AU767112B2 - Morphogen treatment for chronic renal failure - Google Patents

Description

1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

(ORIGINAL)

C LA rj1~c.

Name of Applicant: r~ie~ooeueIc Actual Inventors: Address for Service: Invention Title: Kuber SAMPATH and Charles COHEN DAVIES COLLISON CAVE, Patent Attoreys, (oO cs0 1 Little Cellins Street, Melbeurne, 300200 Morphogen treatment for chronic renal failure The following statement is a full description of this invention, including the best method of performing it known to Q: OPER PDB-DIVISIONALS*2410647 DI\ DOC 26 401

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MORPHOGEN TREATMENT FOR CHRONIC RENAL FAILURE This application is a divisional application derived from Australian Patent Application No. 71420/00, the entire contents of which are incorporated herein by reference.

Field of the Invention The present invention relates generally to methods of treatment for renal disease.

In particular, the invention relates to methods of treatment for conditions which place mammals, including humans, in, or at risk of, chronic renal failure. The methods preferably involve the administration of certain proteins of the osteogenic protein/bone morphogenetic protein (OP/BMP) family within the TGF-0 superfamily of proteins.

More generally, the methods involve the administration of certain morphogens, inducers of those morphogens, or agonists of the corresponding morphogen receptors, or implantation of renal cells induced with those morphogens.

Background of the Invention ~Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

The mammalian renal system serves primary roles both in the removal of catabolic waste products from the bloodstream and in the maintenance of fluid and electrolyte balances in the body. Renal failures are, therefore, life-threatening conditions in which the build-up of catabolites and other toxins, and/or the development of significant imbalances in electrolytes or fluids, may lead to the failure of other major organs systems and death. As a general matter, renal failure is classified as "acute" or "chronic". As detailed below, the differences between these two conditions are not S merely a matter of severity or rapidity but, rather, reflect differences in etiology, prognosis, and treatment.

•O 1B- Acute Renal Failure Acute renal failure is defined as an abrupt cessation or substantial reduction of renal function and, in as many as 90-95% of cases, may be secondary to trauma, surgery or another acute medical condition. Acute renal failure may be due to pre-renal causes decreased cardiac output, hypovolemia, altered vascular resistance) or to post-renal causes obstructions or constrictions of the ureters, bladder or urethra) which do not directly involve the kidneys and which, if treated quickly, will not entail significant loss ofnephrons or other damage to the kidneys. Alternatively, acute renal failure may be due to intrinsic renal causes which involve a more direct insult or injury to the kidneys, and which may entail permanent damage to *o/ o/° *oo *oooo WO 97/41881 PCT/US97/07816 -2the nephrons or other kidney structures. Intrinsic causes of acute renal failure include but are not limited to infectious diseases various bacterial, viral or parasitic infections), inflammatory diseases glomerulonephritis, systemic lupus erythematosus), ischemia renal artery occlusion), toxic syndromes heavy metal poisoning, side-effects of antimicrobial treatments or chemotherapy), and direct traumas.

The diagnosis and treatment of acute renal failure is as varied as its causes. In human patients, oliguria (urine output 400 ml/day) or anuria (urine output 50 ml/day) may be present in 50-70% of cases, BUN levels may climb 10-20 mg/dL/day or faster, plasma creatinine levels may climb 0.5-1.0 mg/dL/day, and metabolic acidosis is almost always present. If not treated, the electrolyte and fluid imbalances hyperkalemia, acidosis, edema) associated with acute renal failure may lead to life-threatening arrhythmia, congestive heart failure, or multiple organ system failures. Present therapies are typically directed at the underlying causes of the acute renal failure pre-renal, post-renal, or infectious causes) and management of the complications. Due to the severity of acute renal failure, episodes rarely last longer than several weeks without mortality and are treated on an in-patient basis.

Chronic Renal Failure Chronic renal failure may be defined as a progressive, permanent and significant reduction of the glomerular filtration rate (GFR) due to a significant and continuing loss of nephrons.

*Chronic renal failure typically begins from a point at which a chronic renal insufficiency a permanent decrease in renal function of at least 50-60%) has resulted from some insult to the renal tissues which has caused a significant loss of nephron units. The initial insult may or may not have been associated with an episode of acute renal failure. Irrespective of the nature of the initial insult, chronic renal failure manifests a "final common path" of signs and symptoms as nephrons are progressively lost and GFR progressively declines. This progressive deterioration in renal function is slow, typically spanning many years or decades in human patients, but seemingly inevitable.

The early stage of chronic renal failure typically begins when GFR has been reduced to approximately one-third of normal 30-40 ml/min for an average human adult). As a result of the significant nephron loss, and in an apparent "attempt" to maintain the overall GFR with fewer nephrons, the average single nephron GFR (SNGFR) is increased by adaptations of the remaining nephrons at both the structural and functional level. One structural manifestation of this adaptation, readily detectable by microscopic examination of biopsy samples, is a WO 97/41881 PCT/US97/07816 -3- "compensatory hypertrophy" of both the glomeruli and the tubules of the kidney, a process which literally increases the volume of filtrate which can be produced by each remaining nephron by literal enlargement of the glomeruli and tubules. Indeed, as a result of the hypertrophy or dilation of the collecting ducts, the urine of subjects with chronic renal failure often contains broad "casts," typically 2-6 times normal diameter, which aid in diagnosis and have also been referred to as "renal failure casts." At the same time, there are functional changes in the remaining nephrons, such as decreased absorption or increased secretion of normally excreted solutes, which may be responses to hormonal or paracrine changes elsewhere in the body increasing levels of parathyroid hormone (PTH) in response to changes in serum levels of calcium and phosphate).

These adaptations in early stage chronic renal failure are not successful in completely restoring GFR or other parameters of renal function and, in fact, subject the remaining nephrons to increased risk of loss. For example, the increased SNGFR is associated with mechanical stresses on the glomerulus due to hypertension and hyperperfusion. The loss of integrity of podocyte junctures leads to increased permeability of the glomerulus to macromolecules or 15 "leakiness" of the glomerular capsule. Proliferative effects are also observed in mesangial, epithelial and endothelial cells, as well as increases in the deposition of collagen and other matrix proteins. Sclerosis of both the glomeruli and tubules is another common symptom of the hypertrophied nephrons and the risk of coagulation in the glomerulus is increased. In particular, these adaptations of the remaining nephrons, by pushing the SNGFR well beyond its normal level, actually decrease the capacity of the remaining nephrons to respond to acute changes in water, solute, or acid loads and, therefore, actually increase the probability of additional nephron loss.

00.0 As chronic renal failure progresses, and GFR continues to decline to less than 10% of normal 5-10 ml/min), the subject enters end-stage renal disease (ESRD). During this phase, the inability of the remaining nephrons to adequately remove waste products from the blood, while retaining useful products and maintaining fluid and electrolyte balance, leads to a rapid decline in which many organ systems, and particularly the cardiovascular system, may begin to fail. For example, BUN and creatinine levels may be expected to rise and, at BUN levels of 100 mg/dL and serum creatinine levels of 8-12 mg/dL, a uremic syndrome will typically develop in which the kidneys can no longer remove the end products of nitrogen metabolism. At this point, renal failure will rapidly progress to death unless the subject receives renal replacement therapy chronic hemodialysis, continuous peritoneal dialysis, or kidney transplantation).

WO 97/41881 PCT/US97/07816 -4- Approximately 600 patients per million receive chronic dialysis each year in the United States, at an average cost approaching S60,000-$80,000 per patient per year. Of the new cases of end-stage renal disease each year, approximately 28-33% are due to diabetic nephropathy (or diabetic glomerulopathy or diabetic renal hypertrophy), 24-29% are due to hypertensive nephrosclerosis (or hypertensive glomerulosclerosis), and 15-22% are due to glomerulonephritis.

The 5-year survival rate for all chronic dialysis patients is approximately 40%, but for patients over 65, the rate drops to approximately Morphoens and Growth Factors A great many proteins have now been identified which appear to act as morphogenetic or growth factors, regulating cell proliferation or differentiation. Typically these growth factors exert their effects on specific sets or subsets of cells 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 subgroup of cells or tissues.

One group of morphogenetic proteins, referred to herein as "morphogens," includes Smembers of the family of osteogenic proteins/bone morphogenetic proteins (OP/BMPs) which were initially identified by their 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 TGF-P superfamily of growth factors. Members of this morphogen family have now been shown to include the mammalian osteogenic protein-1 (OP-1, also known as BMP-7), osteogenic protein-2 osteogenic protein-3 BMP-2 (also known as BMP-2A or CBMP-2A), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-6, Vgr-1, and GDF-1, as well as the Xenopus homologue Vgl and the Drosophila homologues DPP and 60A. Members of this family encode secreted polypeptides that share common structural features and that are similarly processed from pro-proteins to yield carboxy terminal mature proteins having a conserved pattern of cysteines. The active forms of these proteins are either disulfide-bonded homodimers of a single family member, or heterodimers 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 in a variety of tissues during development. BMP-3 for, example, has been shown to be expressed in developing human lung and kidney (Vukicevic et al. (1994) J. Histochem. Cvtochem. 42:869-875), BMP-4 has been WO 97/41881 PICT/US97/07816 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 OP-1 (BMP-7) has been shown immunohistochemically to be associated with basement membranes in human embryos, including those of the developing lungs, pancreas, skin, and convoluted tubules of kidneys (Vukicevic, et al. (1994) Biochem Biophvs Res. Commun. 198:693-700). Some of the morphogens OP-2 and BMP-2) 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). In contrast, high levels of murine OP-1 expression have been observed in adult mouse kidneys (Ozkaynak, et al. (1991) Biochem. Biophvs. Res. Commun. 179:116-123). This suggests a possible role for OP-I synthesized in the kidney as a paracrine regulator of bone growth, and would be consistent with the role of the kidneys in both calcium regulation and bone homeostasis.

A great variety of growth factors have been considered which may participate in the regulation of the growth and repair of renal tissues (reviewed in, Toback (1992) Kidney Intl.

15 41:226-246). For example, EGF, TGF-ae, TGF-0, IGF-I, IGF-II, PDGF, FGF, Renin/Angiotensin SII, IL-1 and OP-1 have all been found to be expressed by various adult renal cells or tissues and to have effects on renal cell proliferation or differentiation (see, Toback (1992) supra, Ozkaynak, et al. (1991) supra). In addition, several of these have been found to be expressed in the developing .kidney, including IGF-I, TGF-P and OP-I (reviewed in, Bard, et al. (1994) Mech. Develop.

48:3-11).

Interestingly, TGF-P has been shown in a murine metanephric organ culture system to retard overall growth and segmental differentiation of all segments of developing nephrons except the thick ascending limb-early distal tubules (Avner and Sweeney (1990) Pediatr. Nephrol. 4:372- 377). In addition, TGF-P expression has been found to be increased in several models of renal 25 disease, suggesting that TGF-p mediated increases in the synthesis of extracellular matrix components may be involved in the etiology of diabetic nephropathy (or diabetic glomerulopathy or diabetic renal hypertrophy), renal fibrosis, glomerulosclerosis and glomerulonephritis, interstitial fibrosis, and hypertensive nephrosclerosis (Shankland, et al. (1994) Kidney Intl.

46:430-442; Yamamoto, et al. (1994) Kidney Intl. 45:916-927; Yamamoto, et al. (1993) PNAS 90:1814-1818; Tamaki, et al. (1994) Kidney Intl. 45:525-536; Border, et al. (1990) Nature 346:371-374; Hamaguchi, et al. (1995) Hypertension 26:199-207).

Also of interest is the fact that serum levels of human growth hormone (GH) are elevated in subjects with chronic renal failure (Wright et al. (1968) Lancet 2:798; Samaan and Freeman (1970) Metabolism 19:102). Recombinant GH has been shown to help maintain protein balance in malnourished chronic renal failure patients, and to promote "catch-up" growth in children with chronic renal failure. It has been suggested that these effects are mediated by IGF-I (see, Kopple (1992) Miner. Electrolyte Metab. 18:269-275). Although some studies have found that the administration of IGF-I increases renal plasma flow and GFR in chronic renal failure patients Guler, et al.

(1989) PNAS 86:2868-2872; Hirschberg. et al. (1993) Kidney Intl. 43:387-397), other studies have found that this effect is merely transient (Miller, et al. (1994) Kidney Intl.

46:201-207).

Thus, although some growth factors have been shown to be expressed in both developing and adult renal tissues, and although at least one has been shown to increase renal function in the short term, none has yet been shown to be of therapeutic benefit in preventing, inhibiting or delaying the progressive loss of renal function that characterizes chronic renal failure. A need remains, therefore, for treatments which will prevent the progressive loss of renal function which causes hundreds of thousands of patients to become dependent upon chronic dialysis, and which results in the premature deaths of tens of thousands each year.

.00 Summary of the Invention 0 According to a first aspect, the present invention provides a method of treating a mammal in, or at risk of, chronic renal failure comprising administering to said mammal a therapeutically effective amount of a morphogen.

According to a second aspect, the present invention provides a method of treating a mammal in, or at risk of, chronic renal failure comprising administering to said omammal a therapeutically effective amount of an inducer of endogenous morphogen expression.

S:According to a third aspect, the present invention provides a method of treating a mammal in, or at risk of, chronic renal failure comprising administering to said mammal g 30 a therapeutically effective amount of an agonist of a morphogen receptor.

6a- According to a fourth aspect, the present invention provides a method of treating a mammal in, or at risk of, chronic renal failure comprising introducing within the kidney of said mammal a therapeutically effective amount of renal mesenchymal progenitor cells.

According to a fifth aspect, the present invention provides a method of treatment to delay the need for, or reduce the frequency of, chronic dialysis treatments comprising administering to a mammal a therapeutically effective amount of a morphogen.

According to a sixth aspect, the present invention provides a method of treatment to delay the need for, or reduce the frequency of, chronic dialysis treatments comprising administering to said mammal a therapeutically effective amount of an inducer of endogenous morphogen expression.

According to a seventh aspect, the present invention provides a method of treatment to delay the need for, or reduce the frequency of, chronic dialysis treatments comprising administering to said mammal a therapeutically effective amount of an agonist of a morphogen receptor.

According to an eighth aspect, the present invention provides a method of promoting metanephric differentiation of renal mesenchymal progenitor cells comprising the step of contacting said cells with a morphogen in an amount effective to induce said differentiation.

According to a ninth aspect, the present invention provides use of a morphogen q o for the manufacture of a medicament for treating a mammal in, or at risk of, chronic renal failure.

According to a tenth aspect, the present invention provides use of an inducer of endogenous morphogen expression for the manufacture of a medicament for treating a mammal in, or at risk of, chronic renal failure.

According to an eleventh aspect, the present invention provides use of an agonist of a morphogen receptor for the manufacture of a medicament for treating a mammal in, o or at risk of, chronic renal failure.

S•According to a twelfth aspect, the present invention provides use of renal 30 mesenchymal progenitor cells for the manufacture of a medicament for treating a **•mammal in, or at risk of, chronic renal failure.

6b According to a thirteenth aspect, the present invention provides use of a morphogen for the manufacture of a medicament to delay the need for, or reduce the frequency of, chronic dialysis treatments.

According to a fourteenth aspect, the present invention provides use of an inducer of endogenous morphogen expression for the manufacture of a medicament to delay the need for, or reduce the frequency of, chronic dialysis treatments.

According to a fifteenth aspect, the present invention provides use of an agonist of a morphogen receptor for the manufacture of a medicament to delay the need for, or reduce the frequency of, chronic dialysis treatments.

According to a sixteenth aspect, the present invention provides use of a morphogen for the manufacture of a medicament to promote metanephric differentiation of renal mesenchymal progenitor cells.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

The present invention is directed to methods of treatment, and pharmaceutical preparations for use in the treatment, of mammalian subjects in, or at risk of, chronic :9 renal failure, or at risk of the need for renal replacement therapy. Such subjects include 20 subjects already afflicted with chronic renal failure, or which have already received renal replacement therapy, as well as any subject reasonably expected to suffer a progressive loss of renal function associated with progressive loss of functioning nephron units.

S 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. Subjects in, or at risk of, chronic renal failure, or at risk of the need for renal replacement therapy, include but are not limited to the following: subjects which may be regarded as afflicted with chronic renal failure, end-stage renal disease, chronic diabetic nephropathy, hypertensive nephrosclerosis, chronic glomerulonephritis, hereditary nephritis, and/or renal dysplasia, subjects having a biopsy indicating glomerular hypertrophy, tubular hypertrophy, O: 30 chronic glomerulosclerosis, and/or chronic tubulointerstitial sclerosis; subjects having an 0*e*

S

•5.55 WO 97/41881 PCT/US97/07816 -7ultrasound, MRI, CAT scan, or other non-invasive examination indicating renal fibrosis; subjects having an unusual number of broad casts present in urinary sediment; subjects having a GFR which is chronically less than about 50%, and more particularly less than about 40%, 30% or of the expected GFR for the subject; human male subjects weighing at least about 50 kg and having a GFR which is chronically less than about 50 ml/min, and more particularly less than about 40 ml/min, 30 ml/min or 20 ml/min; human female subjects weighing at least about 40 kg and having a GFR which is chronically less than about 40 ml/min, and more particularly less than about 30 ml/min, 20 ml/min or 10 ml/min; subjects possessing a number of functional nephron units which is less than about 50%, and more particularly less than about 40%, 30% or 20%, of the number of functional nephron units possessed by a healthy but otherwise similar subject; subjects which have a single kidney; and subjects which are kidney transplant recipients.

The methods and compositions of this invention capitalize in part upon the discovery that o certain proteins of eukaryotic origin may be used as renal therapeutic agents in the treatment of Ssubjects at risk, as defined herein, of chronic renal failure or the need for renal replacement S* 15 therapy. Generally, these renal therapeutic agents are proteins, or are based upon proteins, which are members of the osteogenic protein/bone morphogenetic protein (OP/BMP) family of proteins.

Thus, useful OP/BMP renal therapeutic agents of the invention include polypeptides, or functional variants of polypeptides, comprising at least the C-terminal six- or seven-cysteine domain of a mammalian protein selected from the group consisting of OP-l, OP-2, OP-3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP9, and proteins which exhibit at least 70% or, more preferably. or 80% amino acid sequence homology with the amino acid sequence of the seven-cysteine domain of human OP-1; and which are capable of inducing chondrogenesis in the Reddi- Sampath ectopic bone assay (Sampath and Reddi (1981), Proc. Natl. Acad. Sci. (USA) 78:7599- 7603) or a substantially equivalent assay, capable of significantly preventing, inhibiting, delaying or alleviating the progressive loss of renal function in a standard animal model of chronic renal failure, or capable of causing a clinically significant improvement in a standard marker of renal function when administered to a mammal in, or at risk of, chronic renal failure. More generally speaking, the invention provides for the use of"morphogens" which are dimeric proteins that induce morphogenesis of one or more eukaryotic mammalian) cells, tissues or organs.

Of particular interest herein are morphogens that induce morphogenesis at least of mammalian renal tissue, including formation of functional renal epithelium and, in particular, functional glomerular and tubular epithelium. Morphogens comprise a pair of polypeptides that, when WO 97/41881 PCT/US97/07816 -8folded, adopt a configuration sufficient for the resulting dimeric protein to elicit morphogenetic responses in cells and tissues displaying receptors specific for said morphogen. That is, morphogens generally induce all of the following biological functions 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. "Progenitor" cells are uncommitted cells that are competent to differentiate into one or more specific types of differentiated cells, depending on their genomic repertoire and the tissue specificity of the permissive environment in which morphogenesis is induced. Morphogens further can delay or mitigate the onset of senescence- or quiescence-associated loss of phenotype and/or tissue function. Morphogens still further can stimulate phenotypic expression of differentiated cells, including expression of metabolic and/or functional, secretory, properties thereof In O. .addition, morphogens can induce redifferentiation of committed cells under appropriate .environmental conditions. As noted above, morphogens that induce proliferation and/or 15 differentiation at least of mammalian renal tissue, and/or support the growth, maintenance and/or functional properties of mammalian nephrons, are of particular interest herein.

In preferred embodiments, the pair of morphogen polypeptides have amino acid sequences each comprising a sequence that shares a defined relationship with an amino acid sequence ofa reference morphogen. Herein, 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: 4. Preferably, morphogen polypeptides share a defined relationship with at least the Cterminal 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.

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.

WO 97/41881 PCT/US97/07816 -9- 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 (or "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" in Dayhoff et al. (1978), 5 Atlas of Protein Sequence and Structure, 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 20 mean amino acid sequence similarity. Homologous sequences share identical 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 o 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 25 substitutions of the corresponding residues in a reference sequence.

0* Of particular interest herein are morphogens, which, when provided to the kidney of a mammal, induce or maintain the normal state of differentiation and growth of nephron units. Of still more particular interest herein are morphogens which, when administered to a mammal, prevent, inhibit or delay the development of compensatory hypertrophy, including glomerular hypertrophy and/or tubular hypertrophy. Such morphogens can be used to treat a mammal in, or at risk of, chronic renal failure by preventing, inhibiting or delaying the progressive loss of functional nephron units and the consequent progressive loss of renal function.

WO 97/41881 PCT/US97/07816 The present invention alternatively can be practiced with methods and compositions comprising a morphogen stimulating agent or morphogen inducer in lieu of a morphogen. A "morphogen inducer" is a compound that stimulates in vivo production, expression, of a therapeutically effective concentration of an endogenous morphogen in the body of a mammal sufficient to regenerate or maintain renal tissue and/or to inhibit additional loss thereof Such compounds are understood to include substances which, when administered to a mammal, act on cells of tissue(s) or organ(s) that normally are competent to produce and/or secrete a morphogen encoded within the genome of the mammal, and which cause the endogenous level of the morphogen in the mammal's body to be altered. Endogenous or administered morphogens can act as endocrine, paracrine or autocrine factors. That is, endogenous morphogens can be synthesized by the cells in which morphogenetic responses are induced, by neighboring cells, or by cells of a distant tissue, in which circumstances the secreted endogenous morphogen is transported to the site of morphogenesis, by the individual's bloodstream. In preferred S embodiments, the agent stimulates expression and/or secretion of an endogenous morphogen so S 15 as to increase amounts thereof in renal tissues.

