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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) Internationa] Patent Oassification ^ 
A61K 37/00 



A2 



(11) International Publication Number: WO 94/06449 

(43) International Publication Date : 3 1 March 1 994 (3 1 .03.94) 



(21) International Application Number: PCT/US93/08808 

(22) International Filing Date: 16 September 1993 (16.09.93) 



(30) Priority data: 

946,238 
029.335 
040,510 



16 September 1992 (16.09.92) US 
4 March 1993 (04.03.93) US 
3 1 March 1 993 (3 1 .03.93) US 



(71) Applicant: CREATIVE BIOMOLECULES, INC. [US/ 

US]; 45 South Street, Hopkinton, MA 01748 (US). 

(72) Inventors: KUBERASAMPATH, Thangavel ; Six Spring 

Street, Medway, MA 02053 (US). RUEGER, David, C. ; 
19 Downey Street, Hopkinton, MA 01748 (US). OPPER- 
MANN, Hermann ; 25 Summer Hill Road, Medway, 
MA 02053 (US). PANG, Roy, H., L. ; 15 Partridge 
Road, Etna. NH 03750 (US). COHEN, Charles^ M; ; 98 
Winthrop Street, Medway, MA 02053 (US). OZKAY- 
NAK, Engin ; 44 Purdue Drive, Milford, MA 01757 
(US). SMART, John, E. ; 50 Meadow Brook Road, Wes- 
ton, MA 02193 (US). 



(74) Agent: KELLEY, Robin, D.; Testa, Hurwitz & Thibeault, 
Exchange Place. 53 State Street, Boston, MA 02109 
(US). 



(81) Designated States: AT, AU, BB, BG, BR, CA, CH. C2, 
DE, DK, ES, Fl, GB, HU, JP. KP. KR, LK, LU, MG, 
MN, MW, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SK, 
UA, European patent (AT. BE, CH, DE, DK, ES, FR, 
GB, GR, IE. IT, LU, MC, NL, PT. SE), OAPI patent 
(BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE. SN. 
TD, TG). 



Published 

Without international search report and to be republished 
upon receipt of that report. 



(54) Title: MORPHOGEN INDUCED LIVER REGENERATION 



(57) Abstract 

Disclosed are therapeutic treatment methods, compositions and devices for maintaining liver function in a mammal, in- 
cluding means for regenerating lost or damaged hepatic tissue, means for enhancing viability and integration of hepatic tissue 
and organ transplants, and means for correcting liver function deficiencies, including means for enhancing diminished liv«r func- 
tion due to tissue injury or disease. The methods, compositions and devices on this invention all provide a therapeutically effec- 
tive morphogen concentration to the hepatic cells to be treated. Also disclosed are methods and compositions useful in a gene 
therapy or drug delivery protocol for correcting a protein deficiency in a mammal. 



FOR THE FURPOSES OF INFORMATiON ONLY 

Codes used lo identify States party to the PCT on the front pages of pamphlets publishing international 
applications under the PCT. 



AT 


Austria 


AU 


Ausiralia 


BB 


Barbados 


BE 


Belgium 


BP 


Burkina Faso 


BG 


Bulgaria 


BJ 


Benin 


BR 


Brazil 


BY 


Belarus 


CA 


Canada 


CF 


Central African Republic 


CG 


Congo 


CH 


Switzerland 


CI 


Cote d'lvoire 


CM 


Cameroon 


CN 


China 


OS 


CEcchoslovakia 


cz 


Czech Republic 


DE 


Germany 


DK 


Denmark 


ES 


Spain 


Fl 


Fmland 



FR 


France 


CA 


Gabon 


GB 


United Kingdom 


GN 


Guinea 


GR 


Greece 


HV 


Hungary 


IE 


Ireland 


IT 


luly 


JP 


Japan 


KP 


Dcmocraiic Pcopte^s Republic 




of Korea 


KR 


Republic of Korea 


KZ 


Kazakhstan 


U 


Uechtensiein 


LK 


Sri LanLa 


tu 


L4iu:mbourg 


LV 


Latvia 


MC 


Monaco 


MG 


Madagascar 


ML 


Mali 


MN 


Mongolia 



MR 


Mauritania 


MW 


Malawi 


NE 


Niger 


NL 


Netherlands 


NO 


Norway 


NZ 


New Zealand 


PL 


Poland 


PT 


Portugal 


RO 


Romania 


RU 


Russian Federation 


SD 


Sudan 


SE 


Sweden 


SI 


Slovenia 


SK 


Slovak Republic 


SN 


Senegal 


TD 


Chad 


TC 


Togo 


UA 


Ukraine 


US 


United States of America 


uz 


Uztvekistan 


VN 


Viet Nam 



wo 94/06449 



PCr/US93/08808 



Morphoqen- Induced Liver Regeneration 

FIELD OF THE INVENTION 

The present invention relates generally to liver 
treatment methods. 

5 

BACKGROUND OF THE INVENTION 

The present invention relates to methods and 
compositions for regenerating lost or damaged liver 

10 tissue in vivo and to methods and compositions for 

maintaining normal liver function which may be reduced 
or lost as a result of such tissue damage. The 
invention further relates to methods and compositions 
for correcting one or more liver function deficiencies 

15 in a mammal, particularly a human. 

The liver is the largest viceral organ in the body 
and consists of two main lobes, a larger right lobe and 
a smaller left lobe. The right lobe also contains two 
20 smaller segments referred to as the cuadata and 

guadrata lobes. The liver has a dual blood supply, 
consisting of the hepatic artery and the portal vein. 
The hepatic lymphatics drain principally into lymph 
nodes of the porta hepatis and celiac axis. 

25 

The liver is responsible for a wide variety of 
functions, broadly characterized as metabolic, storage, 
synthetic, catabolic and excretory. Specifically, the 
liver is the central organ of glucose homeostasis, 
30 responsible for both storing excess blood glucose as 
glycogen and restoring blood glucose by glycogenolysis 



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and gluconeogenesis and by converting free fatty acids 
to triglycerides and lipoproteins • The liver also 
stores triglycerides, iron, copper and lipid-soluble 
vitamins and synthesizes many of the binding proteins 
5 for iron, copper and Vitamin A. 

In addition, most senim proteins, with the 
exception of immunoglobulins, are synthesized in the 
liver, including albumin, the principal source of 

10 plasma oncotic pressure, blood clotting factors such as 
prothrombin, fibrinogen and Factor VIII, as well as 
complement and other acute phase reactants involved in 
an immune response. The liver also functions as a 
catabolic site for hormones, serum proteins, and other 

15 endogenous proteins, as well as acting as the 

detoxification site for foreign compounds, including 
drugs (pharmaceuticals), industrial chemicals, 
environmental contaminants, and various bacterial 
metabolism byproducts. Finally, the liver excretes 

20 bile, which provides a repository for the products of 
hemecatabolism and also is vital for fat absorption in 
the small intestine. 

Not surprisingly, liver function disorders, whether 
25 resulting from a particular protein deficiency or from 
hepatic tissue damage and/or loss, has serious and far- 
reaching consequences. For example, reduced albiunin 
levels in chronic liver disease contribute to the 
development of edema and ascites; liver failure also is 
30 characterized by severe and often life-threatening 
bleeding, due to the reduced production of essential 
blood clotting factors. Hepatic failure also can 
induce neurological dysfunction, characterized broadly 
as hepatic encephalopathy, as well as associated renal 
35 failure, jaundice, pulmonary complications, and a host 
of disorders associated with hormonal imbalances. 



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Unlike most other organs in the body the liver has 
a defined regenerative capacity following hepatic 
tissue damage or cell death. Specifically, while 
5 hepatocytes do not proliferate actively following fetal 
and post natal liver growth, normally quiescent 
hepatocytes do divide in response to cell death or loss 
of liver tissue. However, where tissue damage is 
extensive and/or chronic, permanent tissue damage can 

10 result, reducing the organ's viability and functional 
capacity. Permanent hepatic tissue damage typically is 
characterized by extensive necrosis and/or fibrogenesis 
or scarring (cirrhosis). Another source of 
nonreparative damage results from hepatic neoplasms and 

15 metastatic carcinomas. 

Where either the mass of liver cells is 
sufficiently diminished or their function sufficiently 
impaired, hepatic failure ensues. The etiology of 

20 hepatic failure may be metabolic (e.g., altered 

bilirubin metabolism or fatty acid storage), infectious 
(e.g., induced by viral hepatitis, hepatic 
schistomiasis, syphilis, or ascariaris), toxic (e.g., 
induced by ethanol, ammonia, phenol, and other 

25 environmental toxins, fatty acids, drugs and/or their 
metabolites), autoimmune, ischemic or nutritional 
(e.g., alcoholic liver disease). 

Another source of hepatic failure results from 
30 malignant tumors. The tumor cells may be derived from 
hepatic tissue cells (as in hepatocellular carcinoma, 
bileduct carcinomas, hepatoblastomas or 
hemangiosarcoma) or may be derived from distant tissue 
as part of a metastatic cancer. In fact, metastatic 
35 cancers are by far the most common malignant neoplasms 
of the liver, most notably derived from cancers of the 
gastrointestinal tract, breast and lung. 



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Another source of diminished liver function arises 
from hepatic protein deficiencies, which may result 
from a genetic defect (so called "inborn errors of 
5 metabolism") or may be induced by, for example, a 
pharmaceutical, infectious agent byproduct, or the 
like. For example, hemophilia is believed to be 
associated with diminished Factor VIII production, 
Wilson's disease, a copper metabolism disorder, is 
10 associated with deficient ceruloplasmin production by 
the liver, altered albumin production affects bilirubin 
metabolism, and Cj^-antitrypsin deficiency, normally 
produced in the liver, can result in fatal neonatal 
hepatitis • 

15 

To date, the only viable treatment for hepatic 
failure or for patients at risk for hepatic failure due 
to, for example, chronic acute hepatitis, biliary 
atresia, idiopathic cirrhosis, primary biliary 

20 cirrhosis, sclerosing cholangitis, inborn errors of 

metabolism or malignancy, is liver transplantation. To 
date, liver transplantation also is the only viable 
alternative for correcting significant liver function 
deficiencies that result from inborn errors of 

25 metabolism. Liver transplantation as a treatment 
method suffers from donor scarcity, particularly of 
pediatric livers, technical surgical complexity, 
postoperative complications including organ rejection, 
and continuing difficulties in maintaining organ 

30 viability throughout the transplant process. 

Selective cell transplantation of only those 
parenchymal elements necessary to replace lost function 
has been proposed as an alternative to whole or partial 
35 organ transplantation that avoid major surgery with its 
attendant blood loss, anesthetic difficulties, and 
complications (P.S.Russell, Ann. Surg. 201(3), 255-262 



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(1985). Replacing only those cells which supply the 
needed function reduces problems with passenger 
leukocytes, antigen presenting cells r and other cell 
types which may promote the rejection process. The 
5 ability to expand cell numbers with proliferation of 
cells in culture, in theory, allows autotransplantation 
of one's own tissue. In addition, transplantable cells 
may be used as part of a gene therapy to correct a 
liver protein deficiency, and/or as in vivo drug 

10 delivery vehicles. WO88/03785 published June 2, 1988, 
and WO90/12640 published November 1, 1990, both 
describe methods for attaching hepatocytes to matrices 
and implanting the matrices at sites in vivo that are 
capable of providing the cells with adequate nutrition 

15 or gas exchange, such as within mesentery folds or the 
odentum. To date, the existing protocols suffer from a 
variety of limitations. Typically, partial hepatectomy 
is required to stimulate cell proliferation of the 
synthetic tissue in vivo. In addition, cell 

20 implantation typically is accompanied by significant 

cell loss, requiring a substantial seed cell population 
for implantation, which may further require lengthy in 
vitro incubation periods. The delay in in vivo 
integration of the implanted cell-matrix structure also 

25 places significant restrictions on the matrix scaffold 
composition. Finally, the implanted cell-matrix 
structures also are at risk for destruction by the 
implant host's immune response mechanisms. 

30 It is an object of this invention to provide 

methods and compositions for regenerating lost or 
damaged hepatic tissue in vivo in an existing liv^r 
without requiring organ or tissue transplant. Another 
object is to provide means for maintaining normal liver 

35 function following hepatic tissue injury or in 

anticipation of such injury. Another object is to 



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provide means for enhancing or increasing a depressed 
liver function level which may result from a tissue 
injury or disease. Still another object is to provide 
methods and compositions for correcting a liver 
5 function deficiency in a mammal. Yet another object is 
to provide gene therapy protocols and compositions 
useful for correcting a protein deficiency in a mammal. 
Yet another object is to enhance integration of a liver 
tissue implant. These and other objects and features 
10 of the invention will be apparent from the description^ 
drawings and claims which follow. 



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Siunmarr of the Invention 

The present invention provides methods and 
compositions for maintaining liver function in a 
5 mammal. The invention provides means for correcting 
one or more liver function deficiencies in a mammal 
that may arise, for example, from an inborn metabolism 
defect, and means for regenerating lost or damaged 
hepatic tissue in a mammal, including means for 

10 protecting the tissue from damage thereto. The 
invention also provides means for enhancing the 
viability of a hepatic tissue or organ to be 
transplanted and means for enhancing the integration of 
the transplanted tissue. The methods and compositions 

15 of this invention include providing to hepatic cells a 
therapeutically effective concentration of a 
morphogenic protein ( "morphogen", as defined herein) 
upon hepatocellular injury, or in anticipation of such 
injury, or following diagnosis of a liver function 

20 defect in a mammal, for a time and at a concentration 
sufficient to maintain or regain liver function in 
vivo. 

In one aspect, the invention features compositions 
25 and therapeutic treatment methods that include 

administering to a mammal a therapeutically effective 
amount of a morphogenic protein ("morphogen"), as 
defined herein, upon hepatocellular injury, or in 
anticipation of such injury, or following diagnosis of 
30 a liver function deficiency, for a time and at a 

concentration sufficient to maintain normal and/or to 
regain lost liver function in vivo , including 
regenerating lost or damaged hepatic tissue, and/or 
inhibiting additional damage thereto. The morphogens 
35 described herein also are capable of enhancing the 

level of a liver function which may be depressed as a 
result of a tissue injury or disease. 



