ala

ACC

Thr

GCC

Al a 255

CGG

Arg

GCG

Ala

CCC

Pro

GTG

Val1

CCC

Pro 335

GAG

Asp

GAC

CCG

Pro

TCA

Ser

CCT

Pro

CTG

Leu 240

GAA

Glu

CTG

Leu

CCG

Pro

GTC

Val1

CTG

Leu 320

TGC

Gys

AAG

Asn

CCA

Pro

TGG

Trp

GCC

Al a 225

GAG

Asp

CCC

Pro

TAC

Tyr

CGC

Arg

GG

Ala 305

CGC

Arg

TGC

Gys

AGC

Ser

GTG

Val1

CG

Pro 210

GCC

Ala

GCG

Pro

GTG

Val

GTG

Val1

GGG

Gly 290

GTG

Leu

GG

Ala

GTG

Val1

GAG

Asp

GGC

Arg 195

CGC

Arg

TGG

Gys

CGG

Arg

TTG

Leu

AGG

Ser 275

TTG

Phe

TCG

Ser

GTG

Leu

GGG

Pro

AAG

As n 355

C

Ala

AGC

Ser

GG

Al a

GTG

Leu

GGG

Gly 260

TTC

Phe

CTG

Leu

GGG

C ly

ATG

Met

GG

Ala 340

GTG

Val

GAG

Glu

GTG

Leu

GGC

Arg

TGG

Gys 245

GGG

G ly

GGC

Arg

GCC

Ala

TGG

Ser

CAG

His 325

CGG

A\rg

GTG

Leu

GGC

Arg

GTG

Leu 230

GAG

His

GGC

Gly

GAG

Glu

AAC

As n

GGG

Gly 310

GG

Ala

GTG

Leu

CTG

Leu

GTG

Leu 215

GGG

Ala

CCC

Pro

GGG

Pro

GTG

Val1

TAG

Tyr 295

GGG

Gly

GCG

Al a

TPG

Ser

GGG

Gly 200

GG

Ala

GAG

Glu

CTG

Leu

GGG

Gly

GGC

Gly 280

TGG

Gys

CG

Pro

GCG

Al a

CG

Pro

GCG

Ala

CTG

Leu

GGG

Aia

GCC

Ala

GGG

Gly 265

TGG

Trp

GAG

Gin

CCG

Pro

CG

Pro

ATC

Ile 345 686 734 782 830 878 926 974 1022 1070 1118 1166 1219 1247 3TG GTG CGC GAG TAT Val Leu Arg Gln Tyr Glu Asp Met Val Val Asp Glu Gys 365 GGG TGG CGG Gly Gys Arg 370 TAACCGGGG GGGGAGGGA GGGGGGGGCA ACAATAAATG

CGGCGTGG

INFORMATION FOR SEQ ID NO:3i: SEQUENGE GHARAGTERISTIGS: LENGTH: 372 amino acids TYPE: amino acid TOPOLOGY: linear WO 98/27995 PCT/US97/23611 -87- (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:31: Met Pro Pro Pro Gin Gin Gly Pro Cys Gly His His Leu Leu Leu Leu Leu Pro Pro Leu Thr Val Arg Val Ala 145 Gly Asp Pro Trp Ala 225 Asp Pro Tyr Arg Ale Gly Gin Phe Ser Ala Ala Val 130 Arg Gly Pro Val Pro 210 Ala Pro Val Val Gly 290 Leu Pro Gly Arg Pro Gly Ser 115 Phe Leu Trp Gly Arg 195 Arg Cys Arg Leu Ser I 275 Phe I Leu 2C Ala Ala Arg Gly Asn 100 Glu Asp Glu Glu Pro 180 Ala Ser Ala Leu Gly 260 Phe Leu

I

Leu Pro Ser Leu Pro SAla Pro Arg Val Ile Pro Leu Leu Leu 165 Val Glu Leu Arg Cys 245 Gly Arg Ala Ala Arg Asp 70 Thr Val Val Ser Arg 150 Ser Leu Leu Arg Leu 230 His Gly Glu Asn Leu Leu 55 Pro Leu Arg Ser Ala 135 Phe Val Leu Leu Leu 215 Ala Pro Pro Val Tyr 295 SLeu 40 SArg SGin Gin His Ala 120 Val Ala Ala Arg Gly 200 Ala Glu Leu Gly Gly 280 Cys 25 Gin Pro Glu Pro Ile 105 Ala Glu Ala Gin Gin 185 Ala Leu Ala Ala Gly 265 Trp Gin Leu Ala Val Thr Cys 90 Pro Gly Pro Ala Ala 170 Leu Ala Ala Ser Arg 250 Ala His 1 Gly Thr Leu Pro Arg 75 His Asp His Ala Ala 155 Gly Val Trp Leu Leu 235 Pro Cys Arg 31n Arg Gly Pro Ser Val Arg Cys Glu 140 Ala Gin Pro Ala Arg 220 Leu I Arg 1 Arg Trp Cys 300 Ale Leu Val Gly Glu Gly Pro 125 Arg Ala Gly Ala Arg 205 Pro Leu Arg Ala Val 285 Ala SPro I Arg Met Ser Glu Ala 110 Glu Pro Ala Ala Leu 190 Asn Arg Val Asp Arg 270 Ile Leu Val Asp Trp Arg Leu Pro Trp Ser Pro Gly 175 Gly Ala Ala Thr Ala 255 Arg Ala Pro SPro Glu Arg Arg Gly Thr Thr Arg Glu 160 Ala Pro Ser Pro Leu 240 Glu Leu Pro Val Ala 305 Leu Ser Gly Ser Gly Pro Pro Ala Leu Asn His Ala Val Leu WO 98/27995 PCT/US97/2361 1 88 Arg Ala Leu Met His Ala Ala Ala Pro Gly Ala Ala Asp Leu Pro Cys 325 330 335 Cys Val Pro Ala Arg Leu Ser Pro Ile Ser Val Leu Phe Phe Asp Asn 340 345 350 Ser Asp Asn Val Val Leu Arg Gln Tyr Glu Asp Met Val Val Asp Glu 355 360 365 Cys Gly Cys Arg 370