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 means a compound which binds to the receptor and for which such binding has a similar functional result as binding of the natural, endogenous ligand of the receptor. That is, the compound must, upon 20 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 similar functional 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 25 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 agent may also be referred to as a morphogen "mimic," "mimetic," or "analog." WO 97/41881 PCT/US97/07816 -11 The OP/BMP renal therapeutic agents of the invention, or the morphogens, morphogen inducers and agonists ofmorphogen receptors of the invention, may be administered by any route of administration which is compatible with the selected agent, and may be formulated with any pharmaceutically acceptable carrier appropriate to the route of administration. Preferred routes of administration are parenteral and, in particular, intravenous. intraperitoneal, and renal intracapsular. Treatments are also preferably conducted over an extended period on an outpatient basis. Daily dosages of the renal therapeutic agents are expected to be in the range of about 0.01- 1000 pg/kg body weight, and more preferably about 10-300 pg/kg body weight, although precise dosages will vary depending upon the particular renal therapeutic agent employed and the particular subject's medical condition and history Finally, in yet further embodiments, renal cells may be implanted into the kidney of a subject in, or at risk, chronic renal failure, or at risk of needing renal replacement therapy, in order to serve as a source ofmorphogen and/or to provide a source of additional functional renal tissue.

These cells may be renal mesenchymal progenitor cells, or renal mesenchymal progenitor cells S' 15 which have been induced to undergo metanephric differentiation. The cells may be derived from a donor a tissue-type matched donor, sibling, identical twin), may be derived from a tissue S; culture undifferentiated renal mesenchyme culture, fetal renal tissue culture), or may be explanted from the subject and then be re-implanted after proliferation and/or differentiation.

o ,o Preferably, the cells are induced to undergo metanephric differentiation by treatment with a boo 20 morphogen OP-1) either before or after implantation.

o* The treatments of the present invention are useful in preventing, inhibiting or delaying the o0:::o progressive loss of functional nephron units, and the consequent progressive loss of renal oooo function, which typify chronic renal failure. As such they are of great value in preventing or o odelaying the need for chronic dialysis or renal replacement therapy in subjects with chronic renal 25 insufficiency, or reducing the necessary frequency of chronic renal dialysis in subjects with endstage renal disease. As such, they are useful in prolonging the lives, and in maintaining the quality of life, of subjects at risk of, or already afflicted with, chronic renal failure.

Brief Description of the Figures Figure 1. This figure is a bar graph showing average serum creatinine levels for groups of sham-operated ("SHAM") or partially nephrectomized ("Nx Contr" and rats. 5-6 months WO 97/41881 PCT/US97/07816 13- Detailed Description of the Invention I. Definitions 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.

Renal therapeutic agent. As used herein, the term "renal therapeutic agent" means a polypeptide, or a functional variant ofa polypeptide, comprising at least the C-terminal six- or seven-cysteine domain of a mammalian protein selected from the group consisting of OP-1, OP-2, OP-3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP9, and proteins which exhibit at least 70% or, more preferably, 75% or 80% amino acid sequence homology with the amino acid sequence of the seven-cysteine domain of human OP-1; and which is capable of inducing chondrogenesis in the Reddi-Sampath ectopic bone assay (Sampath and Reddi (1981), Proc. Natl. Acad. Sci.

(USA) 78:7599-7603) or a substantially equivalent assay, capable of significantly preventing, S inhibiting, delaying or alleviating the progressive loss of renal function in a standard animal model S 15 of chronic renal failure, or capable of causing a clinically significant improvement in a S* standard marker of renal function when administered to a mammal in, or at risk of, chronic renal S: failure. As used herein, a percentage "homology" between two amino acid sequences indicates the percentage of amino acid residues which are identical or similar between the sequences and, as used herein, "similar" residues are "conservative substitutions" which fulfill the criteria defined for 20 an "accepted point mutation" in Dayhoffet al. (1978), Atlas of Protein Sequence and Structure Vol. 5 (Suppi. pp. 354-352, Natl. Biomed. Res. Found., Washington, D.C.

~Therapeutic efficacy. As used herein, a renal therapeutic agent of the invention is said to have "therapeutic efficacy," and an amount of the agent is said to be "therapeutically effective," if administration of that amount of the agent is sufficient to cause a clinically significant 25 improvement in a standard marker of renal function when administered to a mammalian subject a human patient) in, or at risk of, chronic renal failure. Such markers of renal function are well known in the medical literature and include, without being limited to, rates of increase in BUN levels, rates of increase in serum creatinine, static measurements of BUN, static measurements of serum creatinine, glomerular filtration rates (GFR), ratios of BUN/creatinine, serum concentrations of sodium urine/plasma ratios for creatinine, urine/plasma ratios for urea, urine osmolality, daily urine output, and the like (see, for example, Brenner and Lazarus (1994), in Harrison's Principles of Internal Medicine, 13th edition, Isselbacher et al., eds., WO 97/41881 PCTIUS97/07816 -14- McGraw Hill Text, New York; Luke and Strom (1994), in Internal Medicine, 4th Edition, J.H.

Stein, ed., Mosby-Year Book, Inc. St. Louis.) Glomerular Filtration Rate (GFR). The "glomerular filtration rate" or "GFR" is proportional to the rate of clearance into urine of a plasma-borne substance which is not bound by serum proteins, is freely filtered across glomeruli, and is neither secreted nor reabsorbed by the renal tubules. Thus, as used herein, GFR preferably is defined by the following equation: GFR U x conC where Ucon, is the urine concentration of the marker, is the plasma concentration of the marker, and IF is the urine flow rate in ml/min. Optionally, GFR is corrected for body surface area. Thus, the GFR values used herein may be regarded as being in units ofml/min/1.73m 2 The preferred measure of GFR is the clearance of inulin but, because of the difficulty of measuring the concentrations of this substance, the clearance of creatinine is typically used in clinical settings. For example, for an average size, healthy human male (70 kg, 20-40 yrs), a :':typical GFR measured by creatinine clearance is expected to be approximately 125 ml/min with 15 plasma concentrations of creatinine of 0.7-1.5 mg/dL. For a comparable, average size woman, a typical GFR measured by creatinine clearance is expected to be approximately 115 ml/min with creatinine levels of 0.5-1.3 mg/dL. During times of good health, human GFR values are relatively stable until about age 40, when GFR typically begins to decrease with age. For subjects surviving to age 85 or 90, GFR may be reduced to 50% of the comparable values at age 20 Expected Glomerular Filtration Rate An estimate of the "expected GFR" or "GFRl," may be provided based upon considerations of a subject's age, weight, sex, body surface area, and degree of musculature, and the plasma concentration of some marker compound creatinine) as determined by a blood test. Thus, as an example, an expected GFR or GFRxp may S* be estimated as: GFR, (1 4 0 age) x weight (kg) 72 x Poe (mgldl) This estimate does not take into consideration such factors as surface area, degree of musculature, or percentage body fat. Nonetheless, using plasma creatinine levels as the marker, this formula has been employed for human males as an inexpensive means of estimating GFR. Because creatinine is produced by striated muscle, the expected GFR or GFLRp of human female subjects WO 97/41881 PCT/US97/07816 is estimated by the same equation multiplied by 0.85 to account for expected differences in muscle mass. (See Lemann, et al. (1990) Am. J. Kidney Dis. 16(3):236-243.) Broad Cast. Microscopic examination of urinary sediment for the oresence of formed elements is a standard procedure in urinalysis. Amongst the formed elements which may be present in urine are cylindrical masses of agglutinated materials that typically represent a mold or "cast" of the lumen of a distal convoluted tubule or collecting tubule. In healthy human subjects, such casts typically have a diameter of 15-25 pm. In subjects with chronic renal failure, however, hypertrophy of the tubules may result in the presence of "broad casts" or "renal failure casts" which are 2-6 times the diameter of normal casts and often have a homogeneous waxy appearance. Thus, as used herein, a "broad cast" means a urinary sediment cast having a diameter of 2-6 times normal, or about 30-150 jpm for human casts.

Chronic. As used herein with respect to clinical indications such as urinary casts, measured GFR, or other markers of renal function, "chronic" means persisting for a period of at least three, and more preferably, at least six months. Thus, for example, a subject with a 15 measured GFR chronically below 50% of GFRC,, is a subject in which the GFR has been measured and found to be below 50% of GFIL,, in at least two measurements separated by at least three, and more preferably, by at least six months, and for which there is no medically sound reason to believe that GFR was substantially 10%) higher during the intervening period.

Subjects in, or at risk of, chronic renal failure. As used herein, a subject is said to be in, or at risk of, chronic renal failure, or at risk of the need for renal replacement therapy. if the subject is reasonably expected to suffer a progressive loss of renal function associated with progressive loss of functioning nephron units. Whether a particular subject is in, or at risk of, chronic renal failure is a determination which may routinely be made by one of ordinary skill in the relevant medical or veterinary art. Subjects in, or at risk of, chronic renal failure, or at risk of the need for 25 renal replacement therapy, include but are not limited to the following: subjects which may be regarded as afflicted with chronic renal failure, end-stage renal disease, chronic diabetic nephropathy, hypertensive nephrosclerosis, chronic glomerulonephritis, hereditary nephritis, and/or renal dysplasia; subjects having a biopsy indicating glomerular hypertrophy, tubular hypertrophy, chronic glomerulosclerosis, and/or chronic tubulointerstitial sclerosis; subjects having an ultrasound, MRI, CAT scan, or other non-invasive examination indicating renal fibrosis; subjects having an unusual number of broad casts present in urinary sediment; subjects having a GFR which is chronically less than about 50%, and more particularly less than about 40%, 30% or WO 97/41881 PCT/US97/07816 -16of the expected GFR for the subject; human male subjects weighing at least about 50 kg and having a GFR which is chronically less than about 50 ml/min, and more particularly less than about 40 ml/min, 30 ml/min or 20 ml/min; human female subjects weighing at least about 40 kg and having a GFR which is chronically less than about 40 ml/min, and more particularly less than about 30 ml/min, 20 ml/min or 10 ml/min; subjects possessing a number of functional nephron units which is less than about 50%, and more particularly less than about 40%, 30% or 20%, of the number of functional nephron units possessed by a healthy but otherwise similar subject; subjects which have a single kidney; and subjects which are kidney transplant recipients.

II. Description of the Preferred Embodiments A. General The present invention depends, in part, upon the surprising discovery that administration of certain protein-based renal therapeutic agents to subjects in, or at risk of, chronic renal failure, can reduce mortality and/or morbidity rates, and prevent, inhibit, delay or alleviate the progressive loss of renal function which characterizes chronic renal failure. Alternatively, or in addition, 15 administration of the renal therapeutic agents of the present invention can prevent, inhibit or delay the progressive loss of functional nephron units and the progressive decline in glomerular filtration rate (GFR) which slowly but inevitably leads to the need for renal replacement therapy renal transplant or chronic dialysis) or death. In preferred embodiments, the therapeutic agents of the invention are members of the osteogenic protein/bone morphogenetic protein (OP/BMP) family within the TGF-P superfamily of proteins.

B. Renal Therapeutic Agents The renal therapeutic agents of the present invention are naturally occurring proteins, or functional variants of naturally occurring proteins, in the osteogenic protein/bone morphogenetic protein (OP/BMP) family within the TGF-P superfamily of proteins. That is, these proteins form 25 a distinct subgroup, referred to herein as the "OP/BMP family," within the loose evolutionary grouping of sequence-related proteins known as the TGF-P superfamily. Members of this protein family comprise secreted polypeptides that share common structural features, and that are similarly processed from a pro-protein to yield a carboxy-terminal mature protein. Within the mature protein, all members share a conserved pattern of six or seven cysteine residues defining a 97-106 amino acid 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, e.g., Massague (1990), Annu. Rev. Cell Biol. 6:597; Sampath et al. (1990), J. Biol. Chem.

WO 97/41881 I'CTLIS97/07816 17- 265:13198). For example, in its mature, native form, natural-sourced human OP-I is a giycosylated dimer typically having an apparent molecular weight of about 30-36 kDa as determined by SDS-PAGE. When reduced, the 30 kDa protein gives rise to two glycosylated peptide subunits having apparent molecular weights of about 16 kDa and 18 kDa. The unglycosylated protein has an apparent molecular weight of about 27 kDa. When reduced, the 27 kDa protein gives rise to two unglycosylated polypeptide chains, having molecular weights of about 14 kDa to 16 kDa.

Typically, the naturally occurring OP/BMP proteins are translated as a precursor, having an N-terminal signal peptide sequence, a "pro" domain, and a "mature" protein domain. The signal peptide is typically less than 30 residues, and is cleaved rapidly upon translation at a cleavage site that can be predicted using the method of Von Heijne (1986), Nucleic Acids Research 14:4683-4691. The "pro" domain is variable both in sequence and in length, ranging from approximately 200 to over 400 residues. The pro domain is cleaved to yield the "mature" C-terminal domain of approximately 115-180 residues, which includes the conserved six- or 15 seven-cysteine C-terminal domain of 97-106 residues. As used herein, the "pro form" of an OP/BMP family member refers to a protein comprising a folded pair of polypeptides, each comprising a pro domain in either covalent or noncovalent association with the mature domains of the OP/BMP polypeptide. Typically, the pro form of the protein is more soluble than the mature form under physiological conditions. The pro form appears to be the primary form secreted from cultured mammalian cells. The "mature form" of the protein refers to mature C-terminal domain which is not associated, eiti.er covalently or noncovalently, with the pro domain. Any preparation of OP-1 is considered to contain mature form when the amount of pro domain in the preparation is no more than 5% of the amount of "mature" C-terminal domain.

•OP/BMP family members useful herein include any of the known naturally-occurring 25 native proteins including allelic, phylogenetic counterpart and other variants thereof, whether naturally-sourced or biosynthetically produced including "muteins" or "mutant proteins"), as well as new, active members of the OP/BMP family of proteins.

Particularly useful sequences include those comprising the C-terminal seven cysteine domains of mammalian, preferably human, human OP-1, OP-2, OP-3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP8 and BMP9. Other proteins useful in the practice of the invention include active forms of GDF-5, GDF-6, GDF-7, DPP, Vgl, Vgr-1, 60A, GDF-1, GDF-3, GDF-6, GDF-7, BMP10, BMP11, BMP13, BMPI5, UNIVIN, NODAL, SCREW, ADMP or WO 97/41881 PCT/US97/07816 -18- NURAL and amino acid sequence variants thereof. In one currently preferred embodiment, the renal therapeutic agents of the invention are selected from any one of: OP-1, OP-2, OP-3, BMP2, BMP3, BMP4, BMP5, BMP6, and BMP9.

Publications disclosing these sequences, as well as their chemical and physical properties, include: OP-1 and OP-2: U.S. Pat. No. 5,011.691, U.S. Pat. No. 5,266,683, and Ozkaynak et al.

(1990), EMBO J. 9:2085-2093; OP-3: W094/10203; BMP2, BMIP3, and BMP4: U.S. Pat.

No. 5,013,649, W091/18098, W088/00205, and Wozney et al. (1988), Science 242:1528-1534; BMPS and BMP6: W090/11366 and Celeste et al. (1991), Proc. Natl. Acad. Sci. (USA) 87:9843-9847; Vgr-I: Lyons et al. (1989), Proc. Natl. Acad. Sci. (USA) 86: 4554-4558; DPP: Padgett et al. (1987), Nature 325:81-84; Vgl: Weeks (1987), Cell 51:861-867; BNMP-9: W095/33830; BNMP10. W094/26893; BMP-l 1: W094/26892; BMP12: W095/16035; BMP-13: W095/16035; GDF-1: W092/00382 and Lee et al. (1991), Proc. Natl. Acad. Sci.

(USA) 88:4250-4254; GDF-8: W094/21681; GDF-9: W094/15966; GDF-10: W095/10539; GDF-1 1: W096/01845; BMP-15: W096/36710; NMP121: W096/01316; GDF-5 (CDMP-1, 15 MP52): W094/15949, W096/14335, W093/16099 and Storm et al. (1994), Nature 368:639- 643; GDF-6 (CDMP-2, BMPI3): W095/01801, W096/14335 and W095/10635; GDF-7 (CDMP-3, BIMP12): W095/10802 and W095/10635; BMP-3b: Takao, et al. (1996), Biochem.

Biophvs. Res. Comm. 219:656-662; GDF-3: W094/15965; 60A: Blaster et al. (1993), Cell 73:687-702 and GenBank accession number L12032. In another embodiment, useful proteins include biologically active biosynthetic constructs, including novel biosynthetic proteins and chimeric proteins designed using sequences from two or more known OP/BMP family proteins.

See also the biosynthetic constructs disclosed in U.S. Pat. No. 5,011,691, the disclosure of which is incorporated herein by reference COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16).

In other preferred embodiments, the renal therapeutic agents useful herein include 25 therapeutically effective proteins in which the amino acid sequences comprise a sequence sharing at least 70% amino acid sequence "homology" and, preferably, 75% or 80% homology with the C-terminal seven cysteine domain present in the active forms of human OP-l residues 330-431, as shown in SEQ ID NO: 2 of U.S. Pat. No. 5,266,683). In other preferred embodiments, the renal therapeutic agents useful herein include therapeutically effective proteins in which the amino acid sequences comprise a sequence sharing at least 60% amino acid sequence identity and, preferably, 65% or 70% identity with the C-terminal seven cysteine domain present in the active forms of human OP-1. Thus, a candidate amino acid sequence thought to have WO 97/41881 PCT/US97/07816 -19therapeutic efficacy in the present invention can be aligned with the amino acid sequence of the C-terminal seven cysteine domain of human OP-1 using the method of Needleman et al. (1970), L Mol. Biol. 48:443-453, implemented conveniently by computer programs such as the Align program (DNAstar, Inc.). As will be understood by those skilled in the art, homologous or functionally equivalent sequences include functionally equivalent arrangements of the cvsteine residues within the conserved cysteine domain, 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 protein, including their ability to form such intra- or inter-chain disulfide bonds as may be necessary for biological activity. Therefore, internal gaps and amino acid insertions in the candidate sequence are ignored for purposes of calculating the 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. Thus, as used herein, a percentage "homology" between two amino acid sequences indicates the percentage of amino acid residues which are identical or 15 similar between the sequences. "Similar' residues are "conservative substitutions" which fulfill the criteria defined for an "accepted point mutation" in Dayhoffet al. (1978), Atlas of Protein Sequence and Structure Vol. 5 (Suppl. pp. 354-352, Natl. Biomed. Res. Found., Washington, D.C. Thus, "conservative substitutions" are residues that are physically or functionally similar to the corresponding reference residues, having similar size, shape, electric charge, and/or chemical 20 properties such as the ability to form covalent or hydrogen bonds, or the like. Examples of conservative substitutions inciude the substitution of one amino acid for another with similar characteristics, substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine, methionine; and phenylalanine, tyrosine. The term 25 "conservative substitution" or "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid in a given polypeptide chain, provided that the resulting substituted polypeptide chain also has therapeutic efficacy in the present invention.

The renal therapeutic agents of the invention are also characterized by biological activities which may be readily ascertained by those of ordinary skill in the art. Specifically, a renal therapeutic agent of the present invention is capable of inducing chondrogenesis in the Reddi-Sampath ectopic bone assay (Sampath and Reddi (1981), Proc. Natl. Acad. Sci. (USA) WO 97/41881 PCTUS97/07816 78:7599-7603) or a substantially equivalent assay, capable of significantly preventing, inhibiting, delaying or alleviating the progressive loss of renal function in a standard animal model of chronic renal failure, or capable of causing a clinically significant improvement in a standard marker of renal function when administered to a mammal in, or at risk of, chronic renal failure.

The Reddi-Sampath ectopic bone assay is well known in the art as an assay of chondrogenic activity. The assay, which can be easily performed, is described and discussed in, for example, Sampath and Reddi (1981), Proc. Natl. Acad. Sci. (USA) 78:7599-7603; and Wozney (1989), "Bone Morphogenetic Proteins," Progress in Growth Factor Research 1:267- 280. Many equivalent assays, using other animals and tissue sites, may be employed or developed by those of skill in the art to evaluate the biological activity of the renal therapeutic agents of the present invention. See, for example, the bioassays described in U.S. Pat. No. 5,226,683.

The renal therapeutic agents of the present invention also may be tested in animal models of chronic renal failure. Mammalian models of chronic renal failure in, for example, mice, rats, 15 guinea pigs, cats, dogs, sheep, goats, pigs, cows, horses, and non-human primates, may be created by causing an appropriate direct or indirect injury or insult to the renal tissues of the animal.

Animal models of chronic renal failure may, for example, be created by performing a partial 5/6) nephrectomy which reduces the number of functioning nephron units to a level which initiates compensatory renal hypertrophy, further nephron loss, and the progressive decline in renal function which characterizes chronic renal failure.

Finally, the renal therapeutic agents of the present invention may be evaluated for their therapeutic efficacy in causing a clinically significant improvement in a standard marker of renal function when administered to a mammalian subject a human patient) in, or at risk of, .chronic renal failure. Such markers of renal function are well known in the medical literature and 25 include, without being limited to, rates of increase in BUN levels, rates of increase in serum creatinine, static measurements of BUN, static measurements of serum creatinine, glomerular filtration rates (GFR), ratios of BUN/creatinine, serum concentrations of sodium urine/plasma ratios for creatinine, urine/plasma ratios for urea, urine osmolality, daily urine output, and the like (see, for example, Brenner and Lazarus (1994), in Harrison's Principles of Internal Medicine. 13th edition, Isselbacher et al., eds., McGraw Hill Text, New York; Luke and Strom (1994), in Internal Medicine, 4th Edition, J.H. Stein, ed., Mosby-Year Book, Inc. St.