In another aspect, the invention features 
compositions and therapeutic treatment methods for 
maintaining liver function in a mammal in vivo which 
include administering to the mammal, upon 
hepatocellular injury or in anticipation of such 
injury, or following diagnosis of a liver function 
deficiency, a compound that stimulates in vivo a 
therapeutically effective concentration of an 
endogenous morphogen within the body of the mammal 
sufficient to increase or enhance the level of a 
depressed liver function, and/or to maintain normal 
and/or regain lost liver function, including 
regenerating damaged or lost hepatic tissue and/or 
inhibiting additional damage thereto. These compounds 
are referred to herein as morphogen-stimulating agents, 
and are understood to include substances which, when 
administered to a mammal, act on cells of tissue(s) or 
organ(s) that normally are responsible for, or capable 
of, producing a morphogen and/or secreting a morphogen, 
and which cause the endogenous level of the morphogen 
to be altered. The agent may act, for example, by 
stimulating expression and/or secretion of an 
endogenous morphogen. 

While the methods and compositions described herein 
are particularly related to liver organ therapies, as 
will be appreciated by those skilled in the art, the 
methods and compositions of this invention can be 
applied, without undue experimentation, to other organ 
applications, including but not limited to, the 
pancreas, lung, kidney and heart. Accordingly, the 
methods and compositions disclosed herein can be used 
to advantage in the repair, regeneration, 
transplantation and/or function level enhancement of 
damaged or lost tissue such as, for example, damaged 



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- 9 - 

lung tissue resulting from emphysema, cirrhotic kidney 
or pancreatic tissue, damaged heart or blood vessel 
tissue, as may result from cardiomyopathies and/or 
atherothrombotic or cardioembolic strokes, damaged 
5 stomach tissue resulting from ulceric perforations or 
their repair, damaged neural tissue as may result from 
physical injury, degenerative diseases such as 
Alzheimer's disease or multiple sclerosis or strokes, 
and damaged dental and/or periodental tissue as may 

10 result from disease or mechanical injury. The methods 
and compositions also may be used to protect these 
tissues from anticipated injury, including unavoidably 
or deliberately induced injury, as may occur in a 
surgical or other clinical procedure. In addition to 

15 the tissue regenerative properties provided herein, the 
gene therapy and drug delivery protocols described 
herein may be used to particular advantage in 
pancreatic tissue, renal tissue and lung tissue 
contexts • 

20 

As embodied herein, the expression "maintaining 
nomral liver function" means both regaining or 
restoring liver function lost due to a hepatocellular 
injury or ixiborn metabolic defect, as well as 

25 protecting the hepatic tissue at risk of damage from 
hepatocellular injury. "Depressed liver function" 
level refers to a diminished or deficient liver 
function as a result of a tissue injury or disease. 
The expression "enhance viability of" transplant 

30 hepatic tissue or organ, as used herein, means 

protection from, reduction of and/or elimination of 
reduced or lost tissue or organ function as a result of 
tissue necrosis and/or fibrosis associated with 
transplantation, particularly immune response-mediated 

35 tissue necrosis and/or fibrosis. "Alleviating" means 



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protection from^ reduction of and/or elimination of 
undesired tissue destruction, particularly immune 
response-mediated tissue destruction. "Transplanted" 
living tissue includes both tissue grafts and cellular 
5 transplants, as in the case of transplanted isolated 
progenitor or stem cells, for example, which may be 
implanted alone or in association with a temporary 
scaffolding. Tissues may be autologous or allogenic 
tissue and/or synthetic tissue created, for example, by 

10 culturing hepatic cells in the presence of an 
artificial matrix. "Morphogenically permissive 
environment" is understood to mean an environment 
competent to allow tissue morphogenesis to occur. 
Finally, "symptom alleviating cof actor" refers to one 

15 or more pharmaceuticals which may be administered 

together with the therapeutic agents of this invention 
and which alleviate or mitigate one or more of the 
symptoms typically associated with liver tissue and/or 
liver function loss. Exemplary cof actors include 

20 antibiotics, antiseptics, non-steroidal anti- 
inflammatory agents, and the like. 

In one aspect of the invention, the methods and 
compositions of this invention are useful in the 

25 replacement of diseased, damaged or lost hepatic tissue 
in a mammal, particularly when the damaged tissue 
interferes with normal tissue or organ function. Where 
hepatic tissue has been lost, remaining hepatocytes are 
capable only of compensatory cell division to retuni 

30 the organ volume essentially to its original size. As 
determined by extensive experimental partial 
hepatectomy studies wherein part of all of a liver lobe 
is excised, this compensatory growth does not involve 
true morphogenisis, and the lost tissue is not itself 



regenerated. Rather, the intact lobe is capable only 
of tissue augmentation to compensate for the lost mass. 
By contrast/ recent studies on toxin- induced tissue 
damage does suggest that this repair involves 
morphogenesis, particularly the infiltration and 
proliferation of progenitor cells. As described in 
Example 3 and i, below, endogenous morphogen expression 
is enhanced following toxin- induced hepatic tissue 
damage, and not following partial hepatectomy. 

When the proteins described herein are provided to, 
or their expression stimulated at, a hepatic tissue 
locus, the developmental cascade of tissue 
morphogenesis is induced, capable of stimulating the 
migration, proliferation and differentiation of hepatic 
progenitor cells, to regenerate viable hepatic tissue, 
including inducing the necessary associated 
vascularization (see below). Thus, in one embodiment 
the invention provides methods and compositions for 
regenerating lost or substantially irreparably damaged 
hepatic tissue. The morphogen preferably is provided 
directly to the locus of tissue regeneration, e.g., by 
injection of the morphogen dispersed in a 
biocompatible, injectable solution, or by topical 
administration, as by painting or spraying a morphogen- 
containing solution on the tissue. Preferably, the 
locus has been surgically prepared by removing existing 
necrotic or cirrhotic tissue. Alternatively, morphogen 
may be provided locally by means of an osmotic pump 
implanted in the peritoneal cavity. At least one 
morphogen (OP-1) is known to be expressed by hepatic 
tissue during liver formation. Accordingly, in the 
alternative, and/or in addition, an agent capable of 
stimulating expression and/or secretion of an 
endogenous morphogen may be administered. As yet 



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another alternative / progenitor hepatocytic cells may 
be stimulated ex vivo by exposure to a morphogen or 
morphogen-stimulating agents and the stimulated cells ^ 
now primed for proliferation and differentiation^ then 
5 provided to the hepatic tissue locus. A morphogen or a 
morphogen-stimulating agent also may be implanted with 
the cells. Alternatively, a suitable local morphogen 
concentration may be maintained by means / for example, 
of an osmotic pump. In all these cases the existing 

10 tissue provides the necessary matrix requirements, 

providing a suitable substratum for the proliferating 
and differentiating cells in a morphogenically 
permissive environment, as well as providing the 
necessary signals for directing the tissue-specificity 

15 of the developing tissue. 

When the morphogens (or progenitor ceils stimulated 
by these morphogens) are provided at a tissue-specific 
locus (e.g., by systemic injection or by implantation 

20 or injection at a tissue-specific locus, or by 

administration of an agent capable of stimulating 
morphogen expression in vivo) , the existing tissue at 
that locus, whether diseased or damaged, has the 
capacity of acting as a suitable matrix. 

25 Alternatively, a formulated matrix may be externally 
provided together with the stimulated progenitor cells 
or morphogen, as may be necessary when the extent of 
injury sustained by the damaged tissue is large. The 
matrix should be a biocompatible, suitably modified 

30 acellular matrix having dimensions such that it allows 
the influx, differentiation, and proliferation of 
migratory progenitor cells, and is capable of providing 
a morphogenically permissive environment (see infra). 



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Currently preferred matrices also are biodegradable. 
Where morphogen and/or progenitor cells are to be 
implanted and the existing liver tissue is insufficient 
to provide the necessary matrix components, the 
5 formulated matrix preferably is tissue-specific. 

Formulated matrices may be generated from a fibrin 
clot or dehydrated organ-specific tissue, prepared for 
example, by treating the tissue with solvents to 

10 substantially remove the non- structural components from 
the tissue. Alternatively, the matrix may be 
formulated synthetically using one or more 
biocompatible, preferably in vivo biodegradable, 
structural carrier materials such as collagen, laminin, 

15 and/or hyaluronic acid which also may be in association 
with suitable tissue-specific cell attachment factors. 
Other biocompatible, in vivo biodegradable components, 
including synthetic polymers, including polybutyric, 
polylactic, polyglycolic acids, polyanhydrides and/or 

20 copolymers thereof. Currently preferred structural 
materials contain collagens. Currently preferred cell 
attachment factors include glycosaminoglycans and 
proteoglycans. The matrix further may be treated with 
an agent or agents to increase the number of pores 

25 and/or micropits on its surfaces, so as to enhance the 
influx, proliferation and differentiation of migratory 
progenitor cells from the body of the mammal. 

In many instances, the loss of hepatic tissue 
30 function results from fibrosis or scar tissue 

formation, formed in response to an initial or repeated 
injury to the tissue. The degree of scar tissue 
formation generally depends on the regenerative 
properties of the injured tissue, and on the degree and 
35 type of injury. In liver, repeated tissue damage 



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- 14 ^ 

results in liver cirrhosis which destroys normal 
hepatic architecture by fiborous septa, causing 
vascular disorganization and perfusion deficits that 
impair liver function and unchecked, lead to hepatic 
5 failure- Thus, in another aspect, the invention 

provides methods and compositions that may be used to 
prevent and/or substantially inhibit the formation of 
scar tissue in hepatic tissue by providing the 
morphogens, or morphogen-stimulated cells, to a newly 
10 injured tissue locus (see below). 



The morphogens of this invention also may be used 
to increase or regenerate a liver progenitor or stem 
cell population in a mammal. For example, progenitor 

15 cells may be isolated from an individual's bone marrow, 
stimulated ex vivo for a time and at a morphogen 
concentration sufficient to induce the cells to 
proliferate, and returned to the bone marrow. Other 
sources of progenitor cells that may be suitable 

20 include biocompatible cells obtained from a cultured 
cell line, stimulated in culture, and subsequently 
provided to the body. Alternatively, the morphogen may 
be provided systemically, or implanted, injected or 
otherwise provided to a progenitor cell population in 

25 an individual to induce its mitogenic activity in vivo. 
For example, an agent capable of stimulating morphogen 
expression in the progenitor cell population of 
interest may be provided to the cells in vivo, for 
example systemically, to induce mitogenic activity. 

30 

In still another aspect of the invention, the 
morphogens also may be used to support the growth and 
maintenance of differentiated cells, inducing existing 
differentiated cells to continue expressing their 
35 phenotype. It is anticipated that this activity will 



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be particularly useful in the treatment of liver 
disorders where loss of liver function is caused by 
cells becoming metabolically senescent or quiescent. 
Application of the protein directly to the cells to be 
5 treated, or providing it by systemic injection, can be 
used to stimulate these cells to continue expressing 
their phenotype, thereby significantly reversing the 
effects of the dysfunction. Al tentatively, 
administration of an agent capable of stimulating 
10 morphogen expression in vivo also may be used. In 

addition, the morphogens of this invention also may be 
used in gene therapy protocols to stimulate the growth 
of quiescent cells, thereby potentially enhancing the 
ability of these cells to incorporate exogenous DNA. 

15 

In another aspect of the invention, the method 
disclosed is useful for redif ferentiating transformed 
cells, particularly transformed cells of parenchymal 
origin, such that the morphogen-treated cells are 

20 induced to display a morphology characteristic of 
untransformed cells. As described in international 
application US92/01968 (W092/15323) and [CRP070PC] the 
morphogens previously have been found to induce 
redifferentiation of transformed embryonic cells and 

25 cells of neuronal origin to a morphology characteristic 
of untransformed cells. Morphogen treatment preferably 
induces cell rounding and cell aggregation (clumping), 
cell-cell adhesion, and CAM production. The methods 
described herein are anticipated to substantially 

30 inhibit or reduce hepatocytic cell tumor formation 
and/or proliferation in liver tissue. It is 
anticipated that the methods of this invention will be 
useful in substantially reducing the effects of various 
carcinomas and sarcomas of liver tissue origin, 
35 including hepatocellular carcinomas, bileduct 



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carcinomas, hepatoblastomas, and hemangiosarcomas. In 
addition, the method also is anticipated to aid in 
inhibiting neoplastic lesions caused by metastatic 
tissue. Metastatic tumors are one of the most common 
5 neoplasms of the liver, as they can reaching the liver 
through the bloodstream or lymph nodes. Metastatic 
tumors may damage hepatic function for example, by 
distorting normal liver tissue architecture, blocking 
or inhibiting blood flow, and/or by stimulating the 
10 body's iimaune response. 

In another aspect of the invention, the morphogens 
described herein are useful for providing 
hepatocellular protective effects to alleviate liver 

15 tissue damage associated with the body's 

immune/inflammatory response to an initial injury to 
the tissue. As described in detail in international 
application US92/07358 (WO93/04692 ) , such a response 
may follow acute or chronic trauma to hepatic tissue, 

20 caused, for example, by an autoimmune dysfunction, 
neoplastic lesion, infection, chemical or mechanical 
trauma, disease or by partial or complete interruption 
of blood flow to hepatocytes, for example following 
ischemia or hypoxia, or by other trauma to the liver or 

25 surrounding material. For example, portal hypertension 
is a significant liver disease caused by reduced blood 
flow through the portal vein and is characterized by 
tissue necrosis and cirrhosis. Application of the 
morphogen directly to the cells to be treated, or 

30 providing the morphogen to the mammal systemically, for 
example, intravenously or indirectly by oral 
administration, may be used to alleviate and/or inhibit 
the immunologically related response to a hepatic 
tissue injury. Alternatively, administration of an 

35 agent capable of stimulating morphogen expression 



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and/or secretion in vivo , preferably at the site of 
injury/ also may be used. Where the injury is to be 
unavoidably or deliberately induced, as during surgery 
or other aggressive clinical treatment, the morphogen 
5 or agent may be provided prior to induction of the 
injury to provide a cytoprotective effect to the liver 
tissue at risk. 