Claims

16. A method as in claim I wherein said morphogen is selected from the group consisting of a pro form of a morphogen, a soluble form of a morphogen, a mature morphogen, and a C-terminal fragment of a morphogen comprising at least the seven cysteine domain of said morphogen.

17. A method as in claim I wherein said morphogen is selected from the group consisting of osteogenic proteins and bone morphogenic proteins.

18. A method as in claim 1 wherein said morphogen induces a cascade of tissue-specific morphogenesis culminating in the formation of t. functional mammalian myocardium; and i comprises a pair of folded polypeptides, the amino acid sequence of each of which comprises a sequence having at least 70% amino acid sequence homology with the C-terminal seven-cysteine domain of human OP-1, mouse OP-I, human OP-2 or mouse OP-2, residues 38- 139 of SEQ ID NOs. 5, 6, 7 or 8, respectively.

19. A method as in claim 1 wherein said morphogen is selected from the group consisting of OP-I, CBMP-2A (BMP-2), and CBMP-2B (BMP-4).

20. A therapeutic composition for promoting the repair of regeneration of mammalian myocardium comprising isolated mammalian myogenic precursor cells, and an amount of a morphogen sufficient to promote proliferation or differentiation of said myogenic precursor cells into functional myocardium in a mbrphogehically permissive environment, wherein said morphogen is selected from the group consisting of OP-1, CBMP-2A (BMP-2) and CBMP-2B(BMP-4).

21. A therapeutic composition for promoting the repair or regeneration of mammalian myocardium comprising isolated mammalian myogenic precursor cells, and WO 98/27995 PCT/US97/23611 -92- 4 an amount of an inducer of a morphogen sufficient to promote proliferation or differentiation of said myogenic precursor cells into functional myocardium in a morphogenically 6 permissive environment. 1 22. A therapeutic composition for promoting the repair or regeneration of mammalian 2 myocardium comprising 3 isolated mammalian myogenic precursor cells, and 4 an amount of an agonist of a morphogen receptor sufficient to promote proliferation or differentiation of said myogenic precursor cells into functional myocardium in a morphogenically 6 permissive environment. 1 23. A therapeutic composition for promoting the repair or regeneration of mammalian 2 myocardium comprising 3 isolated mammalian myogenic precursor cells, and 4 an amount of a small molecule morphogenic activator sufficient to promote proliferation or differentiation of said myogenic precursor cells into functional myocardium in a 6 morphogenically permissive environment. 1 24. A method of culturing mammalian myogenic precursor cells comprising 2 isolating said myogenic precursor cells, and 3 culturing said myogenic precursor cells in a medium comprising an amount of a 4 morphogen sufficient to promote proliferation or differentiation of said myogenic precursor cells into functional myocardium in a morphogenically permissive environment. 1 25. A method of culturing mammalian myogenic precursor cells comprising 2 isolating said myogenic precursor cells, and 3 culturing said myogenic precursor cells in a medium comprising an amount of an inducer 4 of a morphogen sufficient to promote proliferation or differentiation of said myogenic precursor cells into functional myocardium in a morphogenically permissive environment. 1 26. A method of culturing mammalian myogenic precursor cells comprising 2 isolating said myogenic precursor cells, and WO 98/27995 PCT/US97/23611 -93- 3 culturing said myogenic precursor cells in a medium comprising an amount of an agonist 4 of a morphogen receptor sufficient to promote proliferation or differentiation of said myogenic precursor cells into functional myocardium in a morphogenically permissive environment. 1 27. A method of culturing mammalian myogenic precursor cells comprising 2 isolating said myogenic precursor cells, and 3 culturing said myogenic precursor cells in a medium comprising an amount of a small 4 molecule morphogenic activator sufficient to promote proliferation or differentiation of said myogenic precursor cells into functional myocardium in a morphogenically permissive 6 environment. 1 28. A method of inducing myogenic precursor cells, naturally competent to differentiate into 2 skeletal or smooth muscle, to differentiate into cardiomyocytes, said method comprising the steps 3 of 4 contacting said myogenic precursor cells with a morphogen; and maintaining the product of in an environment morphogenically permissive for 6 cardiomyogenesis. 1 29. A method of producing replacement cardiomyocytes in a mammal in need thereof, said 2 method comprising the step of implanting into said mammal myogenic precursor cells induced by 3 the method of claim 28. 1 30. A pharmaceutical composition comprising 2 a morphogenic agent selected from the group consisting of a morphogen, a morphogen 3 inducer, an agonist of a morphogen receptor, and a small molecule morphogenic activator; and 4 a mitogen selected from the group consisting ofbFGF, IGF, PDGF, LIF, ACTH, MSH, and G-CSF.