Louis.) WO 97/41881 PCT/US97/07816 -21 The renal therapeutic agents contemplated herein can be expressed from intact or truncated genomic or cDNA or from synthetic DNAs in prokaryotic or eukaryotic host cells. The dimeric proteins can be isolated from the culture media and/or refolded and dimerized in vitro to form biologically active compositions. Heterodimers can be formed in vitro by combining separate, distinct polypeptide chains. Alternatively, heterodimers can be formed in a single cell by co-expressing nucleic acids encoding separate, distinct polypeptide chains. See, for example, W093/09229, or U.S. Pat. No. 5,411,941, for several exemplary recombinant heterodimer protein production protocols. Currently preferred host cells include, without limitation, prokaryotes including E. coli, or eukaryotes including yeast, Saccharomyces, insect cells, or mammalian cells, such as CHO, COS or BSC cells. One of ordinary skill in the an will appreciate that other host cells can be used to advantage. Detailed descriptions of the proteins useful in the practice of this invention, including how to make, use and test them for chondrogenic activity, are disclosed in numerous publications, including U.S. Pat. Nos. 5,266,683 and 5,011,691, the disclosures of which are herein incorporated by reference.

C. Morphogens. Inducers and Aeonists *Table 1, below, summarizes various naturally occurring members of the OP/BMP family 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 I is intended and should be understood to embrace the therapeutically effective proteins expressed from nucleic acids encoding the identified sequence menuoned below and set fonli in the Sequence Listing, or an active fragment or precursor thereof, or a functional equivalent thereof such as a naturally occurring or biosynthetic variant. Naturally occurring variants include allelic variant forms isolated from other individuals of a single biological species, as well as species variants (homologues) isolated from phylogenetically distinct biological species.

TABLE 1 "OP-1" Refers generically to mammalian proteins equivalent to the human OP-1 protein disclosed in SEQ ID NO: 4 and includes at least mouse OP-1, SEQ ID NO: 5 In each of human and mouse OP-1, SEQ ID NOs: 4 and 5, the conserved C-terminal seven cysteine domain is defined by residues 38 to 139.

cDNA sequences and corresponding amino acid sequences for the full length WO 97/41881 I'CTI/US97/07816 -22proteins are provided in SEQ LD NOs: 15 and 16 (hOP-1) and SEQ ID NOs: 17 and 18 (mOP-1.) The mature proteins are defined by residues 293-431 (hOP-1) and 292-430 (mOP-1). The "pro" regions of the proteins, cleaved to yield the mature proteins are defined essentially by residues 30-292 (hOP-1) and residues 30-291 (mOP-1).

"OP-2" Refers generically to mammalian proteins equivalent to the human OP-2 protein disclosed in SEQ ID NO: 6 and includes at least mouse OP-2 ("mOP- SEQ ID NO: In each of human and mouse OP-2, the conserved C-terminal seven domain is defined by residues 38 to 139 of SEQ ID NOs: 6 and 7. cDNA sequences and corresponding amino acid sequences for the full length proteins are provided in SEQ ID NOs: 19 and 20 (hOP-2) and SEQ ID NOs: 21 and 22 (mOP- The mature proteins are defined essentially by residues 264-402 (hOP-2) and 261-399 (mOP-2). The "pro" regions of the proteins, cleaved to yield the mature proteins are defined essentially by residues 18-263 (hOP-2) and residues 18-260 15 (mOP-l).

"OP-3" Refers generically to mammalian proteins equivalent to the mouse QP-3 protein disclosed in SEQ ID NO: 26 The conserved C-terminal seven domain is defined by residues 298 to 399 of SEQ ID NO: 26, which shares greater than 79% amino acid identity with the corresponding mOP-2 and hOP-2 sequences, and 20 greater than 66%/ identity with the corresponding OP-1 sequences. A cDNA sequence encoding the above-mentioned amino acid sequence is provided in SEQ ID NO: 25. OP-3 is unique among the morphogens identified to date in that the residue at position 9 in the conserved C-terminal seven domain residue 315 of SEQ ID NO: 26) is a serine, whereas other morphogens typically have a tryptophan at this location.

"BMP-2" Refers generically to mammalian proteins equivalent to the BMP-2 proteins, including at least human BMP-2 (or CBMP-2A, SEQ ID NO: The amino acid sequence for the full length proteins, referred to in the literature as BMP-2 or BMP-2A, appear in Wozney, et al. (1988) Science 242:1528-1534. The pro WO 97/41881 PCT/US97/07816 -23domain for BMP-2 (BMP-2A) likely includes residues 25-248; the mature protein, residues 249-396.

"BMP-4" Refers generically to mammalian proteins equivalent to the CBMP-4 proteins, including at least human BMP-4 (or BMP-2B, SEQ ID NO: The amino acid sequence for the full length proteins, referred to in the literature as BMP-4 and BMP-2B, appear in Wozney, et al. (1988) Science 242:1528-1534. The pro domain for BMP-4 (BMP-2B) likely includes residues 25-256; the mature protein, residues 257-408.

"DPP" refers to proteins encoded by a Drosophila DPP gene and defining a conserved Cterminal seven domain (SEQ ID NO: 10). The amino acid sequence for the full length protein appears in Padgett, et al. (19S7) Nature 325:81-84. The pro domain likely extends from the signal peptide cleavage site to residue 456; the mature protein likely is defined by residues 457-588.

"Vgl" refers to proteins encoded by a Xenopus Vgl gene and defining a conserved Cterminal seven domain (SEQ ID NO: 1 The amino acid sequence for the full length protein appears in Weeks (1987) Cell 51:861-867. The prodomain likely extends from the signal peptide cleavage site to residue 246; the mature protein likely is defined by residues 247-360.

"Vgr-l" refers to proteins encoded by a murine Vgr-1 gene and defining a conserved Cterminal seven domain (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; the mature protein likely is defined by residues 300-438.

"GDF-1" refers to proteins encoded by a human GDF-1 gene and defining a conserved Cterminal seven domain (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.

WO 97/41881 PCT/US97/07816 -24- "BMP-3" "BMP-5" refers generically to proteins expressed from a nucleic acid the Drosophila gene) encoding a 60A protein or active fragments thereof (see SEQ ID NOs: 23 and 24 wherein the cDNA and encoded amino acid sequence for the full length protein are provided). The conserved C-terminal seven domain is defined by residues 354 to 455 of SEQ ID NO: 24. The prodomain likely extends from the signal peptide cleavage site to residue 324; the mature protein likely is defined by residues 325-455. The 60A protein is considered likely to be a phylogenetic counterpart of the human and mouse OP-I genes; Sampath, et al. (1993) PNAS 90:6004-6008.

refers to proteins encoded by a human BM.P-3 gene and defining a conserved Cterminal seven domain (SEQ ID NO: 26). 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 site to residue 290; the mature protein likely is defined by residues 291-472.

refers to proteins encoded by a human BMP-5 gene and defining a conserved Cterminal seven domain (SEQ ID NO: 27). 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; the mature protein likely is defined by residues 317-454.

refers to proteins encoded by a human BMP-6 gene and defining a conserved Cterminal seven domain (SEQ ID NO: 28). The amino acid sequence for the full length protein appears in Celeste, et al. (1990) PNAS 87:9843-5847. The pro domain likely includes extends from the signal peptide cleavage site to residue 374; the mature sequence likely includes residues 375-513.

"BMP-6" As shown in Figure 7, the OP-2 and OP-3 proteins have an additional cysteine residue in the conserved C-terminal region see residue 41 of SEQ ID NOs: 6 and The GDF-1 protein has a four amino acid insert within the conserved C-terminal cysteine domain (residues 44-47 of SEQ ID NO: 13). Further, the BMP-2 and BMP-4 proteins are missing one amino acid residue within the cysteine domain. Thus, the alignment of these amino acid WO 97/41881 PCTIUS97/07816 sequences in Figure 7 illustrates the principles of alignment used herein with respect to the preferred reference sequence of human OP-1, residues 38-139 of SEQ ID NO: 4.

In addition to the OP/BMP renal therapeutic agents described in the previous section, the present invention may be practiced using "morphogens," as defined herein. Morphogens useful in the present invention include those in which the amino acid sequences ofmorphogen polypeptides comprise a sequence sharing at least 70% amino acid sequence homology or "similarity", and preferably 80% homology or similarity with a reference sequence selected from the foregoing naturally OP/BMP family members. Preferably, the reference protein is human OP-1, and the reference sequence thereof is the C-terminal seven cysteine domain present in active forms of human OP-1, residues 38-139 of SEQ ID NO: 4. Morphogens useful herein accordingly include allelic, phylogenetic counterpart 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 OP/BMIP family of proteins set forth and identified above, in S* connection with Table I. 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 morphogen polypeptides useful in the present invention are defined by a generic amino acid sequence. For example, Generic Sequence 7 (SEQ ID NO: I) and Generic Sequence 8 (SEQ ID NO: 2) disclosed below, accommodate the 20 homologies shared among preferred OP/BMP protein family members identified to date, including at least OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, 60A, DPP, Vgl, BMP-5, BMP-6, Vgr-1, and GDF-1 (SEQ ID NOs: 4-15, 24, and 26-29). 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 domains (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 domain 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 active sequences of OP-2 and OP-3.

Generic Sequence 7 Leu Xaa Xaa Xaa Phe Xaa Xaa WO 97/41881 WO 971881PCTIUS97/07816 26 Xaa Gly Trp Xaa Xaa Xaa Xaa Cys Xa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Me: Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Ty r Cys Xaa Pro Xaa Xaa Xaa Asn. His Ala Xa a Xzaa Xaa Xaa Xaa Xaa Xaa Cyrs Cys Xaa Pro Xaa Xaa Xaa Leu Xaa Xaa Val Xaa Leu Xaa Xaa Val Xaa Xaa Cys Xaa Xaa Pro Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 4 wherein each Xaa independently is selected from a group of one or more specified aino acids defined as follows: "Res." means "residue" and Xaa at res.2 (Tyr or Lys);- Xaa at res.3 Val or lie); Xaa at res.4 (Ser, Asp or Giu);, Xaa at res.6 =(Arg, Gin, Ser, Lys or Ala);- Xaa at res.7 (Asp or Giu); Xaa at res.8 (Leu, Val or Ile); Xaa at res. 11I (Gin, Leu, Asp, His, Asn or Ser); Xaa at res. 12 (Asp, Arg, Asn or Giu); Xaa at res. 1 3 (Trp or Ser); Xaa at res.i14 (le or Val); Xaa at res. 15 (lie or Val); Xaa at res. 16 (Ala or Ser); Xaa at res. 18 (Giu, 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, Hs, 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 atl res.28 (Giu, Lys, Asp, Gin or Ala); Xaa at res.30 (Ala, Ser, Pro, Gin, Ilie 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, Hs, Ser or lie); Xaa at res.37 =(Met, Phe, Gly or Leu); Xaa at res.38 (Asn, Ser or Lys); Xaa at res.39 (Ala, Ser, Giy or Pro); Xaa at res.40 (Thr, Leu or Ser); Xaa at res.44 (Ilie, Val or Thr); Xaa at res.45 (Val, Leu, Met or fie); Xaa at res.46 (Gin or Mg); Xaa at res.47 =(Thr, Ala or Ser); Xaa at res.48 (Leu or Ilie); Xaa at res.49 (Val or Met); Xaa at res.5O (His, Asn or Mrg); Xaa at res.51 (Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52 (lie, Met, Asn, Ala, Val, Gly or Leu); Xaa at res.53 (Asn, Lys, Ala, Giu, Gly or Phe); Xaa at res.54 (Pro, Ser or Val), Xaa at res.55 (Giu, Asp, Asn, Giy, Val, Pro or Lys), Xaa at res.56= 4* *4 WO 97/41881 PCT/US97/07816 -27- (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 (Gin, Lys, Arg or Glu); Xaa at res.67 (Leu, Met or Val); Xaa at res.68 (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 (Aso, Thr or Lys); Xaa at res.82 (Ile, Val or Asn); Xaa at res.84 (Lys or Arg); Xaa at (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 Ile); 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 shified bv ive in GeCeric Sequence 8 Thus. "Xaa at res.2 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 preferred reference sequence of hOP-1. These particularly preferred sequences include allelic and phylogenetic counterpart variants of the OP-i and OP-2 proteins, including the Drosophila 60A protein. Accordingly, in certain particularly preferred embodiments, useful morphogens include active proteins comprising pairs of polypeptide chains within the generic amino acid sequence herein referred to as "OPX" (SEQ ID NO: which defines the seven cysteine domain and accommodates the homologies between several identified WO 97/41881 PCT/US97/07816 -28variants of OP-1 and OP-2. 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- or OP-2 (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, Cterminal sequences defining the conserved C-terminal seven domains of OP-1 or OP-2, 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°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 15 this invention to produce heterodimers. Thus, members of a folded pair of morphogen i. polypeptides in a morphogenically active protein can be selected independently from any of the specific morphogen polypeptides mentioned above. 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. The morphogens generally are competent to induce all of the following biological functions 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.

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 structure. Accordingly, such active forms are considered the equivalent of the specifically described constructs disclosed herein. The proteins WO 97/41881 PCT/US97/07816 -29may include forms having varying glycosylation patterns, varying N-termini, a family of related proteins having regions of amino acid sequence homology, and active truncated or mutated forms of native or biosynthetic proteins, produced by expression of recombinant DNA in host cells.

Figure 7 herein sets forth an alignment of the amino acid sequences of the active regions of naturally occurring proteins that have been identified or appreciated herein as OP/BMP renal therapeutic agents, including human OP-1 (hOP-1, SEQ ID NOs: 4 and 15-16), mouse OP-I (mOP-1, SEQ ID NOs: 5 and 17-18), human and mouse OP-2 (SEQ ID NOs: 6, 7, and 19-22), mouse OP-3 (SEQ ID NOs: 25-26), BMP-2 (SEQ ID NO: BMP-4 (SEQ ID NO: BMP-3 (SEQ ID NO: 27), DPP (from Drosophila, SEQ ID NO: 10), Vgl, (from Xenopus, SEQ ID NO: 11), Vgr-I (from mouse, SEQ ID NO: 12), GDF-l (from mouse and/or human, SEQ ID NOs: 13, 30 and 31), 60A protein (from Drosophila, SEQ ID NOs: 23 and 24), BMP-5 (SEQ ID NO: 28) and BMP-6 (SEQ ID NO: 29). The sequences are aligned essentially following the .o*o method of Needleman, et al. (1970) J. Mol. Biol., 48:443-453, calculated using the Align Program (DNAstar, Inc.). In Figure 7, three dots indicates that the amino acid in that position is 15 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 of BMP-2 (CBMP-2A) and BMP-4 (CBMP-2B) is "missing." Of course, both of these amino acid sequences in this region comprise Asn-Ser (residues 58, 59), with BMP- 2 then comprising Lys and Ile, whereas BMP-4 comprises Ser and Ile. Figure 7 also illustrates the handling of insertions in the morphogen amino acid sequence: between residues 56 and 57 of BMP-3 is an inserted Val residue; between residues 43 and 44 of GDF-I 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, GDF-1 and hOP-1. As is apparent from the amino acid sequence comparisons set forth in Figure 7, significant amino acid changes can be made from the reference sequence while retaining activity. For example, while the GDF-I protein sequence depicted in Figure 7 shares only about 50% amino acid identity with the hOP-1 sequence described therein, the GDF-1 sequence shares greater than 70% amino acid sequence homology (or "similarity") with the hOP-1 sequence, where "homology" or "similarity" includes allowed conservative amino acid substitutions within the aligned sequence, as defined by Dayhoff, et al. (1979) 5 Atlas of Protein Sequence and Structure Suppl. 3, pp. 345- 362, Dayhoff, ed., Natl. BioMed. Res. Found., Washington WO 97/41881 PCT/US97/07816 -31 morphogen-mediated responses induction of differentiation of metanephric mesenchyme, induction of endochondral 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.

Finally, in other embodiments cells may be implanted into the kidney of a subject in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, in order to serve as a source of an OP/BMP renal therapeutic agent or morphogen and/or to provide a source of additional functional renal tissue. Such cells may be host or donor cells which normally express OP/BMP renal therapeutic agents or morphogens, which have been transformed so as to express OP/BMP renal therapeutic agents or morphogens, or which have been treated with OP/B1MP renal therapeutic agents or morphogens.

0 D. Subjects for Treatment As a general matter, the methods of the present invention may be utilized for any 15 mammalian subject in, or at risk of, chronic renal failure, or at risk of the need for renal o* replacement therapy chronic dialysis or renal transplant). 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 o*o.e* 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 vaiue captive or free specimens of endangered species), or which otherwise have value. In addition, as a general matter, the subjects for treatment with the methods of the present invention need not present indications for treatment with an OP/BMP renal therapeutic agent or morphogen other than those indications associated with risk of chronic 00 renal failure. That is, the subjects for treatment are expected to be otherwise free of indications for treatment according to the present invention. In some number of cases, however, the subjects may present with other symptoms osteodystrophy) for which treatment with an OP/BMP renal therapeutic agent or morphogen would be indicated. In such cases, the treatment should be adjusted accordingly so to avoid excessive dosing.

One of ordinary skill in the medical or veterinary arts is trained to recognize subjects which may be at a substantial risk of chronic renal failure, or at substantial risk of the need for renal replacement therapy. In particular, clinical and non-clinical trials, as well as accumulated WO 97/41881 PCT/US97/07816 -32 experience, relating to the presently disclosed and other methods of treatment, are expected to inform the skilled practitioner in deciding whether a given subject is in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, 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 being in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, if that subject has already been diagnosed as afflicted with, or would be regarded as being afflicted with, a condition which typically leads to progressive loss of renal function associated with progressive loss of functioning nephron units. Such conditions include, but are not limited to, chronic renal failure, end-stage renal disease, chronic diabetic nephropathy, hypertensive nephrosclerosis, chronic glomerulonephritis, hereditary nephritis, renal dysplasia and the like. These, and other diseases and conditions known in the art, typically lead to a progressive loss of functioning nephrons and to the onset of chronic renal failure.

Frequently, one of skill in the medical or veterinary arts may base a prognosis, diagnosis 15 or treatment decision upon an examination of a renal biopsy sample. Such biopsies provide a wealth of information useful in diagnosing disorders of the kidney but, due to the invasiveness of the procedure, and the additional trauma to a presumably unhealthy kidney, may not be appropriate for all subjects. Nonetheless, subjects in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, may be recognized by histological indications from renal 20 biopsies including, but not limited to, glomerular hypertrophy, tubular hypertrophy, glomerulosclerosis, tubulointerstitial sclerosis, and the like.

Less invasive techniques for assessing kidney morphology include MRI, CAT and ultrasound scans. Scanning techniques are also available which employ contrasting or imaging agents radioactive dyes) but, it should be noted, some of these are particularly toxic to renal 25 tissues and structures and, therefore, their use may be ill-advised in subjects in, or at risk of, *o chronic renal failure. Such non-invasive scanning techniques may be employed to detect conditions such as renal fibrosis or sclerosis, focal renal necrosis, renal cysts, and renal gross hypertrophy which will place a subject in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy.

Quite frequently, prognosis, diagnosis and/or treatment decisions are based upon clinical indications of renal function. One such indication is the presence in urinary sediment of an unusual number of "broad" or "renal failure" casts, which is indicative of tubular hypertrophy and WO 97/41881 PCT/US97/07816 -33suggests the compensatory renal hypertrophy which typifies chronic renal failure. A better indication of renal function is the glomerular flow rate (GFR), which can be measured directly by quantifying the rate of clearance of particular markers, or which may be inferred from indirect measurements.

It should be noted that the present invention is not directed to the measurement of GFR or to the diagnosis of chronic renal failure. The methods of treatment of the present invention need not, therefore, be restricted to subjects presenting with any particular measures ofGFR, or any other particular marker of renal function. Indeed, it is not necessary that the GFR of a subject, or any other particular marker of renal function, be determined before practicing the treatments of the present invention. Nonetheless, the measurement of GFR is considered to be a preferred means of assessing renal function.

As is well known in the art, GFR reflects the rate of clearance of a reference or marker compound from the plasma to the urine. The marker compound to be considered is typically one i which is freely filtered by the glomeruli, but which is not actively secreted or reabsorbed by the renal tubules, and which is not significantly bound by circulating proteins. The rate of clearance is typically defined by the formula, presented above, which relates the volume of urine produced in a twenty-four period, and the relative concentrations of the marker in the urine and plasma. To be more accurate, the GFR should also be corrected for body surface area. The "gold standard" reference compound is inulin because of its filtration properties and lack of serum-binding. The 20 concentration of this compound is, however, difficult to quantify in blood or urine. The clearance rates of other compounds, including p-aminohippurate (PAH) and creatinine, are therefore often used instead of inulin. In addition, various formulas are often employed which seek to simplify the estimation of actual GFR by omitting considerations of actual urine concentrations of the ooo marker, actual daily volumes of urine produced, or actual body surface area. These values may be 25 replaced by estimates based on other factors, by baseline values established for the same subject, or by standard values for similar subjects. These estimates should be used with caution, however, as they may entail inappropriate assumptions based upon the renal function of normal or healthy subjects.

Various methods and formulas have been developed in the art which describe an expected value of GFR for a healthy subject with certain characteristics. In particular, formulas are available which provide an expected value of the GFR based upon plasma creatinine levels, age, weight and sex. One such formula for an expected GFR is presented above. Other formulas may, WO 97/41881 PCT/US97/07816 -34of course, be employed and tables of standard values may be produced for subjects of a given age, weight, sex, and/or plasma creatinine concentration. Newer methods of measuring or estimating GFR using NMR or MUR technologies) are also now available in the art and may be used in accordance with the present invention (see, U.S. Pat. Nos. 5,100,646 and 5,335,660).