Similarly, hepatic tissues and organs for 
10 transplantation also are subject to the tissue 

destructive effects associated with the recipient host 
body's inflammatory response following transplantation. 
It is currently believed that the initial destructive 
response is due in large part to reperfusion injury to 
15 the transplanted organ after it has been transplanted 
to the organ recipient. 

Accordingly, the success of liver or hepatic tissue 
transplantation depends greatly on the preservation of 

20 the tissue activity (e.g., tissue or organ viability) 
at the harvest of the organ, during storage of the 
harvested organ, and at transplantation. To date, 
preservation of organs such as lungs, pancreas, heart 
and liver remains a significant stumbling block to the 

25 successful transplantation of these organs. U.S. 

Patent No. 4,952,409 describes a superoxide dismutase- 
containing liposome to inhibit reperfusion injury. 
U.S. Patent No. 5,002,965 describes the use of 
ginkolides, known platelet activating factor 

30 antagonists, to inhibit reperfusion injury. Both of 
these factors are described as working primarily by 
inhibiting the release of and/or inhibiting the 
damaging effects of free oxygen radicals. A number of 
patents also have issued on the use of 

35 immunosuppressants for inhibiting graft rejection. A 



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representative listing includes U.S. Patent Nos. 
5^104,858, 5/008,246 and 5,068,323. A significant 
problem with many immunosuppressants is their low 
therapeutic index, requiring the administration of high 
5 doses that can have significant toxic side effects. 

Thus, in another aspect of the invention, where a 
partial or complete organ transplant is desired, the 
morphogen may be administered to transplant tissue to 

10 enhance the viability of the tissue, to alleviate the 
tissue damage associated with immune response-mediated 
tissue destruction and/or to provide a cytoprotective 
effect to tissue at risk for such damage. Exemplary 
transplant tissues include hepatic tissue grafts which 

15 may be allogenic, autologous and/or synthetic (e.g., 
cultured cells attached to an artificial matrix), and 
whole or partial livers. Where the transplant tissue 
(e.g., liver, lung, kidney, pancreas, heart, etc.) is 
to be harvested from a donor host, the morphogen also 

20 preferably is provided to the tissue prior to, or 
concoramitant with the tissue harvest, e.g., as a 
prophylactic, to provide a cytoprotective effect to the 
tissue* 

25 In another aspect of the invention, the morphogens 

described herein also may be used in a gene therapy 
protocol and/or as part of a drug delivery protocol to 
correct a protein deficiency in a mammal, resulting, 
for example, from a genetic disorder or other 

30 dysfunction to the protein-producing tissue. 

Specifically, the methods and compositions of this 
invention are contemplated for use in providing to the 
mammal an in vivo protein-producing mechanism for 
correcting any protein deficiency in the mammal. These 

35 proteins include proteins normally expressed and/or 



- 19 - 



secreted by hepatic tissue and which play a role in 
liver-related functions, proteins nonnally expressed 
and secreted by the liver and which function elsewhere 
in the body, and proteins not normally expressed by 
hepatic tissue. Cells competent for expressing one or 
more proteins necessary to overcome the protein 
deficiency in vivo may be stimulated to proliferate ex 
vivo, and then implanted at a morphogenically 
permissive site at a liver-specific tissue locus in 
vivo * The competent cells may be attached to a 
scaffold-like structure prior to implantation. 
Alternatively, the competent cells may be attached to a 
synthetic or formulated matrix and implanted together 
with a morphogen at an extra-hepatic site in vivo , such 
as within the folds of the mesentery, or other 
associated vascularized tissue locus capable of 
providing the necessary nutrients and gas exchange to 
the cells. A detailed description of useful 
extra-hepatic loci are described, for example, in 
WO90/12604, published November 1, 1990 to Vacanti et 
al . , the disclosure of which is incorporated herein by 
reference. Exposing primary hepatocytes to a morphogen 
stimulates their proliferation (see below), thereby 
enhancing their cellular viability upon implantation, 
accelerating tissue development, and reducing the 
original cell population required to seed the matrix. 
In addition, implantation with a morphogen eliminates 
the need for partial hepatectomy to stimulate 
proliferation, and enhances cellular viability by 
inhibiting the inflammatory /immune response typically 
associated with such a procedure, overcoming the 
significant hepatocyte cell loss typically seen in this 
procedure . 



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Cells competent for correcting a protein deficiency 
include allogenic primary hepatocytes, preferably from 
a serotypically compatible individual and competent for 
expressing the protein or proteins of interest, and 
5 autologous cells transfected with the genetic material 
necessary to express the protein of interest. For 
example, primary hepatocytes may be removed from the 
patient by biopsy, transfected using standard 
recombinant DNA technology, proliferated, attached to a 

10 matrix and reimplanted together with a morphogeny 
Preferably the morphogen is provided to the cells 
during trans fection and proliferation to enhance the 
mitogenic activity (and nucleic acid uptake) of these 
cells. In a currently preferred embodiment, morphogen 

15 is adsorbed to the matrix surface to which the cells 
are attached and the complex implanted as a single 
entity ("cell-matrix structure".) 

In any treatment method of the invention, 

20 "administration of morphogen" refers to the 

administration of the morphogen, either alone or in 
combination with other molecules. For example, the 
mature form of the morphogen may be provided in 
association with its precursor "pro" domain, which is 

25 known to enhance the solubility of the protein. 

Alternatively, the pro form of the morphogen (e.g., 
defined, for example, by residues 30-431 of OPl, Seq. 
I.D. No. 16, see below) may be used. Other useful 
molecules known to enhance protein solubility include 

30 casein and other milk components, as well as various 
serum proteins. Additional useful molecules which may 
be associated with the morphogen or morphogen- 
stimulating agent include tissue targeting molecules 
capable of directing the morphogen or morphogen- 

35 stimulating agent to hepatic tissue. Tissue targeting 



molecules envisioned to be useful in the treatment 
protocols of this invention include antibodies, 
antibody fragments or other binding proteins which 
interact specifically with surface molecules on nerve 
tissue cells. Still another useful tissue targeting 
molecule may include part or all of the morphogen 
precursor "pro" domain. 

Associated tissue targeting or solubility-enhancing 
molecules also may be covalently linked to the 
morphogen using standard chemical means, including 
acid-labile linkages, which likely will be 
preferentially cleaved in the acidic environment of 
bone remodeling sites. 

The morphogens and morphogen-stimulating agents 
also may be provided to the liver tissue together with 
other molecules ( "cof actors" ) known to have a 
beneficial effect in treating damaged hepatic tissue, 
particularly cofactors capable of mitigating or 
alleviating symptoms typically associated with hepatic 
tissue damage and/or loss. Examples of such cofactors 
include antiseptics, antibiotics, tetracycline, 
aminoglycosides, macrolides, penicillins and 
cephalosporins, and other, non-steroidal 
anti- inflammatory agents. 

Among the morphogens useful in this invention are 
proteins originally identified as osteogenic proteins, 
such as the OP-1, OP-2 and CBMP2 proteins, as well as 
amino acid sequence-related proteins such as DPP (from 
Drosophila), Vgl ( f rom Xenopus ) , Vgr-1 (from mouse, see 
U.S. 5,011,691 to Oppermann et al.), GDF-1 (from mouse, 
see Lee (1991) PNAS 88:4250-4254 ) , all of which are 
presented in Table II and Seq. id Kos.5-14), and the 



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recently identified 60A protein (from Drosophila, Seq. 
ID No. 24, see Wharton et al. (1991) PNAS 
^:9214-9218. ) The members of this family, which 
include members of the TGF-p super-family of proteins, 
5 share substantial amino acid sequence homology in their 
C-terminal regions. The proteins are translated as a 
precursor, having an N- terminal signal peptide 
sequence, typically less than about 30 residues, 
followed by a "pro" domain that is cleaved to yield the 

10 mature sequence. The "pro" form of the protein, 

includes both the pro domain and the mature domain, and 
forms a soluble species that apprears to be the primary 
form secreted from cultured mammalian cells. The 
signal peptide is cleaved rapidly upon translation, at 

15 a cleavage site that can be predicted in a given 

sequence using the method of Von Heijne ((1986) Nucleic 
Acids Research 14 ; 4683-4691. ) Table I, below, 
describes the various morphogens identified to date, 
including their nomenclature as used herein, their Seq. 

20 ID references, and publication sources for the amino 
acid sequences for the full length proteins not 
included in the Seq. Listing. The disclosure of these 
publications is incorporated herein by reference. 

TABLE I 

25 

"OP-1" Refers generically to the group of 

morphogenically active proteins expressed 
from part or all of a DNA sequence 

30 encoding OP-1 protein, including allelic 

and species variants thereof, e.g., human 
OP-1 ("hOP-1", Seq. ID No. 5, mature 
protein amino acid sequence), or mouse 
OP-1 ("mOP-1", Seq. ID No. 6, mature 

35 protein amino acid sequence.) The 



PCr/US93/08808 



- 23 - 

conserved seven cysteine skeleton is 
defined by residues 38 to 139 of Seg. ID 
Nos. 5 and 6. The cDNA sequences and the 
amino acids encoding the full length 
proteins are provided in Seq. Id Nos. 16 
and 17 (hOPl) and Seq. ID Nos. 18 and 19 
(mOPl.) The mature proteins are defined 
by residues 293-431 (hOPl) and 292-430 
(mOPl). The "pro" regions of the 
proteins, cleaved to yield the mature, 
morphogenically active proteins are 
defined essentially by residues 30-292 
(hOPl) and residues 30-291 (mOPl). 

refers generically to the group of active 
proteins expressed from part or all of a 
DNA sequence encoding OP-2 protein, 
including allelic and species variants 
thereof, e.g., human OP-2 ("hOP-2", Seq. 
ID No. 7, mature protein amino acid 
sequence) or mouse OP-2 ("mOP-2", Seq. ID 
No. 8, mature protein amino acid 
sequence). The conserved seven cysteine 
skeleton is defined by residues 38 to 139 
of Seq. ID Nos. 7 and 8. The cDNA 
sequences and the amino acids encoding the 
full length proteins are provided in Seq. 
ID Nos. 20 and 21 (hOP2) and Seq. ID Nos. 
22 and 23 (mOP2.) The mature proteins are 
defined essentially by residues 264-402 
(hOP2) and 261-399 (mOP2). The "pro" 
regions of the proteins, cleaved to yield 
the mature, morphogenically active 
proteins likely are defined essentially by 
residues 18-263 (hOP2) and residues 18-260 



PCT/US93/08808 



• 24 - 

(mOP2). (Another cleavage site also 
occurs 21 residues upstream for both OP-2 
proteins. ) 

refers generically to the morphogenically 
active proteins expressed from a DNA 
sequence encoding the CBMP2 proteins^ 
including allelic and species variants 
thereof, e.g., human CBMP2A ( "CBMP2A(fx) 
Seq ID No. 9) or human CBMP2B DNA 
("CBMP2B(fx)", Seq. ID No. 10). The amino 
acid sequence for the full length 
proteins, referred to in the literature as 
BMP2A and BMP2B, or BMP2 and BMP4, appear 
in Wozney, et al. (1988) Science 242 ; 1528- 
1534. The pro domain for BMP2 (BMP2A) 
likely includes residues 25-248 or 25-282; 
the mature protein, residues 249-396 or 
283-396. The pro domain for BMP4 (BMP2B) 
likely includes residues 25-256 or 25-292; 
the mature protein, residues 257-408 or 
293-408. 

refers to protein sequences encoded by the 
Drosophila DPP gene and defining the 
conserved seven cysteine skeleton (Seq. ID 
No. 11). The amino acid sequence for the 
full length protein appears in Padgett, et 
al (1987) Nature 325 ; 81-84. The pro 
domain likely extends from the signal 
peptide cleavage site to residue 456; the 
mature protein likely is defined by 
residues 457-588. 



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"Vgl(fx)" refers to protein sequences encoded by the 
Xenopus Vgl gene and defining the 
conserved seven cysteine skeleton (Seq. ID 
No. 12). The amino acid sequence for the 
5 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 
10 residues 247-360. 

"Vgr-l{fx)" refers to protein sequences encoded by the 
murine Vgr-1 gene and defining the 
conserved seven cysteine skeleton (Seq. ID 

15 No. 13). 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 

20 mature protein likely is defined by 

residues 300-438. 



"GDF-l(fx)" refers to protein sequences encoded by the 
human GDF-1 gene and defining the 

25 conserved seven cysteine skeleton (Seq. ID 

No. 14). The cDNA and encoded amino 
sequence for the full length protein is 
provided in Seq. ID. No. 32. The 
prodomain likely extends from the signal 

30 peptide clavage site to residue 214; the 

mature protein likely is defined by 
residues 215-372. 



"60A" refers generically to the morphogenically 

35 active proteins expressed from part or all 

of a DNA sequence (from the Drosophila 60A 
gene) encoding the 60A proteins (see Seq. 



PCT/US93/08808 



- 26 - 

ID No. 24 wherein the cDNA and encoded 
amino acid sequence for the full length 
protein is provided). "60A(fx)" refers to 
the protein sequences defining the 
conserved seven cysteine skeleton 
(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. 

refers to protein sequences encoded by the 
human BMP3 gene and defining the conserved 
seven cysteine skeleton (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 protein sequences encoded by the 
human BMP5 gene and defining the conserved 
seven cysteine skeleton (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 protein sequences encoded by the 
human BMP6 gene and defining the conserved 
seven cysteine skeleton (Seq. ID No. 28). 
The amino acid sequence for the full 



- 27 - 



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. 

The OP- 2 proteins have an additional cysteine 
residue in this region (e.g., see residue 41 of Seq. ID 
Nos. 7 and 8), in addition to the conserved cysteine 
skeleton in common with the other proteins in this 
family. The GDF-1 protein has a four amino acid insert 
within the conserved skeleton (residues 44-47 of Seq. 
ID No. 14) but this insert likely does not interfere 
with the relationship of the cysteines in the folded 
structure. In addition, the CBMP2 proteins are missing 
one amino acid residue within the cysteine skeleton. 

The morphogens are inactive when reduced, but are 
active as oxidized homodimers and when oxidized in 
combination with other morphogens of this invention. 
Thus, as defined herein, a morphogen is a dimeric 
protein comprising a pair of polypeptide chains, 
wherein each polypeptide chain comprises at least the 
C-terminal six cysteine skeleton defined by residues 
43-13? of Seq. ID No. 5, including functionally 
equivalent arrangements of these cysteines (e.g., amino 
acid insertions or deletions which alter the linear 
arrangement of the cysteines in the sequence but not 
their relationship in the folded structure), such that, 
when the polypeptide chains are folded, the dimeric 
protein species comprising the pair of polypeptide 
chains has the appropriate three-dimensional structure, 
including the appropriate intra- or inter-chain 
disulfide bonds such that the protein is capable of 



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acting as a morphogen as defined herein. Specifically, 
the morphogens generally are capable of all of the 
following biological functions in a morphogenically 
permissive environment: stimulating proliferation of 
5 progenitor cells; stimulating the differentiation of 
progenitor cells; stimulating the proliferation of 
differentiated cells; and supporting the growth and 
maintenance of differentiated cells, including the 
"redif ferentiation" of transformed cells. In addition, 
10 it is also anticipated that these morphogens are 
capable of inducing redif f erentiation of committed 
cells under appropriate environmental conditions. 

In one preferred aspect, the morphogens of this 
15 invention comprise one of two species of generic amino 
acid sequences: Generic Sequence 1 (Seq. ID No. 1) or 
Generic Sequence 2 (Seq. ID No. 2); where each Xaa 
indicates one of the 20 naturally-occurring L-isomer, 
a-amino acids or a derivative thereof. Generic 
20 Sequence 1 comprises the conserved six cysteine 

skeleton and Generic Sequence 2 comprises the conserved 
six cysteine skeleton plus the additional cysteine 
identified in OP-2 (see residue 36, Seq. ID No. 2). In 
another preferred aspect, these sequences further 
25 comprise the following additional sequence at their N- 
terminus : 

Cys Xaa Xaa Xaa Xaa (Seq. ID No. 15) 
1 5 

30 

Preferred amino acid sequences within the foregoing 
generic sequences include: Generic Sequence 3 (Seq. ID 
No. 3), Generic Sequence 4 (Seq. ID No. 4), Generic 
Sequence 5 (Seq. ID No. 30) and Generic Sequence 6 
35 (Seq. ID No. 31), listed below. These Generic 



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- 29 - 

Sequences accommodate the homologies shared among the 
various preferred members of this morphogen family 
identified in Table II, as well as the amino acid 
sequence variation among them. Specifically, Generic 
5 Sequences 3 and 4 are composite amino acid sequences of 
the following proteins presented in Table II and 
identified in Seq. ID Nos. 5-14: human OP-1 (hOP-1, 
Seq. ID Nos. 5 and 16-17), mouse OP-1 (mOP-1, Seq. ID 
Mos. 6 and 18-19), human and mouse OP-2 (Seq. ID 

10 Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), CBMP2B 
(Seq. ID No. 10), DPP (from Drosophila, Seq. ID 
No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 * 
(from mouse, Seq. ID No. 13), and GDF-1 (from mouse, 
Seq. ID No. 14.) The generic sequences include both 

15 the amino acid identity shared by the sequences in 
Table II, as well as alternative residues for the 
variable positions within the sequence. Note that 
these generic sequences allow for an additional 
cysteine at position 41 or 46 in Generic Sequences 3 or 

20 4, respectively, providing an appropriate cysteine 

skeleton where inter- or intramolecular disulfide bonds 
can form, and contain certain critical amino acids 
which influence the tertiary structure of the proteins. 

25 Generic Sequence 3 

Leu Tyr Val Xaa Phe 

1 5 

Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

30 10 

Xaa Ala Pro Xaa Gly Xaa Xaa Ala 

15 20 

Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 



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- 30 - 

25 30 
Xaa Pro Xaa Xaa Xaa Xaa Xaa 

35 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 
5 40 45 

Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 

50 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 
55 60 
10 Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 

65 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 

70 75 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
15 . 80 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

85 90 
Xaa Cys Gly Cys Xaa 
95 

20 wherein each Xaa is independently selected from a group 
of one or more specified amino acids defined as 
follows: "Res." means "residue" and Xaa at res. 4 = 
(Ser^ Asp or Glu); Xaa at res. 6 - (Arg, Gin, Ser or 
Lys); Xaa at res. 7 == (Asp or Glu); Xaa at res. 8 = (Leu 

25 or Val); Xaa at res. 11 = (Gin, Leu, Asp, His or Asn); 
Xaa at res. 12 = (Asp, Arg or Asn); Xaa at res. 14 « (lie 
or Val); Xaa at res. 15 = (lie or Val); Xaa at res. 18 = 
(Glu, Gin, Leu, Lys, Pro or Arg); Xaa at res. 20 = {Tyr 
or Phe); Xaa at res. 21 = (Ala, Ser, Asp, Met, His, Leu 



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or Gin); Xaa at res.23 = (Tyr, Asn or Phe); Xaa at 
res«26 = (Glu, His, Tyr, Asp or Gin); Xaa at res«2B = 
(Glu, Lys, Asp or Gin); Xaa at res.30 - (Ala, Ser, Pro 
or Gin); Xaa at res.31 = (Phe, Leu or Tyr); Xaa at 
5 res. 33 = (Leu or Val); Xaa at res.34 = (Asn, Asp, Ala 
or Thr); Xaa at res. 35 = (Ser, Asp, Glu, Leu or Ala); 
Xaa at res. 36 = (Tyr, Cys, His, Ser or lie); Xaa at 
res. 37 « (Met, Phe, Gly or Leu); Xaa at res.3B = (Asn 
or Ser); Xaa at res. 39 = (Ala, Ser or Gly); Xaa at 

10 res. 40 = (Thr, Leu or Ser); Xaa at res. 44 = (lie or 
Val); Xaa at res. 45 = (Val or Leu); Xaa at res. 46 = 
(Gin or Arg); Xaa at res. 47 = (Thr, Ala or Ser); Xaa at 
res. 49 - (Val or Met); Xaa at res. 50 » (His or Asn); 
Xaa at res. 51 = (Phe, Leu, Asn, Ser, Ala or Val); Xaa 

15 at res. 52 = (He, Met, Asn, Ala or Val); Xaa at res. 53 
= (Asn, Lys, Ala or Glu); Xaa at res. 54 = (Pro or Ser); 
Xaa at res. 55 = (Glu, Asp, Asn, or Gly); Xaa at res .56 
= (Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at 
res. 57 = (Val, Ala or He); Xaa at res. 58 = (Pro or 

20 Asp); Xaa at res. 59 = (Lys or Leu); Xaa at res. 60 = 
(Pro or Ala); Xaa at res. 63 - (Ala or Val); Xaa at 
res. 65 = (Thr or Ala); Xaa at res.66 - (Gin, Lys, Arg 
or Glu); Xaa at res. 67 = (Leu, Met or Val); Xaa at 
res. 68 = (Asn, Ser or Asp); Xaa at res. 69 = (Ala, Pro 

25 or Ser); Xaa at res. 70 = (He, Thr or Val); Xaa at 
res. 71 = (Ser or Ala); Xaa at res. 72 = (Val or Met); 
Xaa at res. 74 = (Tyr or Phe); Xaa at res. 75 = (Phe, Tyr 
or Leu); Xaa at res. 76 = (Asp or Asn); Xaa at res. 77 = 
(Asp, Glu, Asn or Ser); Xaa at res. 78 = (Ser, Gin, Asn 

30 or Tyr); Xaa at res. 79 = (Ser, Asn, Asp or tSlu); Xaa at 
res. 80 = (Asn, Thr or Lys); Xaa at res. 82 = (He or 
Val); Xaa at res. 84 = (Lys or Arg); Xaa at res. 85 = 
(Lys, Asn, Gin or His); Xaa at res. 86 « (Tyr or His); 



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- 32 - 

Xaa at res. 87 - (Arg, Gin or Glu); Xaa at res. 88 ~ 
(Asn, Glu or Asp); Xaa at res. 90 » (Val, Thr or Ala); 
Xaa at res. 92 « (Arg, Lys, Val, Asp or Glu); Xaa at 
res. 93 » (Ala, Gly or Glu); and Xaa at res. 97 « (His or 
5 Arg); 

Generic Sequence 4 

Cys Xaa Xaa Xaa Xaa Leu Tyr Val Xaa Phe 
10 1 5 10 

Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

15 

Xaa Ala Pro Xaa Gly Xaa Xaa Ala 
20 25 
15 Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 

30 35 
Xaa Pro Xaa Xaa Xaa Xaa Xaa 

40 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 
20 45 50 

Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 

55 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 
60 65 
25 Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 

70 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 

75 80 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
30 85 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

90 95 
Xaa Cys Gly Cys Xaa 
100 

35 



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wherein each Xaa is independently selected from a group 
of one or more specified amino acids as defined by the 
following: "Res." means "residue" and Xaa at res.2 = 
(Lys or Arg); Xaa at res. 3 - (Lys or Arg); Xaa at res. 4 
5 = (His or Arg); Xaa at res. 5 = (Glu, Ser, His, Gly, Arg 
or Pro); Xaa at res. 9 = (Ser, Asp or Glu); Xaa at 
res. 11 = (Arg, Gin, Ser or Lys); Xaa at res. 12 - (Asp 
or Glu); Xaa at res. 13 = (Leu or Val); Xaa at res. 16 = 
(Gin, Leu, Asp, His or Asn) ; Xaa at res. 17 « (Asp, Arg, 

10 or Asn); Xaa at res. 19 = (He or Val); Xaa at res. 20 = 
(He or Val); Xaa at res. 23 = (Glu, Gin, Leu, Lys, Pro 
or Arg); Xaa at res. 25 = (Tyr or Phe); Xaa at res. 26 = 
(Ala, Ser, Asp, Met, His, Leu, or Gin); Xaa at res. 28 = 
(Tyr, Asn or Phe); Xaa at res. 31 = (Glu, His, Tyr, Asp 

15 or Gin); Xaa at res. 33 - Glu, Lys, Asp or Gin); Xaa at 
res. 35 = (Ala, Ser or Pro); Xaa at res. 36 = (Phe, Leu 
or Tyr); Xaa at res. 38 = (Leu or Val); Xaa at res. 39 = 
(Asn, Asp, Ala or Thr); Xaa at res. 40 - (Ser, Asp, Glu, 
Leu or Ala); Xaa at res. 41 = (Tyr, Cys, His, Ser or 

20 He); Xaa at res. 42 = (Met, Phe, Gly or Leu); Xaa at 
res. 44 = (Ala, Ser or Gly); Xaa at res. 45 = (Thr, Leu 
or Ser); Xaa at res. 49 = (He or Val); Xaa at res. 50 = 
(Val or Leu); Xaa at res. 51 = (Gin or Arg); Xaa at 
res. 52 = (Thr, Ala or Ser); Xaa at res. 54 = (Val or 

25 Met); Xaa at res. 55 = (His or Asn); Xaa at res. 56 = 

(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res. 57 = (He, 
Met, Asn, Ala or Val); Xaa at res. 58 = (Asn, Lys, Ala 
or Glu); Xaa at res. 59 = (Pro or Ser); Xaa at res .60 = 
(Glu, Asp, or Gly); Xaa at res. 61 = (Thr, Ala, Val, 

30 Lys, Asp, Tyr, Ser or Ala); Xaa at res. 62 = (Val, Ala 
or He); Xaa at res. 63 = (Pro or Asp); Xaa at res. 64 = 
(Lys or Leu); Xaa at res. 65 = (Pro or Ala); Xaa at 
res. 68 = (Ala or Val); Xaa at res. 70 = (Thr or Ala); 
Xaa at res. 71 = (Gin, Lys, Arg or Glu); Xaa at res. 72 = 

35 (Leu, Met or Val); Xaa at res. 73 - (Asn, Ser or Asp); 



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- 34 - 

Xaa at res. 74 = (Ala, Pro or Ser)j Xaa at res. 75 = 
(lie, Thr or Val); Xaa at res. 76 = (Ser or Ala); Xaa at 
res. 77 = (Val or Met); Xaa at res. 79 = (Tyr or Phe); 
Xaa at res. 80 = (Phe, Tyr or Leu); Xaa at res.81 = (Asp 
5 or Asn); Xaa at res. 82 = (Asp, Glu, Asn or Ser); Xaa at 
res. 83 = (Ser, Gin, Asn or Tyr); Xaa at res. 84 = (Ser, 
Asn, Asp or Glu); Xaa at res. 85 « (Asn, Thr or Lys); 
Xaa at res. 87 « (lie or Val); Xaa at res .89 = (Lys or 
Arg); Xaa at res. 90 = (Lys, Asn, Gin or His); Xaa at 
10 res. 91 = (Tyr or His); Xaa at res. 92 = (Arg, Gin or 
Glu); Xaa at res. 93 = (Asn, Glu or Asp); Xaa at res. 95 
- (Val, Thr or Ala); Xaa at res. 97 = (Arg, Lys, Val', 
Asp or Glu); Xaa at res. 98 - (Ala, Gly or Glu); and Xaa 
at res. 102 (His or Arg). 