As a general matter, irrespective of the manner in which GFR is measured or estimated, a subject may be considered to be in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, when the subject has a GFR which is chronically less than about 50% of the expected GFR for that subject. The risk is considered greater as the GFR falls lower. Thus, a subject is increasingly considered at risk if the subject has a GFR which is chronically less than about 40%, 30% or 20% of the expected GFR.

As a general matter, irrespective of the manner in which GFR is measured or estimated, a human male subject weighing at least about 50 kg may be considered to be in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, when the subject has a GFR S* which is chronically less than about 50 ml/min. The risk is considered greater as the GFR falls 15 lower. Thus, a subject is increasingly considered at risk if the subject has a GFR which is chronically less than about 40, 30 or 20 ml/min.

As a general matter, irrespective of the manner in which GFR is measured or estimated, a human female subject weighing at least about 40 kg may be considered to be in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, when the subject has a GFR which is chronically less than about 40 ml/min. The risk is considered greater as the GFR falls lower. Thus, a subject is increasingly considered at risk if the subject has a GFR which is chronically less than about 30, 20 or 10 ml/min.

By a employing a variety of methods, including the histological examinations, non-invasive scanning procedures, evaluations of clinical indicators, and other techniques described above and 25 known in the art, those in the medical and veterinary arts may provide estimates of either the number of functioning nephron units which a subject possesses, or the percentage of functioning nephron.units which a subject possesses relative to a healthy but otherwise similar subject a conspecific subject of approximately the same age, weight, and sex). Thus, for example, a biopsy may reveal a decrease in the density of functional nephrons, or imaging with filtered agents may indicate losses of functional renal tissue and/or filtering capacity. Such measures or estimates provide another means of expressing when a subject is in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy. Thus, as a general matter, a subject may be regarded WO 97/41881 PCT/US97/07816 to be in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, if that subject possesses a number of functional nephron units which is less than about 50% of the number of functional nephron units of a healthy, but otherwise similar, subject. As above, the risk is considered greater as the number of functional nephrons decreases further. Thus, a subject is increasingly considered at risk if the subject has a number of functional nephrons which is less than about 40, 30 or 20% of the number for a similar but healthy subject.

Finally, it should be noted that subjects possessing a single kidney, irrespective of the manner of loss of the other kidney physical trauma, surgical removal, birth defect), may be considered to be prima facie at risk of chronic renal failure, or the need for renal replacement therapy. This is particularly true for those subjects in which one kidney has been lost due to a disease or condition which may afflict the remaining kidney. Similarly, subjects which are already recipients of a renal transplant, or which are already receiving chronic dialysis chronic •hemodialysis or continuous ambulatory peritoneal dialysis) may be considered prima facie to be at risk of chronic renal failure, or the need for further renal replacement therapy.

S. 15 E. Formulations and Methods of Treatment SThe OP/BMP renal therapeutic agents, morphogens, morphogen inducers, or agonists of morphogen receptors of the present invention may be administered by any route which is compatible with the particular morphogen, inducer, or agonist employed. Thus, as appropriate, administration may be oral or parenteral, including intravenous, intraperitoneal, and renal 20 intracapsular routes of administration. In addition, administration may be by periodic injections of a bolus of the agent, 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).

The therapeutic agents of the invention may be provided to an individual by any suitable 25 means, preferably directly locally, as by injection or topical administration to a tissue locus) or systemically parenterally or orally). 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. The solution is physiologically acceptable so that in addition to delivery of the desired agent to the patient, the solution does not otherwise adversely affect the patient's electrolyte and/or volume balance. The aqueous medium for the agent thus may comprise normal WO 97/41881 PCT/US97/07816 -36physiologic saline 9.85% NaCI, 0. 15M, pH Such an aqueous solution containing the agent can be made, for example, by dissolving the agent in 50% ethanol containing acetonitrile in 0.1% trifluoroacetic acid (TFA) or 0.1% HCI, 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.

If desired, an agent may be made more soluble by association with a suitable molecule.

For example, association of the mature OP/BMP or morphogen dimer with the pro domain results in the pro form of the protein which typically is more soluble or dispersible in physiological solutions than the corresponding mature form. In fact, endogenous OP/BMP proteins 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 mammalian cells, cells 0.0* transfected with nucleic acid encoding and competent to express the OP/BMP protein or morphogen. Alternatively, a soluble species can be formulated by complexing the mature dimer 15 (or an active fragment thereof) with a 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 OP-I by 80%. Other components found in milk and/or various sees* serum proteins also may be useful.

Finally, as noted above, in another series of embodiments renal cells may be implanted into the kidney of a subject in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, in order to serve as a source of an OP/BMP renal therapeutic agent or morphogen and/or to provide a source of additional functional renal tissue. These cells may be any compatible mammalian cells, including renal mesenchymal progenitor cells, or renal mesenchymal progenitor cells which have been induced to undergo metanephric differentiation.

The cells may be derived from a donor a tissue-type matched donor, sibling, identical twin), may be derived from a tissue culture undifferentiated renal mesenchyme culture, fetal renal tissue culture), or may be explanted from the subject and then be re-implanted after proliferation and/or differentiation. Preferably, the cells are induced to undergo metanephric differentiation by treatment with an OP/BMP renal therapeutic agent or morphogen OP-1) either before or after implantation. Thus, for example, renal mesenchymal progenitor cells may be explanted from a subject, allowed or caused to proliferate in vitro, be induced to undergo metanephric WO 97/41881 PCTIUS97/07816 -37differentiation by morphogen treatment, and be re-implanted where they may provide a source of morphogen and/or differentiate further into functional renal tissue.

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 Rat Remnant Kidney Model A rat partial nephrectomy or rat remnant kidney model (RRKM) model was employed essentially as described (Vukicevic, et al. (1987) J. Bone Mineral Res. 2:533). Male rats (2-3 months old, weighing about 150-200 g) were subjected to unilateral nephrectomy (either left or right kidney). After approximately one week, 2/3 of the remaining kidney was surgically removed. Immediately following surgery, plasma creatinine and BUN levels rise dramatically due S' to the loss of renal mass and function. Over the next several weeks of this "acute" failure phase, plasma creatinine and BUN levels of surviving animals decline somewhat toward normal values S 15 but remain elevated. Renal function then appears to remain relatively constant or stable for a o period of variable duration. After this point, the animals enter a period of chronic renal failure in which there is an essentially linear decline in renal function ending in death.

As surgical controls, additional rats were subjected to a "sham" operation in which the kidneys were decapsulated but no renal tissue was removed.

Intervention Model for Chronic Renal Failure In this model, both nephrectomized and sham-operated rats were maintained for approximately 5-6 months after surgery. At this point, surviving nephrectomized animals were past the stable phase and had entered chronic renal failure.

~Rats were divided into 8 groups with 12 rats in each group. Two groups of 25 nephrectomized rats were used as controls (Nx controls), with one of those groups receiving no treatment at all, while the other received injections of only the vehicle buffer. In addition, two groups of sham-operated rats were used as controls (sham controls), with one group receiving only the vehicle buffer, while the other received soluble OP-l (sOP-1) at 10 pg/kg body weight.

Four experimental groups of nephrectomized rats were employed, receiving sOP-1 at 1, 3, 10 or 50 pg/kg body weight by intraperitoneal injection (OP-1 Nx animals). OP-1 treated and vehicleonly rats received three injections per week for 4-8 weeks. Total injection volume was 300 pl.

WO 97/41881 PCTIUS97/07816 -38- No statistically significant differences were observed between the two Nx control groups or between the two sham control groups.

Compared to the sham group receiving only vehicle, the Nx control receiving only vehicle demonstrated significantly (p 0.01) elevated serum creatinine (Figure I) at the end of the study, indicating a significant loss of renal function Although nephrectomized rats treated with either 1 or 3 pg/kg body weight sOP-1 did not show significantly reduced serum creatinine when compared to the Nx control, nephrectomized rats treated with sOP-1 at doses of 10 or 50 pg/kg body weight showed significant (p 0.05) reductions in creatinine values (Figure Similar results were observed for serum urea levels: Although nephrectomized rats treated with either I or 3 pg/kg body weight sOP-1 did not show significantly reduced serum urea when compared to the Nx control, nephrectomized rats treated with sOP-1 at doses of 10 or 50 pg/kg body weight showed significant (p 0.01) reductions in serum urea values (Figure All nephrectomized rats showed significantly (p 0.01) higher serum urea when compared to the sham-operated rats (Figure 2).

15 Histological observations indicate that, in contrast to the vehicle treated Nx control group, OP-l treated nephrectomized rats exhibit relatively normal glomerular histology,. Figure 3, for example, shows typical renal samples from normal rat kidney, untreated Nx control animals, and OP-I treated nephrectomized rats under low magnification (10x). Figure 4 shows similar samples under higher magnification (40x). Histomorphometric analysis indicates 20 that OP-I Nx rats showed reduced incidence of glomerular sclerosis and loop collapse, relatively scattered sclerosis and microaneurysms, and more viable glomeruli compared to Nx control rats (Table 2).

None of the rats died in any group during this study.

Prophylactic Model for Chronic Renal Failure 25 Rats were subjected to partial nephrectomies or sham-operated as described above. In this model, in order to test the ability of OP/BMP renal therapeutic agents to prevent, inhibit or delay the initiation of chronic renal failure, the rats were allowed to recover for approximately two weeks after surgery before initiation of OP-1 therapy. At this point, surviving animals were past the acute renal failure phase and had not yet entered chronic renal failure.

Rats were divided into two groups of 15-20 rats. One group received only vehicle buffer (Nx control) whereas the other received OP-1 treatment at 10 pg/kg body weight given WO 97/41881 PCT/US97/07816 -39intraperitoneally three times per week. Administration of OP-1 or vehicle continued for a period of approximately 8-9 weeks.

During weeks 1-5 of treatment, both groups showed elevated serum creatinine 100 pmol/L) relative to sham-operated controls (35 7 pmol/L). At about 5 weeks, both groups began to show a rise in serum creatinine suggesting the onset of progressive or chronic renal failure. The rise in serum creatinine was, however, markedly less rapid in the OP-1 treated group and was significantly lower than in the Nx controls (Figure 5: p 0.02 at weeks 6 and 8, p 0.01 at weeks 7 and Similar results were observed in serum BUN values as well.

More important, measurements of GFR, based on serum and urine creatinine values, showed a highly significant decrease in both groups of nephrectomized rats 1.8 ml'min) relative to sham-operated controls (4.7 1.1 m/min). The GFR in both groups continued to decline during weeks 1-3 of treatment. At approximately three weeks, however, GFR in the OP-1 treated group stabilized whereas the decline in renal function continued in the Nx controls. By week the difference in GFR values between OP-1 treated and Nx control rats had become statistically significant (p 0.02). This difference in GFR continued to increase over time (p 0.01 at week 6; p 0.001 at weeks 7 and as the Nx controls continued to decline but the OP-1 treated rats remained stable (Figure By the end of 9 weeks, 40% of the Nx control rats were dead whereas none of the OP-I treated rats had died.

Histological evaluation of tissue sections confirmed that OP-1 treated rats showed greater preservation or maintenance of glomeruli. as well as proximal and distal tubule structures. There were also signs in the OP-1 treated rats of nephrogenic mesenchymal condensations and the :appearance of developmental nephrogenic structures. Table 2 reports results of several standard quantitative PAS-staining of extracellular matrix) and semi-quantitative visual ranking) histomorphometric measures obtained for tissue slices from Nx control and OP-I treated Nx rats. These results indicate that OP-1 treatment of nephrectomized rates resulted in overall improvement (or reduced degeneration) of kidney tissue morphology, increased mesangial or perivascular thickening, decreased glomerular sclerosis and loop collapse, decreased presence of "scattered" sclerosis and microaneurysms, and an increase in viable glomeruli.

WO 97/41881 PCT[US97/07816 40 TABLE2 Equivalents 0e

S

0 S S

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-S

t 0 6

*SSS

0*

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@0 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 97/41881 PCT/US97/07816 -41 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: CREATIVE BIOMOLECULES, INC.

STREET: 45 SOUTH STREET CITY: HOPKINTON STATE: MA COUNTRY: USA POSTAL CODE (ZIP): 01748 TELEPHONE: 1-508-435-9001 TELEFAX: 1-508-435-0454

TELEX:

(ii) TITLE OF INVENTION: MORPHOGEN TREATMENT FOR CHRONIC RENAL FAILURE (iii) NUMBER OF SEQUENCES: 31 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: CREATIVE BIOMOLECULES, INC.

STREET: 45 SOUTH STREET CITY: HOPKINTON STATE: MA COUNTRY: USA ZIP: 01748 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC comoatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION

DATA:

APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/643,321 FILING DATE: C6-;1*.-i996 (viii) ATTORNEY/AGENT INFORMATION: NAME: TWOMEY, MICHAEL J REGISTRATION NUMBER: 38,349 REFERENCE/DOCKET NUMBER: CRP-118PC (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 WO 9714 1881 P'CTIUS97/07816 42 (ix) FEATURE: NA101E/KEY: Protein LOCATION: 1..97 OTHER INFORMATION: /note= "wherein from a acup cf /label= Generic-Seq-7 each Xaa is independently selected cne or more specified amino acids as defined in the specification." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:!: Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Giv Trp Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asr. Pis Ala Xaa Xaa Xaa so Xaa Xaa Xaa Xaa Xaa Xaa ):aa Xaa Cys Cys Xaa Prc a, Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Met Val Xaa Xaa Cvs Xaa Cys INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARA.CTERISTICS: LENGTH: 102 amino acids B) TYPE: amino acid STPANDEDNESS: 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 WO 97/4 1881 PCTIUS97/078 16 43 25 Xaa Cvs Xaa Xaa Pro Xaa Xaa Xaa Xaa X.1aa Xaa Xaa Zaa Asn His Ala 40 XXaa Xaa Xaa aa Xaa aa Xaa N a Nsa Xaa Xaa a3 Xaa Xaa Xaa so 55 6 Xa:a CO..s vs N a XNa a.s s As aa ,.aa as XNsa Xaa 70 75 Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xsa Xaa Xsa Xaa Xaa i~e t X aa val1 90 Xaa Xsa Cys Xaa Cvs Xaa 100 1NFORNAtTIO'11 FOR SEQ: 20D NO: 3: SEQUENCE CHAZRACTE RIICS: LENGTH: 102 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: orotein (ix) FEATURE: NAME/KEY: Protein LOCATION: 1. .102 OTHER INFORMATION: /label= OPX /note= 111HEREIN EACH XAA IS INDEPENDENTLY SELECTED FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS AS DEFINED IN THE SPEC:FICATION" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xaa Asp Leu Gly Trp Xaa 1 5 10 *.Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala 40 Ile Xaa Gin Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys 55 Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 70 75 Asp Xaa Ser Xaa Asn Vai Xaa Leu Xaa Lys Xaa Arg Asn Met Vai Val 90 WO 97/41881 PCTIUS97/07816 -44- 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 OTHER INFORMATION: /label= hOPl-MATURE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Ser Thr Gly Ser Lys Gln Arg Ser GIn Asn Arg Ser Lys Thr Pro Lys 1 5 10 Asn GCn 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 35 40 Asp Leu Gly Trp Gin Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 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 85 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: WO 97/41881 ~~7Q97/488 1PCT[US97/07816 45 LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: sinule TOPOLOGY: linear (ii) IXOLECULE TYPE: protein (vi ORIGINAL SOURCE: ORGA-'qISY,: MNI TISSUE TYPE: EMBRYO (ix) FEATURE: NAM E/KEY: Protein LOCATION: 1..139 OTHER INFORYIATION: /label= rOPI-MATURE SEQUENCE DESCRIPTION: SEQ 7D Ser Thr Gly Gly Lys Gin Arc S=er 0--n -Sn z _c Se Thr Pro Lys 1 5 i0 Asn Gin Glu Ala Leu Arg Mvet Ala Set Val Ala Glu Asn Set Set Set 25 *Aso Gin Arc Gin Ala Cys Lys Lys His Giu Leu Tyr Val Set Phie Arg 40 Asp Leu Gly Trp Gin Asp Trp Ile Ile Ala Pro Giu Gly Tyr Ala Ala 55 Tyr Tyr Cys Giu Gly Glu Cys Ala Phe Pro Leu Asn Set Tyr Met Asn 6S 70 75 Ala Thr Asn His Ala Ile Val Gin Thr Leu Val His Phe Ile Asn Pro Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala Ile 100 105 110 Set Val Leu Tyr Phe Asp Asp Set Set 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 NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: WO 97/41881 PCT/US97/07816 -46- ORGANISM: HOMO SAPIENS TISSUE TYPE: HIPPOCAMPUS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATICN: /label= HOP2-tATURE (xii SEQUENCE DESCRIFTION: SEQ ID NO:6: Ala Val Arg Pro Leu Arc Arg Arg Gin Pro Lys Lys Ser Asn Glu Leu 10 Pro Gin Ala Asn Arg Leu Pro Gly Ile Phe Asp Asp Val His Gly Ser 25 His Gly Arg Gin Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Gin 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 Ser Phe Pro Leu Asp Ser Cys Met Asn 65 70 75 Ala Thr Asn His Ala ie Leu Gin Ser Leu Val His Leu Met Lys Pro 90 Asn Ala 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: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 NVO 97/41881PCIS7O81 PCTIUS97/07816 47 (xi) SEQUENCE DESCRIPTION: SEQ 1D NO:7: Ala Ala Arg Pro Leu Lys Arq Arc Giln Pro Lys Lys Tlhr Asn C-lu Leu 1 5 10 Pro His Pro As,- L,.s Lcu 'Pz Sly -1 1e 7-L e Asp Aso Sly !H-s GSlyr Se r 25 Arc Gly Arg C-lu Val Cys Arc- Arc- is C-l-u Leu Tyr Val Ser Phe Arg 40 Asp Leu Gly Trp Leu Asp Trp Val ile Ala Pro C-in Cly Tyr Ser Ala 55 Tyr Tyr Cys C-lu Gly C-lu Cys Ala P)he Pro Leu Asp Ser Cys Met Asn 70 75 Ala TrAsn His Ala Ile Leu Gin Ser Leu Val His Leu Met Lys Pro 8z 92 9 Aso Val Val Pro Lys Ala C-vs Cys Ala ?Pro Thr Lys Leu Ser Ala Thr 100 105 110 Ser Val Leu Tyr Asp Ser Ser Asn Asn Val le Leu Arg Lys His *115 120 125 Asn Met Val Val Lys Ala Cys Gly Ci's His **130 135 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 101 amino acids TYPE: amino acid D; TOPOLOGY: lnear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: bovinae

FEATURE:

NAME/KEY: Protein LOCATION: I. .101 OTHER INFORMATION: /label= CBMP-2A-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Ci's Ly's 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 TPyr Cys His Gly 25 WO 97/41881 PCT/US97/078 16 48 Giu 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 0 5 5 Cvs Cys Val Pro Thr G0 Leu Ser Ala lie Ser Met Leu Tyr Leu Asp 70 75 Glu Asn Clu Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu 90 Gly Cys Gly Cys Arg 100 INFORMATION FOR SEQ ID 10:9: SEQUENCE CHARA~CTERISTICS: LENGTH: 101 amino acids (B T7YPE: amino acid STR-ANDEDNESS: single fD) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) OR7GINAL SOURCE: A) ORGANISM: HOMO SAPIENS TISSUE TYPE: hippocampus (ix) FEATURE: NAME/KEY: Protein LOCATION: 1. .101 OTHER INFORM.ATION: /label= CBMP-2B-FX (xi) SEQUENCE DESCRIPTION: SEQ ID 1"0:9: Cys Arg Ara- His Sec Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asm 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 .35 40 Ile Val Gin Thr Leu Val Asm Ser Val Asn. Ser Ser Ile Pro Lys Ala 55 Cys Cys Val Pro Thr Giu 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 WO 97/41881 WO 97/41881PCTIUS9 7/0 78 16 49 INFORMIATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid STRAN DEDNESS: sinole TOPOLOGY: linear (ii) 14OLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: DROSOPHILA NIELAI/ODASTER (ix) FEATURE: NAmE/KEY',: Protein LOCATION: 1.-10I OTHER INFORMATION: /label= DP-FX )xf) SEQUENCE DESCRIPTION: SEQ ID Cys Arg Ara His Ser Leu Ty'r Val Asp Phe Ser Asp Val Gly Tro Asp i 10 Asp Trp Ile Val Ala Pro Leu Gly Tyr Asp Ala Tyvr 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 Me t Leu Tyr Let- Asn Asp Gin Ser Thr Val Val Leu Lys Asn 7yr Gin Glu Met Thr Val *85 90 Val Gly Cys Giy Cl's Arg 100 INFORY±.ATION FOR SEQ ID NO:ii: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: XENOPUS (ix) FEATURE: WO 97/41881 PCTJUS97/078 16 50 NAME/KEY: Protein LOCATION: 2..-102 OTHER INFORMATION: /label= VGL-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1!: Cys Lys Lx's Arg His Leu Ty'r Val Glu Phe Lys Asp Val Gly Tro Gln 20 1 Asn Trp Val Ile Ala Pro Gin Gly Tyr mez Ala Asn Tyr Cys Tyr Gly 25 Giu Cys Pro Tyr Pro Leu Thr Glu Ile Leu Asn Gly Ser Asn His Ala 40 Ile Leu GIn Thr Leu Val His Ser lie 3-1u Pro G'u Aso Ile Pro Leu 55 G0 Pro Cys Cx's Val. Fro T-r Lys Met Ser Zlr3 T'e Ser Ie Leu F-e Ty r 70 75 Asp Asn Asn Asp Asn Val Val Leu Ara His Tyr Glu Asn Met Ala Val 90 Asp Glu Cys Gly Cys Ara 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: NME/KEY: Protein LOCATION: 1. .102 OTHER INFORMATION: /label= VOR-l-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Cys Lys Lys His Glu Leu Tyr Val Ser Phe Gln 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 97/41881 PCT/US97/07816 51 Ile Val Gin Thr Le'j Val His Val Met Asn Pro Glu Tyr Val Pro Lys 55 Pro Cys Cx',s Ala Prc Thr Lys Val A.sr A I Ie Ser Val1 Leu h Phe 70 75 Asp As:) Asn, Ser Asn Val I--e Leu 7-2s Lys Aro Asin Me: Val Va 1 Ara Al a Cys Sly Cys His 100 iNFOR;'.ATION FCR SEQ 1D NO: 13: SEQUENCE CHARACTERISTICS: A LENGTHq: 106 aminc acids B) TYFE: amino acid (C2 STRANDEDNESS: single TOPOLOGY: linear (ili) %MOLECULE TYPE: oroteln (iii; HYPCTHErTCkL: N (iv)ANTI-SENSE: NO vi~ RIGIINAL- SOURCE: ORGANISM: Homc sapiens TISSUE TYPE: brain (ix) FEATURE: NANE/KEY: Protein LOCATION%1 1.-106 COTHER INOMTO:/rote= "GDF-l (fx)" SEQUENCE DESCRIPTION: SEQ ID %'0:13: Cys Arg Ala Arg Ara Leu Tyr Val Ser Phe Arg Gin Vai- Gly Trp His l 5 10 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 Len Met His Ala Ala Ala Pro Gly 55 Ala Ala Asp Len Pro Cys Cys Val Pro Ala Ary Len Ser Pro Ile Ser 70 75 Val Leu Phe Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gin Tyr Glu 90 WO 97/41881 P'CT[US97/078 16 52 Asp Met Val Val Asp Glu Cys Gly Cys Arc 100 105 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LEN4GTH: 5 amino acids TYPE: amino acid (C STANE> S: s r l le TOPOLOGY: linear (ii) MOLECULE TYPE: pepride (xi) SEQUENCE DESCRIPTION: SEQ _D NO:14: C,.s Xaa Xaa Xaa Xaa 1 INFORMATION FOP. SEQ N0:15: SEQUENCE CHARACTERISTICS: LENGTH: 1822 base pairs TYPE: nucleic acid STRANDEDNESS: sincle TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPO'HETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: HOM~O SAPIENS TISSUE TYPE: H-IPPOCIAMPUS (ix) FEATURE: NAME/KEY: CDS LOCATION: 49. .1341 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN" /product= "OPi" /evidence= EXPERIMENTAL /standard-names "0P1" (xi) SEQUENCE DESCRIPTION: SEQ ID GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG 57 Met His Val 1 CGC TCA CTG CGA GCT GCG GCG CCC CAC AGC TTC GTG GCG CTC TOG GCA 105 Arg Ser Leu Arq Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala 10 WO 97/41881 WO 971881PCTIUS97/07816 53 CCC CTC TTC C7C CTG CGC TCC CCC CTG CCC GAC TTC AGC CT'S GAC AAC Pro Leu Phe Leu Leu Arg Ser 25 Ala Leu Ala Asp Phe 3 C Ser Leu Asp Asn GAG CCC CAC TCC Glu Val His Ser TTC ATC CAC CSG Phe -ile His Arc CGC CTC CC Arg Leu Arg ACT CCC CC Tie Leu Clv CGS GAG ATG Ara Glu Me: CCG CGC CCS Pro Ara- Pro CGC GAG ATC CTC Arg Glu Ile Lei ACC CAC GAG CGC Ser Gin Glu Arg TTC CCC CAC CC Leu Pro HIs Ara CCC ATC TTC ATG Pro Mezi Phe Met 8 0 GGC CCC CCC GCC Gly Gly Pro Gly CAC CTC CAG GCC His Leu Gin Gly CAC PAC TCC GCA His Asn Ser Ala CTC CAC CTG CAC AAC CCC P73 SC GTC GAG GAG Leu Asp Leu Tyr Asn Ala Met Ala Val Clu Ci -u S.

S

S

CAG GGC CCC TCC GIn Gly Phe Ser CCC CAC AC GCC Pro Tyr Lys Al.-a 77C ACT ACC CAC GC Phe Ser T1hr Cic- Cly 115 CCC CCT CCC GCC Pro Pro Leu Ala CTC CAA GAT AC Leu Gin Asp Ser T7C CCC ACC GC Phe Leu Thr As:) SCC CAC AlJa Asp 130 ATC GCC ACC AOC TTC CCC AAC CCC CCC CPA CAT GAC AC CPA CCC TC Met Val Met Ser Phe Val Asr. Leu Val Ciu His Asp Lys Glu Phe The CAC CCA CC His Pro Arg TAO CAC CAT CCA Tyr His H is Arg -CC CGC TCT CAT Phe Arq The Asp CCC AC AC Se: Lvs Ile CCA CA Pro Glu 165 CCC CPA GC GC Cly Glu Ala Val CCA CCC CPA TCC Ala Ala Clu Phe ATC TAC AC CAC Ile Tyr Lys Asp ATC AGC CCC TAT Ile Ser Val Tyr 195

S.

*5

S

S.

ATC CCC CPA CC Ile Arg Clu Arg CCC CAC Phe Asp 185 PAT GAG ACG TTC CCC Asn Ciu Thr Phe Arg 190 CAC CCC CCC CAC GAG Gin Val Leu Gin Ciu 200 CAC TTIC CCC ACC His Leu Cly Arg TCG CAT CCC TCC Ser Asp Leu Phe CTC CC Leu Leu 210 CAC AGC CCC ACC CTC TCG GCC Asp Ser Arg Thr Leu Trp Ala CC GAG Ser Ciu 220 GAG CCC CCC CCC CCC TTC CAC Clu Cly Trp Leu Val Phe Asp 225 ACC ACA GCC ACC AGOC PAC CAC CCC CTC CCC PAT CCG CCC CAC AAC CTC Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu 230 235 240 WO 97/41881 PCT/US97/078 16 54 GGC CTG Gly Leu 245 CAG CTO TCG Gin Leu Ser TTG GCG GGC CO Leu Ala Gi1% Leu GTG GAG AOG CTG GAT GGS CAG AGC ATC AAC CCC Val Glu Thr Leu Asp Giy Gin Ser Ile Asn Pro 250 255 ATT GGS CGG CAC GSG CCC CzAG zAC AAG CAG CCC Ile Gly Arg His Giy Pro Gin Asn Ly's Gin Pro 265 270 275 =TC _1i G GCC ACC GASGT- 072 Tr7 C__2 AGO ATC Phe Lys Ala Thr Glu Val His Phe Arg Ser Ile 285 290 825 873 921 770 ATS GTG GCT -77O Phe Met Val Ala Phe 280 CGG TCC ACG GGG AsC AAA Arg Ser Thr Gly Ser Lys 295 CAG 000 AGC CAG AAO Gin Ara Ser Gin Asn 300 CC-C TCC A.AG AOG CCC Ara Ser Lys Thr Pro 305 AAG AAC CAG Lys Asn Gin 310 GAA GCC CTG CO Glu Ala Leu Arc "CC ?,AC GTG OCA Ala Asn Val Ala AAO AGO ACC -Asn Ser Ser 1017 AGO GAO CAS AGG CAG 300 TST Scr Asp 325 Gin Arg Gin Ala AAG AAG CAC GAG C7S Lys Lys His Glu Leu 335 TAT GTO AGC TTC Tyr Val Ser Phe p. p.

*0 p p p.

p PP Pp Pp

P

pp..

9 GAO CTG GGC TGG Asp Leu Gly Trp GAO TGG ATO ATC Asp Trp Ile le COT GAA GCO TAC Pro Glu Gi yr 1065 11-3 1161 1209 000 TAO TAO TGT Ala Tyr Tyr Cys GG3 GAG TGT 300 Gly Glu Cys Ala COT 0T3 AAC TOO Pro !,eu Asn Ser TAO ATG Tyr Met 37 0 AAO GOC ACC Asn Ala Thr 003 GAA ACG Pro Glu Thr 390 CAC GOC ATO GTG His Ala Ile Val ACG 073 GTC CAC Thr Leu Val His TTO ATO AAC Phe Ile Asn 385 GTG COO AAS 000 Val Pro Lys Pro TGT 303 COO ACG CAG 070C AAT G00 Cys Ala Pro Thr Gin Leu Asn Ala 400

P.

p p Pp P P ATO TOO Ile Ser 405 GTO CTC TAO TTOC Val Leu Tyr Phe GAO AGO TOO AAC Asp Ser Ser Asn ATO 073 AAG AA Ile Leu Lys Lys TAGCTCCToC 1257 1305 1351 AGA AAO ATG GTG Arg Asn Met Val CGG GOC TGT GGC Arg Ala Cys Gly TGO CAC Cys His 430 GAGAATTOAG ACCOTTTGGG GCCAAG1rTT TCTGGATCCT CCATTGCTOG

CCTTOGGCAG

GAACCAGOAG ACCAACTGCC TTrT-GTGAGA CCTTOCCOTC COTATCCCCA

ACTTTAAAGG

TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGOTTT'ro ATCAGTT'TTT

CAGTGGCAGC

ATCCAATGA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGOAGGAAA

AAAAAACAAC

GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAJA GTOTCAGCCA

TGCACGGACT

1411 1471 1531 1591 1651 WO 97/41881 WO 97/1881 CT/US97/07816 55 CGTTTCCA-A GOTA.A7TATG AGCCCCTACC AGCCAC-3CCA CCCAGCCC-TO GGA-GAGG- GGCCGTGC-"PA G--:GGG'CA CA7TC-GTC-TC T3TCCGAAAC GAAAi-ATTGAC CCG--AAGTTC CTGTAATA TGCACAATA1 ;AT CGAATGZ A AA AkkAAA SEQUENCE CHAPJ'CTERISTICS: LENGTH,: 431 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein 17 11 1771 1822 (Xi) SEQUENC-E Met H is Val Ara Ser Leu Trp Ala Pro Leu Leu Aso Asn C-lu Val C-in Glu Arg Arg C-lu Pro His Arg Pro Arg Met Plhe met Leu Asp Gly Pro Gly C-ly Gin 100 Thr C-in Gly Pro Pro 115 Asp Ala Asp Met Val 130 Glu Phe Phe His Pro 145 Ser Lys Ile Pro Glu 165 Tyr Lys Asp Tyr Ile 180 Phe His Mje t Pro Leu C-ly Leu Met Arg 150 Gly Arg Le-u Ser His Ph~e Al a Ser 135 Tyr C-lu C-lu Leu Ser 40 ArC- Leu As n Se r S er 120 Pi-e His Ala Arg A rg 25 Phe C-lu Al a 105 Leu Val1 His Val1 Phe AlI.a Arc Ile Asn C-Ilu Ala Hi.s 125 C-lu ArC- Glu Aso L e L L eu Ser C-Lu Val '110 ?he His Phe Phe DESCRIPTIC.N: SEQ D :O16 F, l Aa~oHi e 175 Aso Asn C-lu Thr Phe Arg Ile 190 185 Ser Val Tyr C-in Val Leu C-in C-lu His Leu Gly ArC- SrAs e Ser Asp Leu WO 97/41881PC/S7081 PCTIUS97/07816 56 Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Giu Clii Gy Trp Leu 210 Val Phe 225 Hi,'s Asn lie Asn Lys Gin Arg Ser 290 Lys Thr 305 Asn Ser Val Ser Sly Ty~r Ser Tyr1 370 Phe Ile 385 Leu Asn Leu Lys 220 Ile Sly Lvs 260 Phe Arg Lysc Ser Arg 340 Ala Asn Pro Ile Tyr 420 Th r Leu 245 Leu Mer Ser Asr Asp 325 As p Ala Ser 405 Arg Ala Tlhr Ser Asn His Trp Val Val Asn Pro Arg 230 Sin Ala Val Th r G 310 C-in Leu Tyr Thr Thr 390 Val1 As n Leu Ci y Ala GCIy 295 C-lu Ara G Iy Cys As n 375 Val1 Leu Met Val1 Ile 265 Phe _v Le u Ala Gin 345 Cl y Al a Lys Phe Val1 425 235 Glu Thr 250 Gly Ara Lvs Ala Sin Arq 315 Cys Lys 330 Asp Trp C-lu Cys Ile Val Pro Cys 395 Asp Asp 410 Arg Ala Leu Asp dly GSin Ser 255 His Sly Pro GIn Asn 270 T'hr Clii Val His Phe 285 Ser Giln Asn Arg Ser 300 a Asn Val AlIa Cli 320 Lys His GCliu Leu Tyr 335 Ile Ile Ala Pro Clii 350 Ala Phe Pro Leu Asn 365 Gin Thr Leu Val His 380 Clys Ala Pro Thr Gin 400 Ser Ser Asn Val Ile 415 Cys Sly Cys His 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 PCTIUS97/07816 WO 97/41881 57 ORIGINAL SOURCE: ORGANISM: MURIDAE TISSUE TYPE: EMBRYO (ix; FEATURE: NAME/KEY: CDS LOCATION: 104. .1393 OTHER INFORMATION: /functicr- "OSTEOGENIC PROTEIN" rprciuc~ -op! /note= "MOP! (CDNA)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CTGCAGCAAS TGACOITCGGG TO1GTGGACCS CTGCCTGC CO'CGCTS CCACCTCGOOG CGGCGCGO CCGSTGOCO' GSATOGCGCG TAGAGCCGGOO SCG ATG CAC GTG CGC Met His Val Ara TCG CTG COO Ser Leu Arcs GOT CG GCG Ala Ala Ala CCA CAC AGC TTC OTG GOG CTC TGS GC Pro His Ser Phe Val Ala Leu Trp Ala 15 T-C AGC C-O Phe Ser Leu CTG TTC TTO CTG OGC 0CC CO Leu P1he Leu Leu Arg Ser Ala CTG CO GAT -eu Ala Asp 2C GAC AAC GAG Asp Asn Glu GAG CGG CGG Glu Arg Ara CAT COO CCG His Ara Pro GTG CAC :00 Val His Ser GAG A-G CAO Glu Met Gin TOO ATC CA" CGS Pne Ile His Arg CTC CGC AGC CAC Leu Arg Ser Gin CGG GAG ATC OTG Arg Cli Ile Leu ATC 7TA 'OGG TTO Ile Leu Gly Leu COO COO Arg Pro C7,- CTO CASGOGA Hius Leu Gin Gly CAT AkT TOO 000 His Asn Ser Ala A-G TTC ATS TOG Met Phe Met Leu COG TAO AAO Leu Tyr Asn CO ATG Aia Met 90 COO TAO Pro Tyr 105 GCC 000 GAG GAO Ala Val Glu Giu AGO- G00 CCC Ser Gly Pro 95 GAO GGA CAG Asp Gly Gin 100 CCC TTO TOO TAO Gly Phe Ser Tyr AAG GOC 000 TTO AGT ACC CAG 000 COO COT Lys Ala Val Phe Ser Thr Gin Gly Pro Pro 110 115 TTA CO AGO Leu Ala Ser CAG GAO AGO CAT Gin Asp Ser His TTO CTO ACT GAO GC Phe Leu Thr Asp Ala 125 GAO ATG CTC Asp Met Val 130 ATG AGO TO GCO Met Ser Phe Val 135 AAO OTA COG CAA OAT GAO AAA CAA i-rC TOO CAC COT Asn Leu Val Glu His Asp Lys Glu Phe Phe His Pro OGA TAO CAC CAT OGO GAG TO OGO TTT GAT OTT TOO AAG ATO CCC GAG WVO 97/41881 PTU9/71 PCTfUS97/07816 58 Arg Tiyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys lie Pro Glu i s co 160 GGC GAA Gly Glu 165 C3G GTG A.CC C-CA GGCC SAA -TC ASS ATC TAT AAG SAO TAC ATC Arg Val Thr Ala Glu Phe Arg ie T1yr Lys Asp Tyr Ile 175 180 CGG GAG CGA AraclGu Ara CTC CAG GAG Leu Gin Glu 030 ACC ATC Arg Thr Ile 215 =7T GAC P.*ne Asp 185 AAC GAG ACC TTO Asn Glu Thr Phe CAG ATC Gini Il 190 ACA OTC TAT CAG G'TG Th r Val SirGl Val 195 TCA 330C AGO Ser Gly Arg GAG TCG Giu Ser 205 GAG GGC Glu Gly 220 GAC CTC TTC TTG Asp Leu Phe Leu T33 TTG GTG TTT Trp Leu Val Phe 225 CTG GAO AGC Leu Asp Ser 210 GAT ATC ACA Asp Ile Thr TOG OCT TCT GAG Trp Ala Ser Glu 300 ACC Ala Thr 230 AGO-' AAO CAC 7OG Ser Asn His Trp 370 AAO COT 03S Val Asn ?ro Arg AAO CTS GGO TTA Asn Leu Gly Leu 070 TOT GTG GAG Leu Ser Val Glu CTG GAT 333 CAG Leu Asp Gly Gin ATO AAO 000 AAG ile Asn Pro Lys GOA GGO 073 ATT GGA 033 OAT GGA 000 Ala Gly Leu Ilie Gly Arg His Gly Pro 265 AAO AAG CAA 000 Asn Lys Gin Pro 770 ATO Phe Met 275 373 300 770 Val Ala Phe AOG 333 330 Thr Gly Gly 295 AAG 300 AOG GAA Lys Ala Thr Glu OAT CTC 037 AST His Leu Arg Ser ATO COG 7CC Ile Arg Ser 290 COA A.AG AAO Pro Lys Asn A.AG CAG 030 AGO Lys Gin Arg Ser AAT 030 700 AAG Asn Arg Ser Lys 979 1027 1075 CAA GAG Gin Giu 310 300 073 AGG ATG 300 AST 373Q GOA GAA Ala Leu Arg Met Ala Ser Val Ala Giu 315 AGO AGO AST GAO Ser Ser Ser Asp AGG OAG 300 TGC Arg Gin Ala Oys AAA OAT GAG 073 Lys His Giu Leu GTC AGO TTO OGA Val Ser Phe Arg 1123 C117 330 TOO OAG GAO TG Leu Gly Trp Gin Asp Trp 345 ATO ATT GOA Ile Ile Ala 300 770 007 Ala Phe Pro 365 007 GAA Pro Glu 350 330 TAT GOT 300 TAO Gly Tyr Ala Ala Tyr 355 1171 TAO TGT GAG Tlyr Oys Giu GGA GAG 730 Gly Giu Oys 360 073 AAO TOO TAO ATG AAC GOO Leu Asn Ser Tyr Met Asn Ala 370 1219 ACC AAO CAC 300 ATO 370 CAG ACA 073 377 CAC TTC ATOC AAO OCA GAO Thr Asn His Ala Ile Val Gin Thr Leu Val His Phe Ile Asn Pro Asp 1267 WO 97/41881 WO 97/ 1881PCT/US97/078 16 59 ACA GTA CC) Thr Val Prc 390 GTC CTC TAC Val. Leu Tyi 405 AAC ATG GTC Asn Met Val

ACCTTTGCGG

CCCACCTTCGG

AAGCATGTAA

GGCACGT-AC

GTCTGCCAGG

AATCGCAAGC

TCTGTC-TGA

GAATGAAAAA

380 CAAG CCC TGC TGT GCG 0 Lys Pro Cys Cys Ala 395 TTC GAC GAC AGC TCT Phe Asp Aso Ser Ser 410 GTC CGG GCC TGT GCC Val Arg Ala Cys Gly 425 GGCCACACC7 TTCCAAATCT CGAGC-ACAAC AGACCAACC: CGGTTCCAGA AACCTGAGCG GGACAAGATC CTACCAG CTA AAAGTGTCCA GTGT-CCACAT.

CTCGTTCAGC TGAGCAGAA AGGC-AAACCA ASCAGAACC CCC ACC CAG CTC AAC c-CC ATC TCT Pro Thr Gin Leu Asn Ala Ile Ser 400 A.AT GTC ATC CTG AAG AAG TAC AGA Asn Val Ile Leu Lys Lys T1'r Arg 415 420 TGC CAC TAGCTCTTCC 7GAGACCCTG Cys His 430 TCGATG'rTTC ACCATCTAAG TCTCTCACT C7CCTGAGCC TTCCC7CACC TCCCAACCG TGCAGCAGCT GATGAGCGCC CTTTC1CTTC CCACAGCAAA CGCCTAAGAG CAGGAAAAA.