15 

Similarly, Generic Sequence 5 (Seg. ID No. 30) and 
Generic Sequence 6 (Seq. ID No. 31) accommodate the 
homologies shared among all the morphogen protein 
family members identified in Table II. Specifically, 

20 Generic Sequences 5 and 6 are composite amino acid 

sequences of human OP-1 (hOP-1, Seq. ID Nos. 5 and 16- 
17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), 
human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-22), 
CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID No. 10), DPP 

25 (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, 
Seg. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), 
and GDF-1 (from mouse, Seq. ID No. 14), human BMP3 
(Seq. ID No. 26), hximan BMP5 (Seq. ID No. 27), human 
BMP6 (Seq. ID No. 28) and 60(A) (from Drosophila, Seq. 

30 ID Nos. 24-25). The generic sequences include both the 
amino acid identity shared by these sequences in the 
C-terminal domain, defined by the six and seven 
cysteine skeletons (Generic Sequences 5 and 6, 
respectively), as well as alternative residues for the 

35 variable positions within the sequence. As for Generic 



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Sequences 3 and 4, Generic Sequences 5 and 6 allow for 
an additional cysteine at position 41 (Generic Sequence 
5) or position 46 (Generic Sequence 6), providing an 
appropriate cysteine skeleton where inter- or 
5 intramolecular disulfide bonds can form, and containing 
certain critical amino acids which influence the 
tertiary structure of the proteins. 



10 



Generic Sequence 5 



Leu Xaa Xaa Xaa Phe 

Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

10 

15 Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala 

15 20 
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 

25 30 
Xaa Pro Xaa Xaa Xaa Xaa Xaa 
20 35 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 

40 45 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

50 

25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

55 60 
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
65 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 



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70 75 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
80 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 
5 85 90 

Xaa Cys Xaa Cys Xaa 
95 

wherein each Xaa is independently selected from a group 
of one or more specified amino acids defined as 

10 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 Glu); Xaa at res. 6 = (Arg, Gin, Ser, Lys 
or Ala); Xaa at res. 7 = (Asp, Glu or Lys); Xaa at res. 8 
= (Leu, Val or lie); Xaa at res. 11 = (Gin, Leu, Asp, 

15 His, Asn or Ser); Xaa at res. 12 « (Asp, Arg, Asn or 
Glu); Xaa at res. 14 = (He or Val); Xaa at res. 15 = 
(He or Val); Xaa at res. 16 (Ala or Ser); Xaa at res. 18 
= (Glu, Gin, Leu, Lys, Pro or Arg); Xaa at res. 19 = 
(Gly or Ser); Xaa at res. 20 = (Tyr or Phe); Xaa at 

20 res. 21 = (Ala, Ser, Asp, Met, His, Gin, Leu or Gly); 
Xaa at res. 23 = (Tyr, Asn or Phe); Xaa at res. 26 = 
(Glu, His, Tyr, Asp, Gin or Ser); Xaa at res. 28 = (Clu, 
Lys, Asp, Gin or Ala); Xaa at res. 30 = (Ala, Ser, Pr^, 
Gin or Asn); Xaa at res. 31 = (Phe, Leu or Tyr); Xaa at 

25 res. 33 = (Leu, Val or Met); Xaa at res. 34 = (Asn, Asp, 
Ala, Thr or Pro); Xaa at res. 35 = (Ser, Asp, Glu, Leu, 
Ala or Lys); Xaa at res. 36 = (Tyr, Cys, His, Ser or 
He); Xaa at res, 37 = (Met, Phe, Gly or Leu); Xaa at 
res. 38 = (Asn, Ser or Lys); Xaa at res. 39 = (Ala, Ser, 

30 Gly or Pro); Xaa at res. 40 = (Thr, Leu or Ser); Xaa at 
res. 44 = (He, Val or Thr); Xaa at res. 45 = (Val, Leu 
or He); Xaa at res. 46 = (Gin or Arg); Xaa at res. 47 = 
(Thr, Ala or Ser); Xaa at res. 48 = (Leu or He); Xaa at 



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res. 49 = (Val or Met); Xaa at res. 50 = (His, Asn or 
Arg); Xaa at res. 51 = (Phe, Leu, Asn, Ser, Ala or Val); 
Xaa at res. 52 = (He, Met, Asn, Ala, Val or Leu); Xaa 
at res. 53 = (Asn, Lys, Ala, Glu, Gly or Phe); Xaa at 
5 res. 54 = (Pro, Ser or Val); Xaa at res. 55 = (Glu, Asp, 
Asn, Gly, Val or Lys); Xaa at res. 56 = (Thr, Ala, Val, 
Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at res. 57 = 
(Val, Ala or He); Xaa at res. 58 = (Pro or Asp); Xaa at 
res. 59 = (Lys, Leu or Glu); Xaa at res. 60 = (Pro or 

10 Ala); Xaa at res. 63 = (Ala or Val); Xaa at res. 65 = 
(Thr, Ala or Glu); Xaa at res. 66 = (Gin, Lys, Arg or 
Giu); 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 = (He, Thr, Val or Leu); Xaa at 

15 res. 71 = (Ser, Ala or Pro); Xaa at res. 72 = (Val, Met 
or He); 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 or Ser); Xaa at 
res. 78 = (Ser, Gin, Asn, Tyr or Asp); Xaa at res. 79 = 

20 (Ser, Asn, Asp, Glu or Lys); Xaa at res. 80 = (Asn, Thr 
or Lys); Xaa at res. 82 = (He, Val or Asn); Xaa at 
res. 84 = (Lys or Arg); Xaa at res. 85 = (Lys, Asn, Gin, 
His or Val); Xaa at res. 86 = (Tyr or His); Xaa at 
res. 87 = (Arg, Gin, Glu or Pro); Xaa at res. 88 = (Asn, 

25 Glu or Asp); Xaa at res. 90 = (Val, Thr, Ala or He); 
Xaa at res. 92 = (Arg, Lys, Val, Asp 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). 

30 Generic Sequence 6 

Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe 
1 5 10 

Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 
35 15 

Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala 



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20 25 
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 
30 35 
Xaa Pro Xaa Xaa Xaa Xaa Xaa 
5 40 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 
45 50 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

55 

Id Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

60 65 
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
70 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 
15 75 80 

Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
85 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 
90 95 
20 Xaa Cys Xaa Cys Xaa 

100 

wherein each Xaa is independently selected from a group 
of one or more specified amino acids as defined by the 

25 following: "Res." means "residue" and 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); Xaa at res. 5 = 
(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr); Xaa at 
res. 7 = (Tyr or Lys); Xaa at res. 8 «= (Val or lie); Xaa 

30 at res. 9 = (Ser, Asp or Glu); Xaa at res. 11 - (Arg, 
Gin, Ser, Lys or Ala); Xaa at res. 12 = (Asp, Glu, or 
Lys); Xaa at res. 13 = (Leu, Val or lie); Xaa at res. 16 
= (Gin, Leu, Asp, His, Asn or Ser); Xaa at res. 17 = 
(Asp, Arg, Asn or Glu); Xaa at res. 19 = (He or Val); 

35 Xaa at res. 20 = (He or Val); Xaa at res. 21 = (Ala or 



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Ser); Xaa at res. 23 = (Glu, Gin, Leu, Lys, Pro or Arg); 
Xaa at res. 24 = (Gly or Ser); Xaa at res. 25 = (Tyr or 
Phe); Xaa at res. 26 = (Ala, Ser, Asp, Met, His, Gin, 
Leu, or Gly); Xaa at res. 28 = (Tyr, Asn or Phe); Xaa at 
5 res. 31 = (Glu, His, Tyr, Asp, Gin or Ser); Xaa at 
res. 33 = Glu, Lys, Asp, Gin or Ala); Xaa at res. 35 = 
(Ala, Ser, Pro, Gin or Asn); Xaa at res. 36 = (Phe, Leu 
or Tyr); Xaa at res. 38 = (Leu, Val or Met); Xaa at 
res. 39 = (Asn, Asp, Ala, Thr or Pro); Xaa at res. 40 = 

10 (Ser, Asp, Glu, Leu, Ala or Lys); Xaa at res. 41 = (Tyr, 
Cys, His, Ser or lie); Xaa at res. 42 - (Met, Phe, Gly 
or Leu); Xaa at res. 43 = (Asn, Ser or Lys); Xaa at 
res. 44 = (Ala, Ser, Gly or Pro); Xaa at res. 45 = (Thr, 
Leu or Ser); Xaa at res. 49 = (He, Val or Thr); Xaa at 

15 res. 50 = (Val, Leu or He); Xaa at res. 51 = (Gin or 
Arg); Xaa at res. 52 = (Thr, Ala or Ser); Xaa at res. 53 
= (Leu or He); Xaa at res. 54 = (Val or Met); Xaa at 
res. 55 = (His, Asn or Arg); Xaa at res. 56 = (Phe, Leu, 
Asn, Ser, Ala or Val); Xaa at res. 57 = (He, Met, Asn, 

20 Ala, Val or Leu); Xaa at res. 58 « (Asn, Lys, Ala, Glu, 
Gly or Phe); Xaa at res. 59 = (Pro, Ser or Val); Xaa at 
res. 60 - (Glu, Asp, Gly, Val or Lys); Xaa at res. 61 = 
(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); 
Xaa at res. 62 = (Val, Ala or He); Xaa at res. 63 = (Pro 

25 or Asp); Xaa at res. 64 = (Lys, Leu or Glu); Xaa at 
res. 65 = (Pro or Ala); Xaa at res. 68 = (Ala or Val); 
Xaa at res. 70 = (Thr, Ala or Glu); Xaa at res. 71 = 
(Gin, Lys, Arg or Glu); Xaa at res. 72 = (Leu, Met or 
Val); Xaa at res. 73 = (Asn, Ser, Asp or Gly); Xaa at 

30 res. 74 = (Ala, Pro or Ser); Xaa at res. 75 = (He, Thr, 
Val or Leu); Xaa at res. 76 = (Ser, Ala or Pro); Xaa at 
res. 77 = (Val, Met or He); Xaa at res. 79 = (Tyr or 
Phe); Xaa at res. 80 = (Phe, Tyr, Leu or His); Xaa at 
res. 81 = (Asp, Asn or Leu); Xaa at res. 82 = (Asp, Glu, 

35 Asn or Ser); Xaa at res. 83 = (Ser, Gin, Asn, Tyr or 



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Asp); Xaa at res. 84 = (Ser^ Asn, Asp, Glu or Lys); Xaa 
at res. 85 = (Asn, Thr or Lys); Xaa at res. 87 = (He, 
Val or Asn); Xaa at res. 89 = (Lys or Arg); Xaa at 
res. 90 >= (Lys, Asn, Gin, His or Val); Xaa at res. 91 = 
5 (Tyr or His); Xaa at res. 92 = (Arg, Gin, Glu or Pro); 
Xaa at res. 93 = (Asn, Glu or Asp); Xaa at res. 95 - 
(Val, Thr, Ala or He); Xaa at res. 97 = (Arg, Lys, Val, 
Asp or Glu); Xaa at res. 98 = (Ala, Gly, Glu or Ser); 
Xaa at res. 100 = (Gly or Ala); and Xaa at res. 102 = 
10 (His or Arg) • 

Particularly useful sequences for use as morphogens 
in this invention include the C-terminal domains, e.g., 
the C-terminal 96-102 amino acid residues of Vgl, 

15 Vgr-1, DPP, OP-1, OP-2, CBMP-2A, CBMP-2B, GDF-1 (see 
Table II, below, and Seq. ID Nos. 5-14), as well as 
proteins comprising the C- terminal domains of 6 OA, 
BMP3, BMP5 and BMP6 (see Seq. ID Nos. 24-28), all of 
which include at least the conserved six or seven 

20 cysteine skeleton. In addition, biosynthetic 

constructs designed from the generic sequences, such as 
COP-1, 3-5, 7, 16, disclosed in U.S. Pat. No. 
5,011,691, also are useful. Other sequences include 
the inhibins/activin proteins (see, for example, U.S. 

25 Pat. Nos. 4,968,590 and 5,011,691). Accordingly, other 
useful sequences are those sharing at least 70% amino 
acid sequence homology or "similarity", and preferably 
80% homology or similarity with any of the sequences 
above. These are anticipated to include allelic, 

30 species variants and other sequence variants (e.g., 
including "muteins" or "mutant proteins"), whether 
naturally-occurring or biosynthetically produced, as 
well as novel members of this morphogenic family of 
proteins. As used herein, "amino acid sequence 

35 homology" is understood to mean amino acid sequence 



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similarity, and homologous sequences share identical or 
similar amino acids, where similar amino acids are 
conserved amino acids as defined by Dayoff et al.. 
Atlas of Protein Sequence and Structure ; vol.5, 
5 Suppl.3, pp. 345-362 (M.O. Dayoff, ed., Nat'l BioMed. 
Research Fdn., Washington D.C. 1978.) Thus, a 
candidate sequence sharing 70% amino acid homology with 
a reference sequence requires that, following alignment 
of the candidate sequence with the reference sequence, 

10 70% of the amino acids in the candidate sequence are 
identical to the corresponding amino acid in the 
reference sequence, or constitute a conserved amino 
acid change thereto. "Amino acid sequence identity" is 
understood to require identical amino acids between two 

15 aligned sequences. Thus, a candidate sequence sharing 
60% amino acid identity with a reference sequence 
requires that, following alignment of the candidate 
sequence with the reference sequence, 60% of the amino 
acids in the candidate sequence are identical to the 

20 corresponding amino acid in the reference sequence. 

As used herein, all homologies and identities 
calculated use OP-1 as the reference sequence. Also as 
used herein, sequences are aligned for homology and 

25 identity calculations using the method of Needleman et 
al. (1970) J.Mol. Biol. 48x443-453 and identities 
calculated by the Align program (DNAstar, Inc.) In all 
cases, internal gaps and amino acid insertions in the 
candidate sequence as aligned are ignored when making 

30 the homology /identity calculation. 

The currently most preferred protein sequences 
useful as morphogens in this invention include those 
having greater than 60% identity, preferably greater 
35 than 65% identity, with the amino acid sequence 

defining the conserved six cysteine skeleton of hOPl 
(e.g., residues 43-139 of Seq. ID No. 5). These most 



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preferred sequences include both allelic and species 
variants of the OP-1 and OP-2 proteins, including the 
Drosophila 60A protein. Accordingly, in another 
preferred aspect of the invention, useful morphogens 
5 include active proteins comprising species of 
polypeptide chains having the generic amino acid 
sequence herein referred to as "OPX", which 
accommodates the homologies between the various 
identified species of OPl and 0P2 (Seq. ID No. 29). 

10 

In still another preferred aspect of the invention, 
useful morphogens include dimeric proteins comprising 
amino acid sequences encoded by nucleic acids that 
hybridize to DNA or RNA sequences encoding the C- 

15 terminal sequences defining the conserved seven 

cysteine domain of OPl or 0P2, e.g., nucleotides 1036- 
1341 and nucleotides 1390-1695 of Seq. ID No. 16 and 
20, respectively, under stringent hybridization 
conditions. As used herein, stringent hybridization 

20 conditions are defined as hybridization 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'^C. 

25 The morphogens useful in the methods, composition 

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 

30 synthetic techniques, and includes allelic and species 
variants of these proteins, naturally-occurring or 
biosynthetic mutants thereof, as well as various 
truncated and fusion constaructs. Deletion or addition 
mutants also are envisioned to be active, including 

35 those which may alter the conserved C-terminal cysteine 



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skeleton^ 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 
5 specifically described constructs disclosed herein. 
The proteins may include forms having varying 
glycosylation patterns , varying N- termini , a family of 
related proteins having regions of amino acid sequence 
homology^ and active truncated, chimeric and/or mutated 
10 forms of native or biosynthetic proteins, produced by 
expression of recombinant DNA in host cells. 

The morphogenic proteins can be expressed from 
intact, chimeric and/or truncated cDNA or from 

15 synthetic DNAs in procaryotic or eucaryotic host cells, 
and purified, cleaved, refolded, and dimerized to form 
morphogenically active compositions. Currently 
preferred host cells include coli or mammalian 
cells, such as CHO, COS or BSC cells. A detailed 

20 description of the morphogens useful in the methods, 

compositions and devices of this invention is disclosed 
in copending US patent application Serial Nos. 752,764, 
filed August 30, 1991, and 667,274, filed March 11, 
1991, the disclosure of which are incorporated herein 

25 by reference. 

Thus, in view of this disclosure, skilled genetic 
engineers can isolate genes from cDNA or genomic 
libraries of various different species which encode 

30 appropriate amino acid sequences, or construct DNAs 
from oligonucleotides, and then can express them in 
various types of host cells, including both procaryotes 
and eucaryotes, to produce large quantities of active 
proteins capable of maintaining liver function in a 

35 mammal, including correcting liver function 



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deficiencies and stimulating hepatic tissue 
regeneration and repair in a variety of inaitunalS/ 
including h\iinans« 

5 The foregoing and other objects ^ features and 

advantages of the present invention will be made more 
apparent from the following detailed description of the 
invention. 



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Brief Description of the Drawings: 

The foregoing and other objects and features of 
this invention^ as well as the invention itself, may be 
5 more fully understood from the following description, 
when read together with the accompanying drawings, in 
which: 

FIGURE 1 is a representation of a Northern blot 
10 identifying OP-l-specif ic mRNA expression in developing 
liver tissue in embryonic and postnatal mouse, wherein 
lanes 2 and 3 contained RNA from 15- and 20-day embryo 
tissue, respectively; lanes 4-8, RNA from 3, 7, 14, 21 
and 28 days post natal animals, respectively; and lanes 
15 1 and 9 were molecular weight marker ladders; 

FIGURE 2 is a photomicrograph showing the effect of 
phosphate buffered saline (PBS, animal 1) or morphogen 
(OP-1, animal 2) on partially hepatectomized rats 
20 (arrow indicates the treated lobe in both animals); 

FIGURE 3 is a representation of a Northern blot of 
mRNA isolated from sham-operated (lanes 3, 5, 7, 9, 11, 
13 and 15) and partially hepatectomized rats (lanes 2, 
25 4, 6, 8, 10, 12, 14) at 6 hr intervals between 12-96 
hours post surgery, probed with an mOP-l-specif ic 
probe, and lanes 1 and 16 are molecular weight marker 
lanes; 

30 FIGURE 4 is a representation of a Northern blot of 

mRNA isolated from galactosamine-treated rats and 
probed with mOP-l-specif ic probe on days 0-7, 10 (lanes 



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1-9/ respectively, and lane 10 contains molecular 
weight markers); 

FIGURE 5 (A and B) are schematic representations of 
5 morphogen inhibition of early mononuclear phagocytic 
cell multinuclearization in vivo ; and 

FIGURE 6 (A-D) graphs the effects of a morphogen 
(e.g., OP-1, Figs. 6A and 6C) and TGF-B (Fig. 6B and 
10 6D) on collagen (6A and 6B) and hyaluronic acid (6C and 
6D) production in primary fibroblast cultures. 



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Detailed Description of the Invention 

It now has been discovered that the proteins 
described herein are effective agents for maintaining 
5 liver function in a mammal. As described herein, these 
proteins ( "morphogens") are capable of inducing hepatic 
tissue regeneration and repair under conditions where 
true tissue morphogenesis typically does not occur, 
including stimulating the proliferation and 

10 differentiation of hepatocytic progenitor cells. The 
proteins also are capable of providing a cytoprotective 
effect to alleviate the tissue destructive effects 
associated with immunologically-related hepatic tissue 
damage- Accordingly, the proteins may be used as part 

15 of a protocol for regenerating damaged or lost hepatic 
tissue, correcting a liver function deficiency, and 
enhancing the viability of a tissue or organ to be 
transplanted in a mammal. The morphogens also may be 
used in a gene therapy protocol to correct a protein 

20 deficiency in a mammal. 

Provided below are detailed descriptions of 
suitable morphogens useful in the methods, compositions 
and devices of this invention, as well as methods for 

25 their administration and application, and numerous, 
nonlimiting examples which 1) illustrate the 
suitability of the morphogens and morphogen-stimulating 
agents described herein as therapeutic agents for 
maintaining liver function in a mammal; and 2) provide 

30 assays with which to test candidate morphogens and 
morphogen-stimulating agents for their efficacy. 
Specifically, the examples demonstrate the expression 
distribution of endogenous morphogen (Example 1), the 
expression of endogenous morphogen during liver 

35 formation in a developing embryo (Example 2), the 



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ability of morphogens to induce proliferation of 
primary hepatocytes (Example 3), morphogen- induced 
liver tissue morphogenesis following partial 
hepatectomy (Example 4); endogenous morphogen 
5 expression during hepatic tissue repair following 
toxin- induced tissue damage (Examples 5); the 
inhibitory effect of morphogens on the body's cellular 
and humoral immune response (Example 6); effect of 
morphogen on fibrogenesis (Example 7); morphogen 
10 utility in liver diagnostic procedures (Example 8), and 
a screening assay for testing candidate morphogen- 
stimulating agents (Example 9). 

15 I. Useful Morphogens 

As defined herein a protein is morphogenic if it is 
capable of inducing the developmental cascade of 
cellular and molecular events that culminate in the 

20 formation of new^ organ-specific tissue and comprises 
at least the conserved C-terminal six cysteine skeleton 
or its functional equivalent (see supra). 
Specifically, the morphogens generally are capable of 
all of the following biological functions in a 

25 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. 

30 Details of how the morphogens useful in the method of 
this invention first were identified, as well as a 
description on how to make, use and test them for 
morphogenic activity are disclosed in USSN 667,274, 
filed March 11, 1991 and USSN 752,764, filed August 30, 

35 1991, the disclosures of which are hereby incorporated 



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by reference. As disclosed therein, the morphogens may 
be purified from naturally-sourced material or 
recombinantly produced from procaryotic or eucaryotic 
host cells, using the genetic sequences disclosed 
5 therein- Alternatively, novel morphogenic sequences 
may be identified following the procedures disclosed 
therein. 

Particularly useful proteins include those which 
10 comprise the naturally derived sequences disclosed in 
Table II. Other useful sequences include biosynthetic 
constructs such as those disclosed in U.S. Pat. 
5,011,691, the disclosure of which is incorporated 
herein by reference (e.g., COP-1, COP-3, COP-4, COP-5, 
15 COP-7, and COP-16). 

Accordingly, the morphogens useful in the methods 
and compositions of this invention also may be 
described by morphogenically active proteins having 
20 amino acid sequences sharing 70% or, preferably, 80% 
homology (similarity) with any of the sequences 
described above, where "homology" is as defined herein 
above . 

25 The morphogens useful in the method of this 

invention also can be described by any of the 6 generic 
sequences described herein (Generic Sequences 1, 2, 3, 
4, 5 and 6). Generic sequences 1 and 2 also may 
include, at their N-terminus, the sequence 

30 

Cys Xaa Xaa Xaa Xaa (Seq. ID No. 15) 
1 5 

Table II, set forth below, compares the amino acid 
35 sequences of the active regions of native proteins that 
have been identified as morphogens, including human 
OP-1 (hOP-1, Seq. ID Nos. 5 arid 16-17), mouse OP-1 
(mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 



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(Seq. ID Nos. 7, 8, and 20-23), CBMP2A (Seq. ID No. 9), 
CBMP2B (Seq. ID No. 10), BMP3 (Seq. ID No. 26), DPP 
(from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, 
Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), 
5 GDF-1 (from mouse, Seq. ID Nos. 14, 32 and 33), 60A 
protein (from Drosophila, Seq. ID Nos. 24 and 25), BMP5 
(Seq. ID No. 27) and BMP6 (Seq. ID No. 28). The 
sequences are aligned essentially following the method 
of Needleman et al. (1970) J. Mol. Biol. , 48:443-453, 

10 calculated using the Align Program (DNAstar, Inc.) In 
the table, three dots indicates that the amino acid in 
that position is the same as the amino acid in hOP-1. 
Three dashes indicates that no amino acid is present in 
that position, and are included for purposes of 

15 illustrating homologies. For example, amino acid 
residue 60 of CBMP-2A and CBMP-2B is "missing". Of 
course, both these amino acid sequences in this region 
comprise Asn-Ser (residues 58, 59), with CBMP-2A then 
comprising Lys and lie, whereas CBMP-2B comprises Ser 

20 and lie. 



TABLE II 



25 



30 



hOF-1 


Cys Lys 


Lys 


His 


Glu 


mOP-1 


* * • • • • 




« • • 


• • • 


hOF-2 


Arg 


Arg 


• • • 




iiiOF-2 


. . ♦ Arg 


Arg 


• • • 


• • • 


DFF 


Arg 


Arg 


• • • 


Ser 


Vgl 




Lys 


Arg 


His 



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- 51 - 





Vgr-1 




• • • 


... 




Gly 




» . . 








CBMP-2A 




• • • 


Arg 


... 


Pro 


... 


• . . 


... 






CBMF-2B 




Arg 


Arg 




Ser 


... 




... 






BHF3 




Ala 


Arg 


Arg 


lyr 




Lys 






5 


GDF-1 




Arg 


Ala 


Arg 


Arg 




... 








60A 




Gin 


Met 


Glu 


Thr 












BHF5 




• • • 


... 


... 


. « « 




... 








BMF6 




Arg 


... 


... 


« • . 




« « • 










1 








5 










10 
























hOP-1 


Ser 


Phe 


Arg 


Asp 


Leu 


Gly 


Trp 


Gin 


Asp 




nOP-1 






















hOP-2 






Gin 










Leu 




15 


inOP-2 


Ser 




. . • 


... 


... 






Leu 






OPP 


Asp 




Ser 




Val 






Asp 






Vgl 


Glu 




Lys 




Val 








Asn 




Vgr-1 






Gin 




Val 












CBHP-2A 


Asp 


... 


Ser 




Val 


... 




Asn 


... 


20 


CBMP-2B 


Asp 




Ser 




Val 






Asn 






BMP3 


Asp 




Ala 




He 






Ser 


Glu 




GDF-1 








Glu 


Val 






His 


Arc 




60A 


Asp 




Lvs 










His 






SUPS 




















25 


BHF6 






GliJ 




















10 










15 








hOF-1 


Trp 


lie 


He 


Ala 


Pro 


Glu 


Gly 


lyr 


Ala 




mOP-1 




... 








... 






• • . 


30 


hOF-2 




Val 


... 






Gin 






Ser 




inDP-2 




Val 


... 






Gin 






Ser 




DPP 






Val 






Leu 






Asp 




Vgl 




Val 








<;in 






Net 




Vgr-1 






.... 






Lys 






. . • 


35 


CBHP-2A 




. • • 


Val 






Pro 






His 



wo 94/06449 



PCr/US93/08808 



- 52 - 





CBMP-2B 




... 


Val 


« « « 




Pro 


... 




Gin 




BHF3 








Set 




Lys 


Ser 


Phe 


Asp 




GDF-1 




Val 








Arg 


... 


Phe 


Leu 




60A 














... 


... 


Gly 


5 


BHF5 






















BHF6 




* • * 








Lys 
















20 










25 




10 


hOP-1 


Ala 


Tyr 


Tyr 


Cys 


Glu 


Gly 


Glu 


Cys 


Ala 




oOP-l 






















bOP-2 


















Ser 




mOP-2 






















DPP 










His 




Lys 




Pro 


15 


Vgl 




Asn 






Tyr 








Pro 




Vgr-l 




Asn 






Asp 








Ser 




CBMP-2A 




Phe 






His 




Glu 




Pro 




CBHP-2B 




Phe 






His 




Asp 




Pro 




BHP3 










Ser 




Ala 




Gin 


20 


GDF-1 




Asn 






Gin 




Gin 




. . • 




60A 




Phe 






Ser 




• . . 