GGCCCCTGGC GC"-CTGAGTC TTTGAGGAG GGA.ACGGCTT AGCCAGGGTG GCGCCTGGC AC:GTAATGA TATG7CACkA TA.ZAAC "CA

AAAAGAATTC

C

T

T

1315 1363 1413 1473 1533 1593 6 53 171-3 1773 :863 3 1873 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: WA LENGTH: 430 ami~no acids T i' c 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 Glu Val His Ser Ser Ph~e Ile His Arg Arg Leu Arg Ser 40 Gln Glu Arg Arg Clu Met Gin Arg Glu Ile Leu Ser ile Leu Gly Leu 55 Pro His Arg Pro Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro 70 75 NVO 97/41881 WO 9741881PCT/US97/07816 60 Met Ala Val Tyr Ala Glu Ser Phe S-r 100 105 110 Gin Ala Phe 145 Lys Lys Val Leu Phe 225 Asn Asn Gln r Ser Thr 305 Ser Ser Tyr Gly Asp 130 Phe Ile Aso Le u 210 A~sp Leu Pro Pro Ile 290 Pro Ser Phe Al a Pro 115 ,ve t H is Pro Tyz Gin 195 Asp 11 Gly Lys Phe 275 Arg Lys Ser Arg Ala Pro Val Pro Glu Ile 180 Val Ser Thr iLeu Leu 260 Met Ser Asn Asp Asp 340 Tyr Leu rMiet Ara G I y Arg Leu Arg Ala Gin 245 Ala Val1 Thr G in Gin 325 Leu 12C Ser Phe Val 135 Ty r Hi's His 150 Giu Ara Val Glu Arg Phe Gin Giu His 200 Thr Ile Trp 215 Thr Ser Asn 230 Leu Ser Val Gly Leu Ile Ala Phe Phe 260 Gly Gly Lys 295 Glu Ala Leu 310 Arg Gin Ala Gly Trp Gin 125 Asn Arg -hr Asp 185 Ser Ala His Giu Gly 265 Lys Gin Arg Cys Asp 345 Le u G_',u Ala 170 Asn Gly Ser Trp Thr 250 Arg Al a Arg Met Lys 330 Trp Va 1 Phe 155 Ala Arc Giu Val 2 35 Leu His Th.r Ser Al a 315 Lys Ile Al a Gi'u 140 Ara Giu Thr Giu C-l1u 220 Vai Asp Gly Giu Gin 300 Ser His I le Phe Hi s Phe Ph e Phe Ser 205 G ly As n "1l Pro ValI 265 As n Val1 Glu Al a Pro Asp Asp Arg Gin Asp 7 ro Pro Gin Gin 270 ,:is Ara Ala Leu Pro 350 Leu Giu Ser 160 Tyr Tb r Phe Val1 His 240 Ile Lys Arg Lys As n 320 Val1 G ly Ser Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp TIyr Cys Glu Gly Glu Cys 360 365 Tyr Met 370 Asn Ala Thr Asn His 375 Ala Ile Val Gin Thr Leu Val His Phe 360 WO 97/41881 PCTIUS97/07816 61 Ilie Asn Pro Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr 'In Leu 385 390 395 400 Asn Ala Ile Ser Val Leu Pe Asp Asp Ser 5cr Asn Val Ile Leu 405 410 415 Lys Lys TIyr Aro Asn met Val Val Ara A-,a Cys 02%, Cys His 420 42S 439, INFORM ATION FOR SEQ ID NO:19: SEQUENCE CFAFJ.CTERISTICS: LENGTH: !723 base pairs TYPE: nucleic acid STRANIOE-DNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: COGANIISM: Homno sapiens TISSUE TYPE: HIP POCk-1PUS (ix) FEATURE: NAME/KEY: CDS LOCATION: 490. .1696 OTHER INFOR;MATION: /function= "STEOGE:JIC PROTEIN', /product= "hOP2-PP" /note= "hOP2 )cDNA), (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i9: *GGCGCCGQGCA GAGOAGGACT GOCTOGAGGA GCTGTGGTTG GAGCAGGAGG TGGCACGGCA CCACACCGCA CCAAGCGGTG GCTGCAGGAG CTCGCCCATC GCCCCTGCGC TGCTCGGQACC 180 GCGGCCACAG CCGGACTGGC GGGTACGGCG GCGACAGAGO CATTGGCCGA GAGTCCCAGT 240 CCGCAGAGTA GCCCCGGCCT CGAGGCGGTG GCGTCCCGGT C'CTCTCC-TC CAGGAGCCAG 300 *GACAGGTGTC GCGCGGCGGG GCTCCAGGGA CCGCOCCTGA GGCCGGCTGC CCGCCCGTCC 360 CGCCCCGCCC CGCCGCCCGC CGCCCGCCGA GCCCAGCCTC CTTGCCGTCG GGGCGTCCCC 420 AGGCCCTGGG TCGGCCGCGG AGCCGATGCG CGCCCGCTGA GCGCCCCAGC TGAGCGCCCC 480 CGGCCTGCC ATG ACC GCG CTC CCC GGC CCG CTC TGG CTC CTG GGC CTG 528 Met Thr Ala Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu 1 5 GCG CTA TGC GCG CTG GGC GGG GOO GGC CCC GGC CTG CGA CCC CCG CCC 576 Ala Leu Cys Ala Leu Cly Gly Gly Gly Pro Cly Leu Arg Pro Pro Pro 20 WO 97/41881 PIl 97081 PCTfUS97/07816 62 000 TGT CCC CAG CGA CGT CTG GGC 0-00 CGC GAG CGC CGG GAC 070 CAG Gly Cys Pro Gin Arq Arg Leu Gly Ala Arg 0Th Arg Arg Asp Val Gin 35 40 COO GAG ATO CTG 000 0-TO CTC GGS CTG CCT COG COO CCC COG CCC CO-C Arg Glu Ile Leu GCG CCA CCC GCC Ala Pro Pro Ala 070 GAO 070 TAO Leu Asp Leu Tyr Val Leu Gly Leu Pro Gly Arc Pro Arg Pro Arg SCC TCZ- COS 070 Ala Ser Arg Leu 7C CC Sr' Ala Ser Ala CCG CCC 77C ATG Prc Leu Phe Met GAG 0-AC 030 000 Glu Asp Gly Ala CAC 0-00 ATG His Ala Met GO-C GAOC GAO GAO 01)' Asp Asp Asp CcL 000 Pro Ala GAG COG 030 070 Giu Aro Ara Leuj CSC 3CC -AC 0-TO Arg Ala Asp Leu ATG AGC 770 GTT et Ser Pi-.re Val ATO- 370 GAG CO-A Met Val 0-lu Arc 037 000 07S 30-C Ara Ala Leu 3>'v CAG GAG CCC OAT Glu Pro His 9*99 9* 9.

9 .9 .9 9 9 9* 9' 9 9 *99* AAG GAG 770 000 Lys 0Th Phe Arg GAO 070 ACC CAG ATO COO GOT GGG GAG Asp Leu Thr Gin ile Pro Ala Giy Glu 000 070 Ala Val 140 ACA OCT GCG GAG 770 000 ATT TAO AAG 073 000 AGO ATO Thr Ala Ala Oiu Phe Arg Ile Tyr Lys Val Pro Ser Ile CAC 070 070 His Leu Leu 155 GAG CAG 700 Glu Gin Ser AAC AGO ACC Asn Arg Thr 160 070 CAC 070 AGO Leu His Val Ser 770 CAG GTO 070 Phe Gin Val Val 1008 AAC AGO Asn Arg 175 GAO TCT GAO 770 Giu Ser Aso Leu 770 777 Phe Phe 180 73 OAT OTT Leu Asp Leu ACO 070 CGA GCT Thr Leu Arg Ala GAO GAO GC TG Asp Glu Gly Trp 070 070 OAT 370 Val Leu Asp Val ACA OCA 000C AGT GAO TOO Thr Ala Ala Ser Asp Cys 200 205 99 9 *9*9 99 9 9e 90 TOG 770 070 PA.O Trp Leu Leu Lys ACT GAG GAO 000 Thr Giu Asp Gly 225 CAA 000 000 OCA Gin Arg Ala Pro 240 CAC AAG GAO 070 His Lys Asp Leu 070 000 070 TAT Leu Arg Leu Tyr 010 GAG Val Giu 220 1056 1104 1152 1200 1248 1296 CAC AGO 070 OAT His Ser Val Asp COT 000 010 GOC 000 CTG CTG GOT Pro Gly Leu Ala Gly Leu Leu Gly 235 ACT TO TO AGO Thr Phe Phe Arg 250 000 TOO' Arg Ser CAA CAG OCT IO GIG 070 Gin Gin Pro Phe Val Val 245 000 AGT 000 AGT 000 ATO 000 ACC OCT 000 GOA GTG AGO OOA 010 AGO WO 97/41881 WO 9741881PCTIUS97/07816 63 Ala Ser Pro Ser 255 Pro Ile Arg Thr Pro Arg Ala Val Arg Pro Leu Arg AGO CAG CCC AAG Ara Gin Pro Lys AAA AGO Lvs S er 275 GA CAC =O Asn Glu CAC SCC AAC CGA GIn Ala Asn Ara 134 4 CCA GCSG ATO TT P ro_ i, 11ie e SCr. t3-2 CACSS CACGSCC CGG CAG rocl GCO TGC Val Cys 300 1392 CST CGC CAC Arg Arg His TGG GTC ATC Trp Val Ile 320 CTC TAC GC ASC Leu V'al Ser GAG SAC CTC GGC Gin Asp Leu G-y TOO CTC GAO Trc Leu Asp 315 GAG CCC GAG CIL., Sly Gl,, 1440 1488 OT CCC CAA GC Ala Pro C1- Ci)' TCC GCC TAT TAC Ser Ala >0r Tyr TGC. TOG Cys Ser 335 770 GA C=G GAC Phe Pro 'eu Asp TOG2 AT Cys Me:l Asr, A Asn His Ala Ile 0I pose*: GAG 7Cr'CT00G GCAO COG ATG PA.G CCA Gin Ser Leu Val His Leu Me: Lys Pro 'S CA Aer. Ala 360 OTC CCC A.AG Val Pro Lys 158 4 1632 TCC TGT GCA CCC Cys Cys Ala Pro AAGCT07 AGC GC L.ys Leu Ser Ala TCT GTG CTC TAO Ser Val Leu Tyr TAO GAO_ Tyr Asp 380 AGO AGC AAC Ser Ser Asr.

GTC ATO CTG OGO Val :ile Leu Arg CAC CGOC AAC ATG His Aro Asn Me=t COG GC AAC Val Val Lys 395 1680 1723 Cr C 030 Gys 1y Cyls 400 4 4 *4*S

S

S

5* INFORMATION FOR. SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 402 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Thr Ala Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys 1 5 10 1s Ala Leu Cly Cly Cly Cly Pro Cly Leu Arg Pro Pro Pro Gly Cys Pro 25 PCTIUS97/078 16 WO 97/41881 64 Gly Ala Arg Gilu Arg Arg Asp Val Gin Arg Giu Ilie 0 0 *0 .0 o o.

Gin Leu Ala Tyr Arc Glu Arc Gilu 145 Leu Ser Giy Lys G Iy 225 Pro Ser Pro Phe Arg Aia Al a His Arg Arg Phe 130 P he H-is Asp Trp Arg 210 Pis Arg Pro Lys Asp Arg

VI

Ser Ala La u Asp 115 Asp Arg

I

Leu Leu 195 H s Ser Ser Ile Lys 275 Asp Le u Leu Ara Me r Glv 100 Arg Leu Ile Se r Phe 180 Val Lys Val Gin Arg 260 Ser Val1 Gly Leu Ala Arg Ala Thr Tyr Met 165 Phe Leu Asp As p Gin 245 Thr Asn Leu Pro 70 G Ily Ala Leu G Inr Lys 150 Phe Leu Asp :,eu 230 Pro Pro Giu P-ro Gly 55 A Ia Ser Aso Asp Asp Leu Gly His 120 1',e Pro 135 Va I Pro G1 n Val Aso Leu Val Thr 200 G ly Leu 215 Gly Le Phe Val Arg Ala Leu Pro 280 Arc Asp Val1 105 Ala Val1 185 A%!a Arg Val1 Vali 265 Pro Glu 90 Met G l Gin 170 Leu Thr 250 Arg Arc 75 Asp Ser 0 1 u His 155 eu Ser Tiyr 235 Phe Pro Pro Ph e Gly Phe Hs Ala 140 Le u G In Arg Asp Val1 220 Le u Phe Leu Arg Met Ala Val Trr 125 Val L eu Ser Ala Cys 205 Giu Gl Iy Arg Arg Leu Ala Leu Pro Asn 110 Lys Thr A4s r Asn Gly 190 Trp Thr G In Al a Arg 270 Pro Prc Asp Ala Met Arg 175 Asp Leu G lu Arc Se r 255 Arg G ly Pro Leu Giu Val Phe Ala Thn r 160 Glu Glu Leu Asp Ala 240 Pro Gin Ile Gin Ala Asn Arg 285 His Gly Ser His Gly Arg Gin Val Cys Arg Arg His 295 300 290 Giu Leu Tyr 305 Ala Pro Gin Val Ser Phe 310 Giy Tyr Ser 325 Asp Leu Gly TIyr Tyr Cys 330 Trp Leu 315 Giu Gly Asp Trp Giu Cys Val Ile 320 Ser Phe 335 NNIO 97/41881 WO 97/ 1881PCT[US97/07816 65 Pro Leu Asp Ser Cys 340 Leu Val His Leu Met 355 met Asn Ala Thr Asn His Ala Ile Leu Gin Ser 345 350 Lys Pro Asn Ala Val Pro Lys Ala Cys Cys Ala 360 365 Pro Thr 370 Lys Leu Ser Ala Ser Val Leu TPyr TPyr 380 Val Val 395 Asp Ser Ser Asn Val Ilie Leu Ara His Arg Asn Met Lys Ala Cys Cys His INFORMVATION FOR SEQ :D NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 1926 base pairs TYPE: nucleic acid STRANDEDNESS: sinole TOPOLOGY: linear (vi) ORIGINAL SOURCE: ORGANISM: MURIDAE T!ISSUE TYPE: EMBRYO (ix) FEATURE: NAME/KEY: CDS LOCATION: 93. .1289 OTHER INFORYATION: /functicr= /product= 11mOP2-PP"1 /note= "rnOP2 cDNA" "OSTEOGENIC PROTEIN" a.

(xi) SEU S:Q: SQ-, :D ',0:21: GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC CCGACCAOCT ACCAGTOGAT GCGCGCCGGC TGAAAGTCCG AG ATG OCT ATO COT CCC 000 CCA Met Ala Met Ara Pro Oly Pro 1 CTC TOO CTA TTO GOC CTT OCT CTG TOC OCO CTG OGA GOC GOC CAC GOT Leu Trp Leu Leu Oly Leu Ala Leu Cys Ala Leu Gly Oly Gly His Gly 15 CCG COT CCC CO CAC ACC TOT CCC CAG COT COC CTG OGA OCO COC GAO Pro Arg Pro Pro His Thr Cys Pro Oln Arg Arg Leu Oly Ala Arg Glu 30 COC COC GAC ATO CGO COT OAA ATC CTO OCO OTO CTC 000 CTA CO OGA Arg Arg Asp Met Gin Arg Olu Ile Leu Ala Val Leu Oly Leu Pro Giy 45 50 COG CCC COA CCC COT OCA CAA CCC 0CC OCT 0CC COG CAG CCA OCO TCC Arg Pro Arg Pro Arg Ala Gin Pro Ala Ala Ala Arg Gin Pro Ala Ser 113 161 209 257 305 WO 97/41881 ~VO 974 1881PCTIUS9 7/0 78 16 66 GOCG CCC CTC TTC ATCQ TTG C-AC CTA TAC CAC CCC ATC- ACC GAT C-AC GAC Ala Pro Leu Phe met Leu Asp Leu Tyr His Ala Met Thr Asp Aso Asp GAC CCC CCC Asp Gly Cly CCA CCA CAG Pro Pro Gln C-CT CAC TTA Ala His Leu 95 GCC CCT C-CC C-AC CTC GC AT- Cly Arg Ala Asp Leu Val Met 100 AC-C TTC Ser Phe 105 C-TC AAC AT- C-TC Val Asn Met Val CC-C C-AC CC-T ACC CTC- C-CC TAC CAS C-AC Arg Asp Arg Thr Leu Cly Tyr C-In C-lu 115 CAC TC-C AAC C-AA His Trp Lys C-lu CAC TTT C-AC His Phe Asp C-TA ACT Leu Thr 130 CA- ATC CCT C-in Ile Pro C-CT C-CC Ala Cly 135 AC-C ACC Ser Thr 150 C-AC C-CT GC ACA C-CT C-CT C-AC TTT CCC- ATC TAC AAA C-AA CCC C-lu Ala Val Thr Ala Ala C-lu Phe Ary Ile Ty'r Lys C-lu Pro CAC CCC- CTC His Pro Leu C-AC- CAC TCC C-lu His Ser 170 ACA ACC CTC CAC Thr Thr Leu His AC-C AT- TTC C-AA Ser Met Phe C-lu C-TC- C-TC CAA Val Val C-in 165 CTT CAC ACC Leu C-in Thr AAC AC-C C-AS TCT Asn Arg C-lu Ser TC FTC TTT 7TC- Leu Phe Phe Leu CTC CC-A Leu Ary 185 TCT C-C-C C-AC C-AC- Ser Gly Asp C-lu ICC- CTC C-TG CTC- Trp Leu Val Leu ATC ACA C-CA C-CC Ile Thr Ala Ala C-AC CC-A FCC- CTC- Asp Arg Trp Leu AAC CAT CAC PLC- Asn His His Lys CT- C-CA CTC CC Leu Gly Leu Arg TAT C-TC C-AA ACC Tyr Val C-lu Thr C-AT C-C-C CAC ACC Asp Gly His Ser C-AT CCT C-CC CCC- Asp Pro Cly Leu C-CT C-CT Ala Cly 230 CCC- CTT C-CA Leu Leu Cly TTC TTC AC-C Phe Phe Arg 250 CAA C-CA CCA CC C-in Ala Pro Arg AC-A CAC CCT 'FTC Arg C-in Pro Phe AT- C-TA ACC Met Val Thr 245 C-CC AC-C CAC- ACT CCT C-TC CC-C C-CC CCT CC-C C-CA CCC AC-A Ala Ser C-in Ser Pro Val Arg Ala Pro Arg Ala Ala Arg 255 260 Be1 CCA CCC- AA- AC-C AC-C CAC CCA AA- AAA AC- AAC C-AC CTT CCC- CAC CCC Pro Leu Lys Arg Arg C-in Pro Lys Lys Thr Asn C-lu Leu Pro His Pro 265 270 275 AAC AAA CTC CCA C-C-C ATC TTT C-AT C-AT C-CC CAC C-CT FCC CC-C C-CC AC-A Asn Lys Leu Pro Cly Ile Phe Asp Asp Cly His Cly Ser Arg Cly Arg 280 285 290 295 NVO 97/41881 WO 97/ 1881PCT[US97/078 16 67 GAG GTT TGC CGC AGG CAT GAG CTC TAC GTC AGC TTC CGT GAC CTT GGC Glu Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Ara Asp Leu Gly 1025 TGG CTG GAC TOG GTC ATC Trp Leu Asp Trp Val Ile GAG GGS GAG Giu Gly Glu 330 315 TGT GCT Cys Ala TTG CA G Leu Gin

TTC

Phe

TCT

Ser CAT GCC His Ala 345 CCC A3AG Pro Lys 360 TAC TAT Tyr Tyr

ATC

Ile GCC CCC CAG GGC TAO TCT GCC TAT TAC TGT Ala Pro Gin Gly Tyr Ser Ala Tyr Tyr Cys 320 325 CCA CTO GAC TCC TOT ATG AAC GCC ACC AAC Pro Leu Asp Ser Cys Mer Asn Ala Thr Asn 335 340 CTG GTG CAC CTG ATG AAG CCA GAT GTT GTC Leu Val His Lei- Met Lys Pro Asp Val Val 350 355 CCC A-C AAA CTG I=T GCC ACC TOT GTG CTG Pro Thr Lys Leu Ser Ala Thr Ser Val Leu 370 375 AAT G'TO ATC CTS CGT AAA CAC CGT AAC ATG Asn Val Ilie Leu Ara Lys His Ara Asn Met 385 390 TGC CAC TGAGGCCCCG CCCAGCATCrO TGCTTCTACT 1073 1121 1169 1217 1265 1319 OCA TGC TOT GCA Ala Cys Cys Ala 365 GAC AGC AGC AAC Asp Ser Ser Asn V 380 AAG GCC TG- Lys Ala Cys GTG GTC Val Val

GGC

Gly Cys His

ACCTTACCAT

CAGACAGGG

CTTTCCCAGT

CCT.AC CC CA

CTGGGGTCAG

AATGGCAAAT

CTCTGCACCA

GATCAATGCA

OCAGGTATAG

CTGTGAGTTC

GGAATTC

CTGGCCGGGC

CAATGGGAGG

TCCTCTGTCC

CACTGAAGGC

TCTGGATGGT

TTCATTGTGG

TCGCTGTACT

CGGTGCATOT

CCCTCTCCAG AGGCAGAAAC CCCTTCACTT CCCCTGGCCA TTCATGGGGT TTCGGGGCTA CCACATGAGG AAGACTGATC CTAAGAAGGC CCTGGAATTC CAGTTGGGAC ATTTTTAGGT CCTTGAAATC AGAGCTAGCT CATTAATCCC AGCGCTAAAG