Asn 




BMP5 




Phe 






Asp 




. « • 




Ser 




BMP6 




Asn 






Asp 








Ser 












30 










35 


25 
























hOP-1 


Phe 


Pro 


Leu 


Asn 


Ser 


Tyr 


Het 


Asn 


Ala 




nOP-l 


• • • 




















hOP-2 








Asp 




Cys 










inOP-2 








Asp 


... 


Cys 








30 


DPP 








Ala 


Asp 


His 


Phe 




Ser 




Vgl 


Tyr 






Thr 


Glu 


He 


Leu 




Gly 




Vgr-l 










Ala 


His 






. • • 




CBMP-2A 








Ala 


Asp 


His 


Leu 




Ser 




CBHP-2B 








Ala 


Asp 


His 


Leu 




Ser 


35 


GDF-1 


Leu 




Val 


Ala 


Leu 


Ser 


Gly 


Ser** 


* • • 



wo 94/06449 



PCr/US93/08808 



- 53 - 



BHF3 






Het 


Pro 


Lys 


Ser 


Leu 


Lys 


Pro 


60A 


• • « 


• • • 


• • • 


• • • 


Ala 


His 


• . • 


. • * 


... 


BHP5 


• • * 


' • • • 


• • • 


• • * 


Ala 


His 


Het 


« « . 


... 


BHP6 


• • • 


• • • 


• • • 


• • • 


Ala 


His 


Het 


... 


. • • 












40 










hOP-1 


Thr 


Asn 


His 


Ala 


He 


Val 


Gin 


Thr 


Leu 


mOP-1 


• • • 








• • • 


• • • 


• • • 


... 


. . • 


hOP-2 












Leu 




Ser 




mOP-2 












Leu 




Ser 




DPP 










Val 












Ser 










Leu 








Vgr-l 


• • • 








... 








... 


CBMP-2A 




















CBHP-2B 


• • • 












... 






BKP3 


Ser 








Thr 


He 




Ser 


He 


GDF-1 


Leu 








Val 


Leu 


Arg 


Ala 




60A 




















BMP5 




















BHP6 




- 


















45 










50 








hOP-1 


Val 


His 


Phe 


He 


Asn 


Pro 


Glu 


Thr 


Val 


mOP-l 














Asp 






hOP-2 




His 


Leu 


Het 


Lys 




Asn 


Ala 




©OP -2 




His 


Leu 


Het 


Lvs 




Asp 


Val 




DPP 




Asn 


Asn 


Asn 


• . * 




Gly 


Lys 




Vgl 




• • • 


Ser 


• • • 


Glu 




... 


Asp 


He 


Vgr-1 




• • « 


Val 


Het 






... 


Tyr 




CBMP-2A 




Asn 


Ser 


Val 


. * • 


Ser 




Lys 


He 


CBMP-2B 




Asn 


Ser 


Val 


. . • 


Ser 




Ser 


He 


BMP3 




Arg 


Ala** Gly 


Val 


Val 


Pro 


Gly 


He 


GDF-1 


Het 


• • • 


Ala 


Ala 


Ala 


* • . 


Gly 


Ala 


Ala 


60A 


• • • 


• « • 


Leu 


Leu 


Glu 


• • . 


Lys 


Lys 


... 



wo 94/06449 



PCT/US93/08808 



- 54 - 



BMP5 
BMP6 



55 



Leu Het Phe 
Leu Het ... 



Asp His 
Tyr 

60 



10 



15 



hOP-1 

mOP-1 

hOP-2 

mOP-2 

DPP 

Vgl 

Vgr-l 

CBHP-2A 

CB1IP-2B 

BHP3 

GDF-1 

60A 

BMP5 

B11P6 



Pro Lys Pro Cys Cys Ala Pro Thr Gin 



Asp 



Leu 



Glu 
Leu 



Ala 
Ala 
Ala 



Ala 
Ala 



Val 
Val 

Val 
Val 
Val 
Val 



20 



65 



Lys 
Lys 

Lys 
Lys 
Glu 
Glu 
Glu Lys 
Ala Arg 
Arg 
Lys 
Lys 

70 



25 



30 



35 



hOP-1 

mOP-l 

hOP-2 

ibOP-2 

Vgl 

Vgr-l 

DPP 

CBHP-2A 

CBMP-2B 

BHP3 

GDF-l 

60A 

B11P5 

BHP6 



Leu Asn Ala He Ser Val Leu Tyr Phe 



Ser ... Thr 

Ser . . . Thr 

Het Ser Pro 

••• 

Asp Ser Val 

• • • Sez* • • • • • • 

• • • Scr • • • • • • 

Met Ser Ser Leu 



Ser Pro 
Gly 



Leu Pro 



• • • • • • 



Het 

Ala Het 
Het 
Het 
He 



75 



Tyr 
Tyr 

Phe Tyr 

• • * • • • 

Leu 
Leu 
... Leu 
Phe Tyr 
Phe 

His 

• • • • • • 

• • • • • • 

60 



wo 94/06449 



PCT/US93/08808 



- 55 - 





hOP-1 


Asp 


Asp 


Ser 


Ser 


Asn 


Val 


He 


Leu 


Lys 




mOP-1 


.'. . 


• • • 


• • • 


• • • 


• • • 








... 




hOP-2 




Ser 


• * * 


Asn 


• • • 




• • • 




Arg 


s 


dOP-2 




Ser 


« • • 


Asn 


• • • 




• • • 




Arg 




DPP 


Asn 




Gin 




Thr 




Val 








Vgl 




Asn 


Asn 


Asp 


• • • 




Val 


... 


Arg 




Vjrr-l 

'6* * 






Asn 
















CBMP-2A 


... 


Glu 


Asn 


Glu 


Lys 




Val 


... 


... 


10 


CBMP-2B 




Glu 


Tyr 


Asp 


Lys 




Val 








BHP3 




Glu 


Asn 


Lys 






Val 












Arti 

AOAl 




Asp 






Val 




Ar? 




60A 




Asn 


Asp 


Glu 






Asn 








BMP5 




















15 


BHP6 






Asn 


























85 












hOP-1 


Lvs 

A* J a 


Tvr 


Arc 


Asn 


Met 


Val 


Val 


Arg 




20 


mOP-1 






















hOP-2 




His 












Lvs 






mOP-2 




His 












Lvs 






DPP 


Asn 




Gin 


Glu 




Thr 




Val 






Vgl 


nxs 




GXU 






A 1 -s 

Axa 




Asp 




25 


Vgr-1 




• • • 


• ■ « 
















CBMP-2A 


Asn 


• • • 


Gin 


Asp 




• • • 




Glu 






CBHP-2B 


Asn 


« • • 


Gin 


Glu 




• • • 




Glu 






BMP3 


Val 


• • • 


Pro 


• • • 




Thr 




Glu 






GDF-1 


Gin 


• • • 


Glu 


Asp 




• • • 




Asp 




30 


60A 




• • • 


• • • 


• • • 




He 




Lys 






BMP5 






















B11F6 


• • • 






Trp 
















90 










95 









wo 94/06449 



PCT/US93/08808 



- 56 - 





hOP-1 


Ala 


Cys Gly 


Cys 


His 




oOP-l 












hOP-2 












inOP-2 


• • • 








5 


DPP 


Gly 






Are 




Vgl 


Glu 






Are 




Vgr-1 














civ 






Arg 




CBHP-2B 


Gly 






Arg 


10 


BHP3 


Ser 


Ala 




Arg 




GDF-1 


Glu 






Arg 




60A 


Ser 










BMP5 


Ser 






• • • 




BliP6 








• • • 


15 






100 








♦♦Between 


residues 


56 and 57 


of BMP3 


is 2 



between residues 43 and 44 of GDF-1 lies 
the amino acid sequence Gly-Gly-Pro-Pro. 



20 



25 



30 



As is apparent from the foregoing amino acid 
sequence comparisons, significant amino acid changes 
can be made within the generic sequences while 
retaining the morphogenic activity. For example, while 
the GDF-1 protein sequence depicted in Table II shares 
only about 50% amino acid identity with the hOPl 
sequence described therein, the GDF-1 sequence shares 
greater than 70% amino acid sequence homology (or 
"similarity") with the hOPl sequence, where "homology" 
or "similarity" includes allowed conservative amino 
acid changes within the sequence as defined by Dayoff , 
et al • , Atlas of Protein Sequence and Structure vol . 5 , 
supp.3, pp. 345-362, (M.O. Dayoff, ed., Nat'l BioMed. 
Res. Fd'n, Washington D.C. 1979.) 



35 



wo 94/06449 



PCT/l)S93/08808 



- 57 - 



The currently most preferred protein sequences 
useful! as morphogens in this invention intj^ude tMose 
having! greater than 601 identity* preferably grealter 
than 615* identity, with the amino acid science 
5 defining the conserved six cysteine slceleton of fiOPl 
(e.gw'i residues 43-X39 of Seq. ID Ho. 5). jThe^e most 
preferred sequences include both allelic ai^ specjiies 
variants ol the OP-1 and OP-2 proteins, including the 
Drosopkila 60A protein. Accordingly, in ^^ill aiijotber 
10 preferred aspoct, the invention includes aorphog^ 
coapri'sing species of polypeptide chains having the 
generic amino acid sequence referred to hetein as 
"OPX",' which defines the seven cysteine sk^etoa!|and 
accosdodates the identities between the various 
15 identified mouse and human OP 1 and 0P2 proteins, ppx 
is presented in Seq. ID Ho. 2». As described: thsjrein, 
each 3^ at a given position independent l^f 3 is i selected 
from the residues occurring at the ; corresponding 
position in the C-terminal sequence of noyse or buman 
20 OFl or! OP2 (see Seq. ID Sos. 5-8 and/or Seq. ID Hos. 
16'-23)i. 

IX. I4atriz considerations 

25 The mocphogeae of this invention may i>f implffnte* 
snrgiJelly, dispersed in a biocompatible, j preferably M 
vivo biodegradable matrix appropriately aoai£|ied|| to 
provide e structure in which the mbrphog^j "ay 
dispersed and which elloirs the influx, differentiation 

30 and p^liferatlon of migrating progenitor! cell*. 
Alternatively, or, in addition, di£ferentiBte4 
hepatocytes and/or hepatocytlc progenitor j cells, 
stimulated by exposure to the morphogen, dky be 
disposed in and attached to a matrix structure ^ 

35 implanted surgically. In certain iapplicaj^loni, fuch as 



wo 94/06449 



PCr/US93/08808 



- 58 - 

where tissue morphogenesis is to be induced in the 
absence of endogenous tissue-specificity directing 
signals, the matrix preferably also provides signals 
capable of directing the tissue specificity of the 
5 differentiating cells, and provides a morphogenically 
permissive enviroiunent , being essentially free of 
growth inhibiting signals • 

Where the matrix is to be incorporated into a 
10 surgically prepared liver, or provided to a 

biocompatible, associated site, the formulated matrix 
on which the morphogen is disposed may be shaped as 
desired in anticipation of surgery or may be shaped by 
the physician or technician during surgery. Where 
15 cells are to be attached to the matrix before 

implantation, the matrix preferably is shaped before 
cells are attached thereto. The matrix preferably is 
biodegradable in vivo , being slowly absorbed by the 
body and replaced by new tissue growth, in the shape or 
20 very nearly in the shape of the implant. 

Details of how to make and how to use preferred 
matrices useful in this invention are disclosed below. 
In addition to these matrices, WO 88/03785, published 
25 June 2, 1988, and WO90/12604, published November 1, 
1990, describe additional polymeric materials and 
matrix scaffold considerations. The disclosures of 
these publications are incorporated herein by 
reference . 

30 

A. Tissue-derived Matrices 

Suitable biocompatible, in vivo biodegradable 
acellular matrices may be prepared from 
35 naturally-occurring tissue. The tissue is treated with 
suitable agents to substantially extract the cellular, 
nonstructural components of the tissue. The agents 



- 59 - 



also should be capable of extracting any growth 
inhibiting components associated with the tissue • The 
resulting material is a porous, acellular matrix , 
substantially depleted in nonstructurally-associated 
components, and preferably containing structural 
molecules such as collagen, laminin, hyaluronic acid, 
and the like. 

The matrix also may be further treated with agents 
that modify the matrix, increasing the number of pores 
and micropits on its surfaces • Those skilled in the 
art will know how to determine which agents are best 
suited to the extraction of nonstructural components 
for different tissues. For example, soft tissues such 
as liver and lung may be thin-sectioned and exposed to 
a nonpolar solvent such as, for example, 100% ethanol, 
to destroy the cellular structure of the tissue and 
extract nonstructural components. The material then is 
dried and pulverized to yield nonadherent porous 
particles. Structural tissues such as cartilage and 
dentin where collagen is the primary component may be 
demineralized and extracted with guanidine, essentially 
following the method of Sampath et al. (1983) PNAS 
80^:6591-6595. For example, pulverized and 
demineralized dentin is extracted with five volumes of 
4M guanidine-HCl, 50mM Tris-HCl, pH 7.0 for 16 hours at 
4^*0. The suspension then is filtered. The insoluble 
material that remains is collected and used to 
fabricate the matrix. The material is mostly 
collagenous in manner. It is devoid of morphogenic 
activity. The matrix particles may further be treated 
with a collagen fibril-modifying agent that extracts 
potentially unwanted components from the matrix, and 



wo 94/06449 



PCT/US93/08808 



- 60 - 

alters the surface structure of the matrix material. 
Useful agents include acids , organic solvents or heated 
aqueous media. A detailed description of these matrix 
treatments are disclosed in U.S. Patent No. 4,975,526 
5 and PCT publication US90/00912, published September 7, 
1990 (WO90/10018) . 