CCTTCTATGT

CTTCCTGCTA

TCACCCCGCC

CTTGGCCATC

TAAACTAGAT

ATAACAGACA

TGTTAGAAAA

AGACAGAGAC

TATCATAGCT

AAATTCTGGT

CTCTCCATCC

ACT OAGAGST

CTCAGCCCAC

GATCTGGGCT

CATACACTTA

AGAATCAGAG

AGGAGAATCT

1379 1439 1499 1619 1679 1739 1799 1859 1919 1926 AAGGCCACAT AGAAAGAGCC TGTCTCGGGA GCAGGAAAAA AAAAAAAAAC INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 399 amino acids TYPE: amino acid WO 97/41881 WO 9741881PCT[US97/078 16 68 TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Met Ala Met Arg Pro Cly Pro L Cu Trp Leu Leu Gly Leu Ala Leu Cys 1 5 10 Ala Leu Ary Ara Ala Val Ala Ala His Ala Gly Arg Arg Thnr Leu Ttr 130 Ile Tyr 145 Ser Met Phe Phe Val Leu Lys Asp 210 Met Asp 225 Arg Gin Gly Gly Gly G In Th r Asp 100 Gly Ile O iu Glu Asp I80 Ile G ly Giy Gly Le u Pro Asp 85 Leu, Tyr Pro Pro Val 165 Leu Thr Leu Leu His Arg Pro Al a 70 Asp Val G"-n Al a Ser 150 Val G In Al a Arg Al a 230 Gly Glu G ly Ser Asp Me Z Glu Gly 135 Thr G-n Thr Ala Leu 215 Gly Arg Pro Pro 25 Ara Asp e Pro Ary Pro Pro Loeu Pte Gly Gly Pro Val Asn 105 His Trp Lys Ala Val Tlhr Pro Leu Asn 155 His Ser Asn 170 Arg Ser Gly 185 Asp Arg Trp Val Glu Thr Leu Gly Arg 235 Cys Pro Ile Gln Pro Asp Leu Ala His G-u Arg 110 H is Phe Glu Phe Leu His Ser Asp 175 Gly Trp 190 Asn His Gly His Pro Arg Pro Phe Met Val Thr Phe Phe Arg Ala Ser Gin Ser Pro Val 250 255 Arg Ala Pro Arg Ala Ala Ary Pro Leu Lys Arg Arg Gin Pro Lys Lys 260 265 270 WO 9714188 1PCUSIO81 PCTIUS97107816 C. a C. C 69 Thr Asn Glu Leu Pro His Pro Asn Lys Leu Pro Gly Ile 275 280 285 Gly His Gly Ser A-rc Gly Arg C lu Val Cys Arg Arg His 290 295 300 Val Ser Phe Arg Asp Leu Giv Trp Leu Asp Trp Valle 305 310 315 Gly PIyr Ser Ala 71 'r Ty r Cys G Iu Gly Glu Cys Ala Phe 325 330 Ser Cys Xet Asn Ala Thr Asn His Ala Ile Leu G Inr Ser 340 345 Leu Met Lys Pro Asp Val Val Pro Lys Ala Cys Cys Ala 355 360 365 Leu Ser Ala Thr Ser Vai Leu Tyr Tyr Asp Ser Ser Asn 37C 375 380 Leu Arg Lys His Ara Asn Vet Val Val Lys Ala Cs's Gly 385 390 395 NFORMATION FOR SEQ ID NO:23: SEQUENCE CHA.RACTERISTICS: LENGTH: 1368 base pairs TYPE: nucleic acid STRANDEONESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDN'A (iZx FETRE A) HN/E:COS LOCATION: 1.-1368 OTHER INFORMIATION: /label= (xi) SEQUENCE DESCRIPTION4: SEQ ID NO:23: ATG TCG GGA CTG CGA AAC ACC TCG GAG GCC GTT GCA GTG Met Ser Gly Leu Arg Asn Thr Ser Giu Ala Val Ala Val 1 5 10 CTG GGA CTC GGA ATG GTT CTG CTC ATG TTC GTG GCG ACC Leu Gly Leu Gly Met Val Leu Leu Met Phe Val Ala Thr 25 GCC GTT GAG GCC ACC CAG TCG GGG ATT TAC ATA GAC AAC Ala Val Giu Ala Thr Gin 5cr Gly Ile Tyr Ile Asp Asn 40 4 CAG ACG ATC ATG CAC AGA GTG CTG AGC GAG GAC GAC AAG Gin Thr le Met His Arg Vai Leu Ser Giu Asp Asp Lys Phe Glu Ala Pro Leu 350 Pro %s Asp Le u Pro Leu 335 Val Th: r Val His Asp Tyr Gin 320 Asp His Lys Ile CTC GCC TCC Leu Ala Ser ACG CCG CCG Thr Pro Pro GGCI RAG GAC Gly Lys Asp CTG GAC GTC Leu Asp Val 48 96 144 192 WO 97141881 WO 9741881PCTIUTS97/078 16 70 TCG TAC GAG ATC CTO GAG Ser Tyr Glu Ilie Leu Giu TTO CTG GGC ATC GCOC GAA COG OCG ACG CAC Phe Leu Gly Ile Ala Glu Arg Pro Thr His CTG AGC AGO CAC CAG TTO TOG 070 AGO Leu Ser Ser His Gin Leu Ser Leu Ar; AO TOG GCT 0CC AAG TTO CTG Lys Ser Ala Pro Lys Phe Leu CTG GAC GTC Leu Asp Val GAT GAG GAC Asp Giu Asp 115 CAC CGC ATO ACG His Ar; Ile Thr GAG GAG GOT 070 AGO GAT CAG Giu Glu Gly Leu Ser Asp Gin 110 GAO GAC TAO GAA Asp Asp Tyr Giu GOC OAT OGO- Gly His Ara TCO AGO Ser Arg 125 ,AC 770 Asn Phe 140 AGO AGO GOO Arg Ser Ala ATO ACC GAO Ile Thr- Asp GAO 070 Asp Leu 130 GAG GAG GAT GAG Glu Glu Asp Giu GAG OAG CAG AAG Glu Gin Gin Lys 070 GAO AAG COG GOO Leu Asp Lvs Ara Ala 145 AAO A.AO 000 CAC CAC Asn Tys Arg His His 165 GAO GAG AGO GAO Asp Giu Ser Asp AT 0 ATG ACC 770 Ilie Met Thr Phe AAT GTG GAO GPA Asn Val Asp Giu COT CAC GAG CAC Arc His Giu His C COT Gly Arg 175 000 OTO TOG 770 GAO GTO TOO AAO Ar; Leu Trp Phe Asp Val Ser Asn 180 000 AAO GAO AAO Pro Asn Asp Asn TAO 070 070 Tyr Leu Val 190 AAC TGG CTG Lys Trp Leu ATG GOC GAG Met Ala Glu 195 070G OGO ATO TAT Leu Arg Ile Tyr AAO CO AA0 GAG Asn Ala Asn Glu 624 ACO GOO Thr Ala 210 AAO AGG GAG 770 Asn Ara Clii Phe ATC AC OTA TAO Ile Thr Val Tyr ATT GO ACC GGO Ile Gly Thr Gly OTG GGO CAG CAC Leu Gly Gin His ATG GAG COG 070 Met Ciu Pro Leu TOG GTG AAO ACC Ser Val Asn Thr GOG GAO- TAO GTG Gly Asp Tyr Val TGG TTG GAG 070 Trp Leu Giu Leu GTG ACC GAG GCO Val Thr Giu Gly OTG CAC Leu His 255 GAG TGG OTG Glu Trp Leu CAC GOT 070 His Ala Val 275 AAG TOG AAG GAO Lys Ser Lys Asp OAT GGO ATO His Gly Ile TAO ATT GGA GOA Tyr Ile Gly Ala 270 GAO GAO ATTI GGA Asp Asp Ile Gly 285 AAO OGA 000 GAO Asn Arg Pro Asp OGO GAG GTG AAG OTG Ar; Giu Val Lys Leu 280 PCTJUS97/078 16 WO 97/41881 71 CTG ATC CAC CGC A-AG GTG GAC GAC GAG TTC CAG CCC TTC ATG ATC GGC Leu Ile His 290 Arg Lys Val Asp Aso Clii Phe 295 C-C ATC AAG GC Leu Ile Lys Ala Gin Pro Phe Met Ile Gly 300 ACG GCC CAC ACC ASC CAC TTC T7C CCC CGGA CCS GAG Phe Phe Arg Gly Pro Clu 305 310 Thr Ala 3 His Ser Ser CAC AGG AGC AAG His Ara Ser Lys AG-- CCC ACC CAT Ser Ala Ser His CSC AAG CCC A G Arg Lys Arg Lys AAC TC Lys Ser 335 1008 CCC CC CCC Val Ser Pro AGC TCC CAG Ser Cys Gin 355 ,;AC GTC CCCS CTC Asn, Val Pro Leu GAA CCC ATC GAG Glu Pro Met Giu ACC ACG CC Ser Thr Ara 350 CTG CCSC CCC Leu Cly Trp 105G 1104 ATG CAC ACC CCC Met Gin Chr Leu ACA CAC TCC AAC le Asp Phe Lys CAT CAC His Asp 370 CCC ATC ATC GCA Trp ile ie Ala GAG CC CTAT G.

Glu Gly Tyr Cly TTC TAC CCC AGC Phe 7Tyr Cys Ser GAG CCC AAT TC Civ Cys Asn Phe CTC AAT GC CAC Leu Asn Ala His AAC CCC ACG AAC Asn Ala Thr Asn CC ATC GTC CAG Ala Ile Val Gin CCC GCC CAC CTC Leu Val His Leu GAC CCC AAG AAG CCC CCC Clv Pro Lys Lys Val Pro 415 1152 1200 1248 1296 1344 AAG CCC CCC- Lys Pro Cys CAC CCC AAC His Leu Asn 435 CCC AAA TCC Val Lys Ser 450 GC CCC ACC AG Ala Pro Thr Arg GGA GCA CTA CCC Gly Ala Lev Pro CCC CTC TAC Val Leu Tlyr 430 C AAC ATG AT Asn Met Ile CAC GAG AAT CCC Asp Civ Asn Val CCC AAA AAC TAT Leu Lys Lys Tyr TGCC CCC CCC CAT TGA Cys Gly Cys His 455 1368 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: WO 97/41881 PTU9/71 PCTIUS97/07816 72 Ala Val Met Ser Gly Leu Arg Asri Tbr Ser Glu Ala Val Leu Ala Ser 10 is Leu Gly Ala Val Gln Thr Ser Tyr Leu Ser Leu Asp Asp Glu Leu 130 .0 Leu Asp 145 Asn Lys 0 0000Arg Leu 9...Met Ala 0 Thr Ala 210 Thr Leu 00 225 Gly Asp Gly Ala His 100 Asp 3 lu Ara i s Phe 180 Leu Arg G in Val1 Met r His L eu G1 n His Asp Asp) Al a His 165 Asp Ara G lu His C ly 245 Val Gin Arg 70 Leu Arg Tyr Giu I Ie 150 As n Val1 I Ie Phe Thr 230 Trp Leu Ser Val 55 Phe Ser lie Glu Gly 135 Asp Val1 Ser Tyr Thr 215 Met Leu Leu Gly Leu Le u L eu Tb r Arg 120 C- Iu Glu Asp As n G In 200 Ile G lu Glu Met Phe Val Ala 25 Ile 7yr le Asp Scr Glu Asp Asp G IN Tie Ala Glu 75 Arg Lys Ser Ala Ala Glu Glii Giv 105 Gly His Arg Ser Gin Gin Lys Asn 5cr Asp Ile Tle 155 Clii Leu Ara His 170 Val Pro Asn Asp 185 Asn Ala Asr, Glu Thr Val Tyr Ala 220 Pro Leu Ser Ser 235 Leu Asn Val Thr Tb r Asrn Lys A rg Pro Leu Ara 125 Pb e Met Glu Asn Gly 205 Ile Val1 Glu Tb r Gly Leu Pro Lys Ser Arg 7le T!br His Tyr 190 Ly's Gly Asn Gly Pro Lys Asp Tb.r Phe Asp Ser Tb r Phe Gly 175 Leu Trp Thr Thr Leu 255 Pro Asp Val His Leu Gin Al a Asp Leu 160 Arg Val1 Leu Gly Thr 240 His Glu Trp Leu Val Lys Ser Lys Asp Asn His Gly Ile Tyr Ile Gly Ala 260 270 Ala Ile 290 Asn Arg Arg Glu Val Lys 280 Asp Glu Phe Gin Leu Asp 285 Pro Phe 300 Ile Sly Ile Gly WO 97/41881 PCTJUS97/078 16 73 Phe Phe Arg Gly Pro Glu Leu Ile Lys Ala Thr Ala His Ser Ser His 305 310 315 320 His Arc Ser ys Arg Ser Ala Ser His Pro Arg v s Arg Lys Lys Ser 325 330 325 Val Ser Pro Asn Asn Val Pro Leu Leu Glu Pro Mvet Glu Ser Thr Arq 340 345 350 Ser Cys G-_n met c-in Thr Leu Tyr Ile Asp Phe Lys Asp Leu G-ly Trp 355 360 365 His Asp Trp Ile Ilie Ala Pro Glu Gly T'r Gly Ala Phe Tyr cys Ser 370 375 380 Gly c-lu Cys Asn Phe Pro Leu Asn Ala His Met Asn A!-a Thr Asn His 385 390 395 400 Ala Ilie Val Gin Thr Leu Val His Leu Leu c-u Pro Lys Lys Val Pro 405 410 415 Lys Pro Cys Cys Ala Pro Thr Ara Leu Gliy Ala Leu Pro Val Leu Tyr 420 425 430 His Leu Asn Asp c-lu Asn Val Asn Leu Lys Lys 07rr Ara Asn Met Ilie 435 440 445 *Val Lys Ser Cys Gly C ys His 450 455 INFORMATION FOR SEQ ID 140:25: SEQUENCE CHARACTERISTICS: LENGTH: 1674 base pairs acid :C STR.ANDE!D:ESS: sin~gle TOPOLOGY: linear (ii) MOLECULE TYPE: protein *o 4. (ix) FEATURE: NAMIE/KEY: CDS LOCATION: 69. .1268 OTHER INFORMATION: /note= "mOP3-PP", (xi) SEQUENCE DESCRIPTION: SEQ ID GGATCCGCGG CGCTGTCCCA TCCTTGTCGT CGAGGCGTCG CTGGATGCGA GTCCGCTAAA CGTCCGAG ATG c-CT c-Cc- Cc-T CCG c-GA CTC CTA TGG CTA CTG Gc-c CTG c-CT 110 Met Ala Ala Arg Pro Gly Leu Leu Trp Leu Leu Gly Leu Ala 1 5 CTG TGC GTG TTG c-cC c-cC GGT CAC CTC TCG CAT CCC CCG CAC GTC TTT 158 WO 97/41881 WO 97/188 1PCT[US97/078 16 74 Leu Cys Val Leu Gly Sly Gly His Leu Ser His Pro Pro His Val Phe CCC CAG CC-T CGA Pro Gin Arg Arg CTA GGA GTA CGC Leu Giy Val Arg CTG GGSC CTA GCC Leu Sly L',eu Ala GAG CCC CSC SAC Giu Pro Ary Asp 40 ATT CSG SAG Ile Ara Slu GTC SOS SCT Val Sly Ala CSS CCC CSA Aro Pro 7Arq ATG CAS CGSC SAG Met Gin Arg Siu TCC GA SCA CCG Ser Arg Ala Pro TTT ATS TTS SAC Phe Met Leu Asp SCO CAS CAS CCA Ala Sin Gin Pro TCT SOS CCC CTC Ser Ala Pro Leu CTG TAC CST CCC ATS ACS SAT SAC ACT CCC GST Leu Tyr Arg Ala Met Thr Asp Asp Ser Sly Sly ACC CCC CAG CCT Thr Pro Gin Pro TTS SAC CCT SC7 Leu Asp Arg Ala CTO ArT ATG AC Leu Ile Met Ser G70 AAC ATA GTO Val Asn Tie Val CCC SAC CCT ACC Arg Asp Arg Thr CCC TAC CAS SAC Sly Tyr Sin Siu CAC TGG AAG GAA His Trp Lys Siu TTC CAC Phe His 125 TTT SAC CTA Phe Asp Leu TTC CGC ATC Phe Arg Ile 145 CAS ATC CCT GCT Sin le Pro Ala GAS OCT GTC ACA Siu Ala Val Thr OCT SOT GAS Ala Ala Glu 140 ACA ACC CTC Thr Thr Leu TAC AAA SAA CCC Tyr Lys Slu Pro ACC CAC CCC CTC Thr His Pro Leu CAC ATC His lie 160 ACC ATS TTC SAA Ser Met Phe Siu GTrO CAA GAG CAC Val Sin Siu His AAC ASS GAS TCT Asn Arg Siu Ser TTS 'FTC TTT TTS Leu Phe Phe Leu CTT CAS AG CTC Leu Sin Thr Leu TCT 555 SAC SAG Ser Gly Asp Slu TGG CTO GTS CTS Trp Leu Val Leu GAC ATC ACA SCA Asp Ile Thr Ala 195 SCC AGT GAC CGA TGS CTS CTG AAC Ala Ser Asp Arg Trp Leu Leu Asn 200 205 CTC TAT STG GAA ACC GAS GAT GSS Leu Tyr Val Slu Thr Slu Asp Gly 215 220 CAT CAC AAS GAC CTA GGA CTC CGC His His Lys Asp Leu Gly Leu Arg 210 CAC ASC ATA SAT CCT SOC CTA GOT GOT CTG cTT- GSA GA CAA GCA CCA His Ser Ile Asp Pro Sly Leu Ala Gly Leu Leu Sly Arg Gin Ala Pro 225 230 235 CSC 'FCC ASA CAS CCT TTC ATS OTT GOT TTC 'FTC ASS GCC AAC CAG AGT Ary Ser Arg Sin Pro Phe Met Val Sly Phe Phe Arg Ala Asn Sin Ser 734 782 830 WO 97/41881 WO 97/188 1PCTJUS97/07816 75 240 245 250 CCT GTC- CC-C C-CC CCT CGA ACA GCA AGA CCA CTC- AA- AAC- A.A CAG CTA Pro Val Arg Ala Pr-o Arg Thr Ala Arg Pro Leu Lys Lys Lys Gin Leu 255 260 265 270 AAT CAA ATC AAC CA- CTC- CCC- CAC TCC AA2C APAA CAC CTA C-GA ATC CTT Asn C-in Ile Asn C-lm- Leu Pro H-is Ser Asr. Lys his Leu Gly Ile Leu GAT C-AT GC-C Asp Asp Gly CTC TAT C-TC Leu Tyr Val CCC CAC- GC-C Pro C-in Gly C-CT TCT CAC C-CC AC-A C-AA C-TT TC-C CC-C AC-C CAT C-AG C-iy Ser His Gly Ara C-lu Val Cys Arg Arg His C-lu T7C CGOT C-AC Phe Arg Asp TOG CTG C-AC Trp Leu Asp ATT C-CC Vai Ile Ala ATC TAC CCA Il e Tvr P r o TAC TCC C-CC Tvyr Ser Ala 7OT C-CT C-C-C Cys Ala Gly 320 CTC- AAC Leu Asn TCC TC-T ATG Ser Cys Met TOO ACC AA-C C'C C-CC Ser Thr Asn Hi's Ala 345 C-AT ATC ATC CCC AA- Asp le Ile Prc Lys ATO CIG C-CC Met C-in Ala CAT CTC- ATC- His Leu Met C-GTOC 307T Val Cys Cys C-TO CCT Val Pro 365 ACT C-AC- CTC- Thr C-lu Leu GC ATC 070 Val li 1 1-

AC-T

Ser 370

CC-

ATT TCT CTG Ile Ser Leu TAT C-AT AC-A Tyr Asp Arg AAC ;,AT AAT Asn Asn Asn 360 TOT GC-G TOC -l Cys 974 1022 1070 1113 1166 1214 1262 1315 1375 1435 1495 1555 1615 1674 AC-C C-AC- CC-C AAC C-TA C-TC CAC- C-CC CA" TC-ACTCCCTO CCCAACAC-CC TC-CTCCCATC CCATCTATCT His

AC-TCAGGCCCT

CTCTITCCAAC-

TTCACAC-TCT

ACCCT-CTC-A

C-CATCTATC-'

CTACTCCC

CCATCAC-AAC-

C-CAC-OAAACC AACAAAGA-C TOC-CCCTCTC TC-TTCT TTT T CCTCAC-TAC-C CCC-ATCTCTC CC TTT GGCGAT TOC-OCACAGA ACCTGGACT TGAACCTGC-A ATTrAC-GTGT GTGCAC-ACAT OAAC-CCAC-TG CTTTCAACTC GCCAAC-CCTC- AC-AAC-ATCCT ATCTCCCCAA ACTCCCCAAT AGTCCAATTT ACCAACTTAT ACACAC-OCTA C-ACCTCAGGC GCCACACTC CCCCTAC-CAC

CATGTCCACA

CCTAGTTATA

GCACCAGO-

TCATGAC-TCA

TCTTCA-TAT

TCCATACC

INFORMATION FOR SEQ ID NO:26: WVO 97/41881 76 SEQUENCE CHARACTERISTICS: LENGTH: 399 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: prote~n (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: PCT/US97/078 16 *t Ala G ly Leu Le u Gin Met AlIa Leu 115 Gin~ Lys Phe Leu Asp 195 Arg Gly Gly Gly Gin Asp 100 Gly 71 le G lu Asp 180 Ile Pro 01 y Val Leu Pro Asp Leu Tyr Pro Pro Val 165 Leu Thr Ser Pro 40 Arg Ala Gly Ser Pro 120 Glu his Glu Leu Ser 200 Tyr His 25 Arg Pro Pro I y Phe 105 His Ala Pro Hi S Arg 185 Asp Pro Aso Arg Leu Gly 90 Val1 Trp Val1 Leu Ser 170 Ser Arg ValI Arg 4 Ala -e,J G1 n Val Phe 125 Ala Th r C-lu G iu Leu 205 Phe Glu Pro Aso Pro Glu His Glu Leu Ser Gly 190 Asn G ly Pro Ile Va I Leu His Ara Phe Phe Hi.'s Asp 175 Trp His His Gly Leu Leu Trp Leu Leu Gly Leu Ala Leu Cys Lys Asp Leu Gly Leu Arg Leu 210 215 Val Glu Thr Giu Asp 220 Ile Asp Pro Gly Leu Ala Gly Leu Leu Gly Arg Gin Ala Pro Arg Ser 225 230 235 240 Arg Gin Pro Phe Met 245 Val Gly Phe Phe Ala Asn Gln Ser Pro Val 255 WO 97/41881 WO 97/88 1PCTIUS97/07816 Arg Ile Gly Val 305 Gly Se r L eu Leu Leu a 385 (2) 77 Ala Pro Arg Thr Ala Arg Pro Leu Lys Lys L 260 265 Asn Gin Leu Pro His Ser Asn Lys His Leu G 275 280 His Gly Ser His G-i> Ar g Glu Val CyS Arg A: 290 2953 Ser Phe Arg Aso Leu Gly Trp Leu Asp Ser Va 310 315 Tyr Ser Ala Tyr Tyrf Cys Ala Cly Gl-u Cys _1 1 325 330 Cys Hez Asn Ser Thr Asn His Ala Th r Met G! 340 345 Met Lys Pro Asp Ile Ile Pro Lys Va I C\'s Cy 355 360 Ser Ala Ile Ser Leu Leu Tyr 7\,r Aso Arg As] 370 375 386 Arg Arg Glu Ar a Asn V.etL Val Val Glrn Ala Cy 390 395 :NFORMIATIoN FOR SEQ 1D ICO:27: SEQUENCE CHAR.ACTERISTICS: LENGTH: 104 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 12 OLELIETYPE: pr::e_-n

FEATURE:

NAME/KEY: Protein LOCATION: 104 OTHER INFORMATION: /note= 'BMP311 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe A 1 5 10 Glu Trp Ile Ile Ser Pro Lys Ser Phe Asp A 25 Ala Cys Gin Phe Pro Met Pro Lys Ser Leu L 40 Trhr Ile Gln Ser Ile Val Ala Arg Ala Val G ys; Gin Leu 270 17 l-e Leu 285 rQ His Giu

IC

I1 lIe Ala Pro n Ala Leu 350 s Val Pro 3 6 n A sn Asn 0 s G Iy Cys I Asr Aso Leu Pro Leu 335 Val la 1 Gin Aso Tvr Gin 320 Asn Pis Giu Ile la Asp Ile Gly Trp Ser la Tyr Tyr Cys Ser Gly ys Pro Ser Asn His Ala ;iy Val Val Pro Gly Ile WO 97/41881 PCTIUS97/07816 -78- 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 ASsn Met 90 Thr Val Glu Ser Cys Ala Cys Arc 100 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 1C2 amino acids TYPE: amino acid C) STRANDEDNESS: sinle TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS (ix) FEATURE: NAME/KEY: Protein LOCATION: 02 D) OTHER INFORMATION: /ncte= (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 Prc Glu Gly Tyr Ala Ala Phe Tyr Cys Asp Gly 25 9 Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 40 Ile Val Gln Thr Leu Val His Leu Met Phe Pro Asp His Val Pro Lys 50 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 WO 97/41881 PCT/US97/07816 79 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 4ii "OLECULE TYPE: 7rrrein ORITGINzLL SOURCE: ORGANISM: HOND3 SAPIENS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1.-102 OTHER INFORM1,ATION: /note= 1"BMP6"- (xi) SEQUENCE DESCRIPTION: SEQ ID N0:29: Cy s Arg Lys His 0Th- Leu Tyr Val Ser Phe Cin AErp Leu Gly, Tro Gln C) is Asp e 1e la ro ys ~r Aa Aa Asn Tyr Cys Asp Gly 25 G lu Cys Ser Phe Pro Leu Asn Ala tHis me: As- Ala 7hr Asn H!is Ala 354C Tle Val 0In Thr Leu Val His Leu Met Asn. Pro 0-lu Tj r Val Pro Lys 55 Pro Cys, Cys Ala ?ro Thr Ly's Leu Asn Ala :le Ser Val Lei Tyr Phe 70 75 so Asp Asp Asn Ser Asm Val ile Lei Lys Lys Tyr Arg T rp Met Val Val 90 Arg Ala Cx'.s ':lv Cys His INFORMATION FOR SEQ ID 140:30: SEQUENCE CHARACTERISTICS: LENGTH: 1247 base pairs TYPE: nucleic acid 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-l' /note= "GDF-1 CDNA" WO 97/41881 WO 97/ 1881PCTIUS97/07816 80 (xi) SEQUENCE DESCRIPTION: SEQ ID GGGACACCG GCCCCGCCCT CACCCCACT7C cTCCCGGGCC GCCCCGGACC CTGCGCACTC TCTGGTCATC CCCTGOGAGG AAG ATGQ CCA CCG CCG CAG CAA OCT CCC TGC Met Pro Pro Pro Gin Gin Gly Pro Cys GGC CAC CAC CTC CTC CTC CTC CTG GCC CTG CTG CTG CCC TCO CTC CCC Gly His His Leu Leu Leu Leu Leu Ala Leu Leu Leu Pro Ser Leu Pro CTG ACC CC Leu Tbr Arg OCT CTA GGA Ala Leu Sly CCC CCC Ala Pro GTO CCC CCA GGCC CCA OCC Val Pro Pro Oly Pro Al.a 35 GCC CCC CTG CTC Ala Ala Leu Leu COC CAT GAG CCC Arg Asp Glu Pro CuT GCC CCC AGO S -y Ala Pro Arg CTC COG CCC euArg Prr, OTT CCC CCC GTC ATO TGC CGC CTO TTT CSA CCC CGC CAC CCC CAG GAG Val Pro Pro Val Met Tro Arg Leu Phe Ara Arg Ara Asp Pro Cmn Oiu ACC ACO TCT Thr Arg Ser GGC TCC CGS CGS ACS TCC CCA COO Oly Ser Ara Ara Thr Ser Pro Gly ACC CTO CA.A CCC Th.r Leu GIn Pro CAC OT3 GAO CGAG CTC CCC CTC GCC OGA His Val Olu Oiu Leu Oly Val Ala Oly ATC OTO CCC CAC Ile Val Arg His CCC CAC CCC OCT CC CCC ACC CCC GCC Pro Asp Arg Gly Ala Pro Thr Arg Ala TCC GAO Ser Glu 115 CCT GTC TOG Pro Val Ser CCC C Ala Ala 120 OGC CAT 7CC CCT GAO TOO ACA 070 CTC TTC GAC CTC TCG Oly His Cys Pro Olu Trp Thr Val Val Phe Asp Leu Ser OCT OTO OAA Ala Val Olu 1 *5 CCC OCT GAO Pro Ala OJlu 140 CCC CCC AOC COO Arg Pro Ser Arg 0CC CCC CTC GAO Ala Arg Leu Olu 145 GCC GGC TOG GAG Oly Oly Trp Olu CTO COT TTC C CC Leu Arg Phe Ala Ala 150 CTG AGC 070 C CAA Leu Ser Val Ala Cmn 165 CC GC Ala Ala 155 OCG OCA CCC CCG Ala Ala Ala Pro 0CC CAG GC Oly Cmn Gly GC CCC GC GAC CCC COO CCC OTO CTC CTC Ala Oly Ala Asp Pro Oly Pro Val Leu Leu 175 180 CTO 000 CCC CCA 070 CCC CC GAG CTG CTO Leu Oly Pro Pro Val Arg Ala Olu Leu Leu 190 195 CCC CAG Arg Cln 185 GGC CC Oly Ala 200 638 686 770 070 CCC CC Leu Val Pro Ala WNO 97/41881 PCTJUS97/078 16 81 OCT TOOGCCT Ala Trp Ala AAC GCC TCA TGG Asn Ala Ser Trp CGC AGC CTC CC Arg Ser Leu Arg CTG GCG CTG Leu Ala Leu 215 CCC GAG GCC Ala Glu Ala 734 'CC CTA COC CCC COG CCC CCT CCC GCC 7CC CCC' CCC CTG Ala Leu Ara 220 Pro Arg Ala Pro Ala Cys Ala Arg TCG CTC CTC CC'G CTG ACC Ser Leu Leu Leu Val Thr 235 CTC CAC CCC CCC CTG GC Asp Pro Arg Leu Cys 245 CAC CCC CTC GCC His Pro Leu Ala CCC CCC CCC GC Cly Pro Cly Cly CCC COG CCC CAC Pro Arg Arg Asp GAA CCC GTC TTC Glu Pro Val Lei OCT TGT CCC CC Ala Cys Ara Ala CCC CTS TAC CTG Ara Leu TYr Va 7TC CCC GAG CTC Plhe Arg Clu Val CCC TCC Cly Trp 280 CAC CCC TOC His Arg -rp GOT CAC TC Cly Gln Cys 300 ATC GC CCG CC Ile Ala Pro Arg 77TTC C CC AAC Phe Leu Ala Asr.

TAC 7CC CAC Tyr Cys Gln 295 CCC CCC CCC Cly Pro Pro GC CTC CCC GCC Ala Leu Pro Val CTC 7CG C-CC 7CC Leu Ser Cly Ser GCC CTC Ala Lei 315 AAC_ CAC CCT CTC Asn His Ala Val CCC GC CTC A7C Ara Ala Leu M-et CC CCC CCC CCC Ala Ala Ala Pro 574 1C22 i070 1118 1166 1219 CCC CCC GAC CTG Ala Ala Asp Leu 7CC 7CC GTG CCC Cys Cys Val Pro CCC C7C TCC CCC Arg Leu Ser Pro TCC CTC CTC Ser Val Leu TTC 777 Ph-e Phe 350 CAC AAC ACC CAC Asp Asn Ser Asp CAC GAG 7CC GC Asp Glii Cys Gly 370 C7C CTC CTC CCG Val Val Lei Arg CAG TAT Gin Tyr 360 CAC CAC ATC GTC GTC Glu Asp Met Val Val 365 TGC CCC TAI\CCCGGC CGGGCAGGGA Cys Arg CCCGGGCCCA ACAATAAATC CCGCGTGG INFORM.ATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 372 amino acids TYPE: amino acid TOPOLOGY: linear 1247 (ii) MOLECULE TYPE: protein WO 97/41881 WO 971881PCT[US97/07816 82- DESCRIPTION: SEQ ID NO:31: (xi) SEQUENCE Met Pro Pro Pro Gin Gin Gly Pro Cys Gly His His Leu Leu Leu Leu

S

a Leu Pro Pro Leu Thr Val Ara Val Ala 145 Gly Asp Pro Trp Ala 225 Asp Pro Tyr Ala G 1ly Phe Ser Al a Ala Val 130 Arg Gly Pro Val1 Pro 210 Al a Pro Val Val1 Le u Pro ~35 Gly Arg Pro C-ly Ser 115 Phe ILeu Trp, Gly Arg 195 Arg Cys Arg Leu Ser Leu Al a Ala Arg G ly As n 100 3 lu A.sp 3u Pro 180 Ala Ser Ala Leu Gly 260 Phe Leu Ala Pro Arg Va 1 Pro Leu Leu Leu 165 Val1 Giu Leu Ara Cys 245 Gly Pro Al a Ara Asp 70 Thr V a 1 Val Ser Arg 150 Ser Leu Leu Arg Leu 230 His Gly Ser Leu 55 Pro !,eu Arg Ser Al a 135 Phe Val1 Leu Leu Leu 215 Ala Pro Pro Leu 40 Arg Gin G 1n His Al a 120 Val Al a Al a Ara 0 ly 200 Al a Glu 25 Gin Pro Gi1u- Pro II e 105 Ala G hi Ala Gln Giln 185 Ala Leu Ala Ala Leu G- Val Pro Pro T*r Ara Ser 75 CvfS His Val Pro Asp Arg Gly His Cys Pro Ala Glu 140 Ala Ala Ala 155 Al- a Gly Gin 170 Leu Val Pro Ala Trp Ala Ala Leu Arg 220 Ser Leu Leu 235 Arg Pro Arg 250 L eu Val Gly GlIu G ly Pro 125 Ara Ala Gly Ala Arg 205 Pro Leu Arg Met Ser Glu 110 G lu Pro Ala Ala Leu 190 Asn Arg Val1 Asp Glu Arg Arg Gly Th r Thr Arg Glu 160 Ala Pro Ser Pro Leu 240 Glu Leu Pro Leu Thr Ara Ala Pro Val Pro Leu Ala Gly Gly Ala Cys Arg Ala Arg Arg Leu 265 270 Arg Glu Val Gly Trp His Arg Trp Val Ile Ala Pro 280 285 275 Arg Gly Phe Leu Ala Asn Tyr Cys Gin Gly Gin Cys Ala Leu Pro Val WNO 97/4188 1 PCTJUS97/07816 83 290 295 300 Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala Leu Asn His Ala Val Leu 305 310 315 320 Arg Ala Leu Met His Ala Ala Ala Pro Giy Ala Ala Asp Leu Pro Cys 325 330 335 Cys Val Ala Arc L_ e e P e Se- Vil ILe eSp s 340 345 350 Ser Asp Asn Val V'al Leu 9r Gin Ty r G22w Asp Met Val Val Asp G I 355 36035 Cys C- 1y Cys Arc 370

Claims (54)

1. A method of treating a mammal in, or at risk of, chronic renal failure comprising administering to said mammal a therapeutically effective amount of a morphogen.

2. A method of treating a mammal in, or at risk of, chronic renal failure comprising administering to said mammal a therapeutically effective amount of an inducer of endogenous morphogen expression.

3. A method of treating a mammal in, or at risk of, chronic renal failure comprising administering to said mammal a therapeutically effective amount of an agonist of a morphogen receptor.

4. A method of treating a mammal in, or at risk of, chronic renal failure comprising introducing within the kidney of said mammal a therapeutically effective amount of renal mesenchymal progenitor cells. The method of claim 4 comprising the additional step of inducing metanephric differentiation of said cells by contacting said cells with a morphogen. i' 6. The method of claim 4 comprising the additional step of inducing metanephric differentiation of said cells by contacting said cells with an inducer of a morphogen.

7. The method of claim 4 comprising the additional step of inducing metanephric differentiation of said cells by contacting said cells with an agonist of a morphogen receptor.

8. A method of treatment to delay the need for, or reduce the frequency of, chronic dialysis treatments comprising administering to a mammal a therapeutically effective amount of a morphogen.

9. A method of treatment to delay the need for, or reduce the frequency of, chronic o 25 dialysis treatments comprising administering to said mammal a therapeutically effective S* amount of an inducer of endogenous morphogen expression. A method of treatment to delay the need for, or reduce the frequency of, chronic dialysis treatments comprising administering to said mammal a therapeutically effective amount of an agonist of a morphogen receptor.

11. The method of any one of claims 1-10 wherein said mammal is afflicted with a condition selected from the group consisting of chronic renal failure, end-stage renal disease, chronic diabetic nephropathy, diabetic glomerulopathy, diabetic renal hypertrophy, hypertensive nephrosclerosis, hypertensive glomerulosclerosis, chronic glomerulonephritis, hereditary nephritis, and renal dysplasia.

12. The method of any one of claims 1-10 wherein examination of a renal biopsy of said mammal indicates that said mammal is afflicted with a condition selected from the group consisting of glomerular hypertrophy, tubular hypertrophy, glomerulosclerosis, and tubulointerstitial sclerosis.

13. The method of any one of claims 1-10 wherein examination of said mammal indicates renal fibrosis.

14. The method of claim 13 wherein said examination is an ultrasound, MRI or CAT scan of said mammal. The method of any one of claims 1-10 wherein said mammal possesses a number of functional nephron units which is less than about 50% of a number of functional nephron units present in a mammal having intact healthy kidneys.

16. The method of any one of claims 1-10 wherein said mammal possesses a number of functional nephron units which is less than about 40% of a number of functional nephron units present in a mammal having intact healthy kidneys.

17. The method of any one of claims 1-10 wherein said mammal possesses a number of functional nephron units which is less than about 30% of a number of functional nephron units present in a mammal having intact healthy kidneys.

18. The method of any one of claims 1-10 wherein said mammal possesses a number of functional nephron units which is less than about 20% of a number of functional nephron units present in a mammal having intact healthy kidneys. -86-

19. The method of any one of claims 1-10 wherein said mammal is a kidney transplant recipient. The method of any one of claims 1-10 wherein said mammal possesses only one kidney.

21. The method of any one of claims 1-10 wherein examination of a urinary sediment of said mammal indicates a presence of broad casts.

22. The method of any one of claims 1-10 wherein said mammal has a GFR which is chronically less than about 50% of a GFRxp for said mammal.

23. The method of claim 22 wherein said mammal has a GFR which is chronically less than about 40% of a GFRexp for said mammal.

24. The method of claim 22 wherein said mammal has a GFR which is chronically less than about 30% ofa GFRlxp for said mammal. The method of claim 22 wherein said mammal has a GFR which is chronically less than about 20% of a GFRxp for said mammal. ooe.

26. The method of any one of claims 1-10 wherein said mammal is a human male S/nweighing at least about 50 kg and has a GFR which is chronically less than about mi/min.

27. The method of claim 26 wherein said mammal is a human male weighing at least about 50 kg and has a GFR which is chronically less than about 40 ml/min.

28. The method of claim 26 wherein said mammal is a human male weighing at least S"about 50 kg and has a GFR which is chronically less than about 30 ml/min.

29. The method of claim 26 wherein said mammal is a human male weighing at least about 50 kg and has a GFR which is chronically less than about 20 ml/min. S 30. The method of any one of claims 1-10 wherein said mammal is a human female weighing at least about 40 kg and has a GFR which is chronically less than about ml/min. -87-

31. The method of claim 30 wherein said mammal is a human female weighing at least about 40 kg and has a GFR which is chronically less than about 30 ml/min.

32. The method of claim 30 wherein said mammal is a human female weighing at least about 40 kg and has a GFR which is chronically less than about 20 ml/min.

33. The method of claim 30 wherein said mammal is a human female weighing at least about 40 kg and has a GFR which is chronically less than about 10 ml/min.

34. The method of any one of claims 1-10 wherein said treatment reduces serum creatinine levels in said mammal by at least about 5% over 3 months. The method of any one of claims 1-10 wherein prior to said treatment said mammal presented a chronic decline in a clinical indicator of renal function; and after at least about 3 months of said treatment, said indicator stabilizes.

36. The method of any one of claims 1-3 wherein said administration is oral.

37. The method of any one of claims 1-3 wherein said administration is parenteral.

38. The method of claim 37 wherein said administration is intravenous. 15 39. The method of claim 37 wherein said administration is intraperitoneal. a The method of claim 37 wherein said administration is into the renal capsule.

41. The method of claim 37 wherein a stent has been implanted into said mammal for said administration. a* 20 43. The method of claim 41 wherein said stent is an intraperitoneal stent.

44. The method of claim 41 wherein said stent is a renal intracapsular stent. The method of claim 37 wherein said administration is by an implanted device. -88-

46. The method of any one of claims 1-3 wherein said administration is at least once a week for a period of at least about one month.

47. The method of any one of claims 1-3 wherein said administration is at least once a month for a period of at least about one year.

48. The method of claim 1 wherein said morphogen is administered at a dosage of about 0.01-1000 jtg/kg body weight of said mammal.

49. The method of claim 48 wherein said morphogen is administered at a dosage of about 10-300 pg/kg body weight of said mammal. A method of promoting metanephric differentiation of renal mesenchymal progenitor cells comprising the step of contacting said cells with a morphogen in an amount effective to induce said differentiation.

51. The method of claim 1 wherein said renal therapeutic agent comprises a polypeptide comprising a C-terminal cysteine domain of a protein selected from the group consisting of a pro form, a mature form, and a soluble form of a polypeptide 15 selected from the group consisting of OP-1, OP-2, OP-3, BMP2, BMP3, BMP4, *BMP6, and BMP9.

52. The method of claim 51 wherein said renal therapeutic agent comprises a polypeptide comprising a C-terminal cysteine domain of a protein selected from the group consisting of a pro form, a mature form, and a soluble form of human OP-1.

53. The method of claim 1 wherein said renal therapeutic agent comprises a polypeptide having at least 70% homology with an amino acid sequence of a C-terminal seven-cysteine domain of human OP-1.

54. The method of claim 53 wherein said polypeptide has at least 75% homology with an amino acid sequence of a C-terminal seven-cysteine domain of human OP-1.

55. The method of claim 53 wherein said polypeptide has at least 80% homology with an amino acid sequence of a C-terminal seven-cysteine domain of human OP-1. -89-

56. The method of claim 53 wherein said polypeptide has at least 60% identity with an amino acid sequence of a C-terminal seven-cysteine domain of human OP-1.

57. The method of claim 53 wherein said polypeptide has at least 65% identity with an amino acid sequence of a C-terminal seven-cysteine domain of human OP-1.

58. The method of claim 53 wherein said polypeptide has at least 70% identity with an amino acid sequence of a C-terminal seven-cysteine domain of human OP-1.

59. The method of any one of claims 51-58 wherein said renal therapeutic agent: induces chondrogenesis in an ectopic bone assay; prevents, inhibits, delays or alleviates loss of renal function in an animal model of chronic renal failure; or causes a clinically significant improvement in a standard marker of renal function when administered to a mammal in, or at risk of, chronic renal failure. The method of claim 1 wherein said renal therapeutic agent is selected from the 15 group consisting of human osteogenic proteins and human bone morphogenetic proteins.

61. Use of a morphogen for the manufacture of a medicament for treating a mammal in, or at risk of, chronic renal failure.

62. Use of an inducer of endogenous morphogen expression for the manufacture of a medicament for treating a mammal in, or at risk of, chronic renal failure. 20 63. Use of an agonist of a morphogen receptor for the manufacture of a medicament for treating a mammal in, or at risk of, chronic renal failure.

64. Use of renal mesenchymal progenitor cells for the manufacture of a medicament for treating a mammal in, or at risk of, chronic renal failure. Use of a morphogen for the manufacture of a medicament to delay the need for, or reduce the frequency of, chronic dialysis treatments.

66. Use of an inducer of endogenous morphogen expression for the manufacture of a medicament to delay the need for, or reduce the frequency of, chronic dialysis treatments.

67. Use of an agonist of a morphogen receptor for the manufacture of a medicament to delay the need for, or reduce the frequency of, chronic dialysis treatments.

68. Use of a morphogen for the manufacture of a medicament to promote metanephric differentiation of renal mesenchymal progenitor cells.

69. A method of treating a mammal in, or at risk of, chronic renal failure, substantially as herein described with reference to any one of the examples but excluding comparative examples. A method of treatment to delay the need for, or reduce the frequency of, chronic dialysis treatments, substantially as herein described with reference to any one of the examples but excluding comparative examples.

71. A method of promoting metanephric differentiation of renal mesenchymal 15 progenitor cells, substantially as herein described with reference to any one of the examples but excluding comparative examples. S 72. Use of a morphogen for the manufacture of a medicament, substantially as herein described with reference to any one of the examples but excluding comparative examples. 20 73. Use of an inducer of endogenous morphogen expression for the manufacture of a medicament, substantially as herein described with reference to any one of the examples but excluding comparative examples.

74. Use of an agonist of a morphogen receptor for the manufacture of a medicament, substantially as herein described with reference to any one of the examples but excluding comparative examples. -91- Use of renal mesenchymal progenitor cells for the manufacture of a medicament, substantially as herein described with reference to any one of the examples but excluding comparative examples. DATED this 9 th day of September 2003 BALDWIN SHELSTON WATERS Attorneys for: CREATIVE BIOMOLECULES, INC. 0