The currently most preferred agent is a heated 
aqueous fibril-modifying medium such as water, to 

10 increase the matrix particle surface area and porosity. 
The currently most preferred aqueous medium is an 
acidic aqueous medium having a pH of less than about 
4.5, e.g., within the range of about pH 2 - pH 4 which 
may help to "swell" the collagen before heating. 0.1% 

15 acetic acid, which has a pH of about 3, currently is 
most preferred. 0.1 M acetic acid also may be used. 

Various amounts of delipidated, demineralized 
guanidine-extracted collagen matrix are heated in the 

20 aqueous medium (Ig matrix/30ml aqueous medium) under 
constant stirring in a water jacketed glass flask, and 
maintained at a given temperature for a predetermined 
period of time. Preferred treatment times are about 
one hour, although exposure times of between about 0.5 

25 to two hours appear acceptable. The temperature 

employed is held constant at a temperature within the 
range of about 37*C to 65*C. The currently preferred 
heat treatment temperature is within the range of about 
45**C to eo'^c. 

30 

After the heat treatment, the matrix is filtered, 
washed, lyophilized and used for implant. Where an 
acidic aqueous meditim is used, the matrix also is 
preferably neutralized prior to washing and 
35 lyophilization. A currently preferred neutralization 



wo 94/06449 



PCr/US93/08808 



- 61 - 

buffer is a 200inM sodium phosphate buffer, pH 7.0. To 
neutralize the matrix, the matrix preferably first is 
allowed to cool following theirmal treatment, the acidic 
aqueous medium (e.g., 0.1% acetic acid) then is removed 
5 and replaced with the neutralization buffer and the 
matrix agitated for about 30 minutes. The 
neutralization buffer then may be removed and the 
matrix washed and lyophilized. 



10 Other useful fibril-modifying treatments include 

acid treatments (e.g., trif luoroacetic acid and 
hydrogen fluoride) and solvent treatments such as 
dichloromethane, acetonitrile, isopropanol and 
chloroform, as well as particular acid/solvent 

15 combinations. 

After contact with the fibril-modifying agent, the 
treated matrix may be washed to remove any extracted 
components, following a form of the procedure set forth 
20 below: 



1. Suspend matrix preparation in TBS (Tris- 
buffered saline) lg/200 ml and stir at 4**C for 2 hrs; 
or in 6 M urea, 50 mM Tris-HCl, 500 mM NaCl, pH 7.0 

25 (UTBS) or water and stir at room temperature (RT) for 
30 minutes (sufficient time to neutralize the pH); 

2. Centrifuge and repeat wash step; and 



30 



3. Centrifuge; discard supernatant; water wash 
residue; and then lyophilize. 



wo 94/06449 



PCT/US93/08808 



- 62 - 

B. Synthetic Matrices 

Suitable matrix scaffolds may be created from 
biocompatible/ preferably in vivo biodegradable 
5 synthetic polymers ^ including polylactic acid, 

polyglycolic acid, polyanhydride , polybutyric acid, and 
copolymers thereof, and/or synthetic -inorganic 
materials, such as hydroxyapatite, tricalcium 
phosphate, and other calciiim phospates. These 

10 polymers are well described in the art and are 

available commercially* For example, polymers composed 
of polyactic acid (e.g., MW 100 kDa), 80% 
polylactide/20% glycoside or poly 3-hydroxybutyric acid 
(e.g., MW 30 kDa) all may be purchased from 

15 PolySciences, Inc. The polymer compositions generally 
are obtained in particulate form and the osteogenic 
devices preferably fabricated under nonaqueous 
conditions (e.g., in an ethanol-trif luoroacetic acid 
solution, EtOH/TFA) to avoid hydrolysis of the 

20 polymers. In addition, one can alter the morphology of 
the particulate polymer compositions, for example to 
increase porosity, using any of a number of particular 
solvent treatments known in the art. 

25 For example, osteogenic devices fabricated with 

morphogenic protein, solubilized in EtOH/TFA as 
described below, and a matrix composed of polylactic 
acid, poly 3-hydroxybutyric acid, or 80% 
polylactide/20% glycoside are all osteogenically active 

30 when implanted in the rat model and bioassayed as 
described in U.S. Pat. No. 4,968,590 (e.g., as 
determined by calcium content, alkaline phosphatase 
levels and histology of 12-day implants). 



wo 94/06449 



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- 63 - 

C. S ynthetic Tissue-Specific Matrices 

In addition to the naturally-derived 
tissue-specific matrices described above, useful 
5 tissue-specific matrices may be formulated 

synthetically if appropriately modified. These porous 
biocompatible, in vivo biodegradable synthetic matrices 
are disclosed in PCT publication US9 1/03603, published 
December 12, 1991 (W091/18558), the disclosure of which 

10 is hereby incorporated by reference. Briefly, the 
matrix comprises a porous crosslinked structural 
polymer of biocompatible, biodegradable collagen and 
appropriate, tissue-specific glycosaminoglycans as 
tissue-specific cell attachment factors. Collagen 

15 derived from a number of sources may be suitable for 
use in these synthetic matrices, including insoluble 
collagen, acid-soluble collagen, collagen soluble in 
neutral or basic aqueous solutions, as well as those 
collagens which are commercially available. 

20 

Glycosaminoglycans (GAGs) or mucopolysaccharides 
are hexosamine-containing polysaccharides of animal 
origin that have a tissue specific distribution, and 
therefore may be used to help determine the tissue 
25 specificity of the morphogen-stimulated differentiating 
cells. Reaction with the GAGs also provides collagen 
with another valuable property, i.e., inability to 
provoke an immune reaction (foreign body reaction) from 
an animal host. 

30 

Chemically, GAGs are made up of residues of 
hexoseamines glycosidically bound and alternating in a 
more-or-less regular manner with either hexouronic acid 
or hexose moieties (see, e.g., Dodgson et al. in 
35 Carbohydrate Metabolism and its Disorders (Dickens et 
al., eds.) Vol. 1, Academic Press (1968)). Useful GAGs 



wo 94/06449 



PCr/US93/08808 



- 64 - 

include hyaluronic acid, heparin, heparin sulfate, 
chondroitin 6-sulfate, chondroitin 4-sul£ate, derraatan 
sulfate, and keratin sulfate. Other GAGs are suitable 
for forming the matrix described herein, and those 
5 skilled in the art will either know or be able to 
ascertain other suitable GAGs using no more than 
routine experimentation. For a more detailed 
description of mucopolysaccharides, see Aspinall, 
Polysaccharides , Pergamon Press, Oxford (1970). For 
10 example, as disclosed in U.S. Application Serial 

Ko. 529,852, chondroitin-6-sulf ate can be used where 
endochondral bone formation is desired. Heparin 
sulfate, on the other hand, may be used to formulate 
synthetic matrices for use in lung tissue repair. 

15 

Collagen can be reacted with a GAG in aqueous 
acidic solutions, preferably in diluted acetic acid 
solutions. By adding the GAG dropwise into the aqueous 
collagen dispersion, coprecipitates of tangled collagen 
20 fibrils coated with GAG results. This tangled mass of 
fibers then can be homogenized to form a homogeneous 
dispersion of fine fibers and then filtered and dried. 

Insolubility of the collagen-GAG products can be 
25 raised to the desired degree by covalently cross* 

linking these materials, which also serves to raise the 
resistance to resorption of these materials. In 
general, any covalent cross-linking method suitable for 
cross-linking collagen also is suitable for cross- 
30 linking these composite materials, although 

crosslinking by a dehydrotheraal process is preferred. 

When dry, the crosslinked particles are essentially 
spherical, with diameters of about 500 /jm. Scanning 
35 electron miscroscopy shows pores of about 20 ^m on the 
surface and 40 /jm on the interior. The interior is 



wo 94/06449 



PCr/US93/08808 



- 65 - 

made up of both fibrous and sheet-like structures/ 
providing surfaces for cell attachment. The voids 
interconnect/ providing access to the cells throughout 
the interior of the particle. The material appears to 
5 be roughly 99.5% void volume/ making the material very 
efficient in terms of the potential cell mass that can 
be grown per gram of microcarrier. 

D. Morphoqen Adsorption to Matrix Surfaces 

10 

The morphogens described herein can be combined and 
dispersed in a suitable matrix using any of the methods 
described below: 

15 1. Ethanol Precipitation 

Matrix is added to the morphogen dissolved in 
guanidine-HCl. Samples are vortexed and incubated at a 
low temperature. Samples are then further vortexed. 
20 Cold absolute ethanol is added to the mixture which is 
then stirred and incubated. After centrifugation 
(microfuge/ high speed) the supernatant is discarded. 
The matrix is washed with cold concentrated ethanol in 
water and then lyophilized. 

25 

2. Acetonitrile Trif luoroacetic 
Acid Lyophilization 

In this procedure/ morphogen in an 
30 acetonitrile trif luroacetic acid (ACN/TFA solution is 
added to the carrier material. Samples are vigorously 
vortexed many times and then lyophilized. 



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3. Buffered Saline Lyophilization 

Morphogen preparations in physiological saline 
may also be vortexed with the matrix and lyophilized to 
produce morphogenically active material • 

III. Hepatocytic Cell Considerations 

Primary hepatocytes or progenitor cells may be 
implanted in the mammal in one embodiment of the 
invention. For example, implanted hepatocytes may act 
as gene therapy tools capable of correcting a protein 
deficiency in vivo by expressing and/or secreting the 
deficient protein when implanted at a liver tissue or 
associated locus in a mammal. The liver functions in 
part as a protein- synthesizing organ, responsible for 
the production of myriad proteins which are secreted 
from the liver and transported, e.g., via the 
circulatory system, to function elsewhere in the body. 
Accordingly, hepatic tissue, like renal and pancreatic 
tissue, provides an endogenous system having the 
necessary mechanisms in place to act as a vector for 
the in vivo production of (including secretion of) any 
protein, including proteins not normally expressed by 
hepatic tissue. Thus, protein deficiencies that can be 
treated by this method include proteins involved in 
normal liver functions, proteins normally produced and 
secreted by the liver to function elsewhere in the 
body, and proteins not normally produced by hepatic 
tissue. Where the proteins to be produced are not 
normally expressed by hepatic tissue, the hepatocytes 
must be provided with means for expressing that 
protein. For example, the cell may be genetically 
engineered as described below to induce expression of 
the endogenous genetic sequence encoding the protein. 



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Alternatively, a nucleic acid encoding the protein and 
under control of a suitable promoter (and enhancer), 
may be provided to the cell as described below. In 
addition, the cell may be provided with one or more 
5 regulatory elements so that expression of the protein 
of interest mimics that of the endogenously produced 
protein, particularly where normal protein expression 
depends on changes in the physiological concentration 
of a molecule. For example, insulin production is 
10 regulated by blood glucose levels in the body. 

The protein deficiency to be corrected may result 
from defective endogenous protein production, including 
protein expression and/or secretion, or the protein's 

15 efficacy may be reduced due to a preexisting condition 
in the individual. The defect may be genetic or may be 
induced by, for example, damage to the 
protein- synthesizing tissue. Exemplary hepatic 
proteins that may be used in a gene therapy include, 

20 but are not limited to, albumin and albumin synthesis 
proteins, blood clotting factors, including fibrinogen 
and thrombin. Factor VIII, iron or copper binding 
proteins, and vitamin A binding proteins. Exemplary 
non-hepatic proteins that may be used in a gene therapy 

25 include, but are not limited to, insulin, tissue 

plasminogen activator (TPA), erythropoietin, growth 
hormones, and the like. Similarly, the cells also may 
act as in vivo drug delivery vehicles, capable of 
producing and secreting one or more therapeutic drugs 

30 when implanted at a suitable locus in a mammal. The 
cells further may be manipulated to modify antigen 
expression on the cell surface, and limit the in vivo 
immune response typically induced by foreign material. 



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Where cells act as gene therapy tools, the cells 
may be obtained from a donor competent for providing 
the protein of interest. Cells can be obtained by 
biopsy or surgical excision from a donor, or from 
5 established cell lines. Preferably, allogenic cells 
are obtained from a biocompatible donor. 
Alternatively, autologous cells may be obtained from 
the patient and modified by recombinant DNA technology 
to incorporate genetic sequences sufficient to allow 

10 the cells to produce the protein or proteins of 

interest in vivo when the cells are reimplanted in the 
patient. Protocols and detailed discussions of 
considerations for introducing foreign genetic material 
into cells, particularly human cells, are well 

15 described in the art. A representative, but by no 
means exhaustive list, includes US Pat. No. 4,868,116, 
issued September 19, 1989, US Pat. No. 4,980,286, 
issued December 25, 1990, both to Morgan et al., and US 
Pat. No. 4,396,601, issued August 2, 1983, to Salser et 

20 al., Anderson, WF (1992) Science 256:808-813, Karson et 
al., (1992) J. Reprod Med 37:508-514, and Hoeg et al., 
(1990) Trans Assoc. Am Physicians 103 :73-79, these 
disclosures of which are incorporated herein by 
reference. 

25 

A currently preferred protocol for isolating 
primary hepatocytes from liver tissue is described in 
Example 3 below. Other methods known in the art also 
are envisioned to be useful, such as those described, 

30 for example, in WO 88/03785. Where pluripotential 

hemopoietic stem cells are to be used, a useful method 
for their isolation is described in international 
application US92/01968 (W092/15323) . Briefly, and as 
described in detail therein, a biocompatible matrix 

35 material able to allow the influx of migratory 



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progenitor cells may be implanted at an in vivo site 
long enough to allow the influx of migratory progenitor 
cells. For example, a bone-derived^ guanidine- 
extracted matrix, formulated as disclosed for example 
5 in Sampath et al. ((1983) PNAS 80:6591*6595) , or U.S. 
Patent No. 4,975,526, may be implanted into a rat, 
essentially following the method of Sampath et al. 
(ibid). After three days the implant is removed, and 
the progenitor cells associated with the matrix 
10 dispersed and cultured. Another method is described, 
for example, in US Pat. No. 5,061,620, issued 10/29/91, 
to Tsukamoto et al. 

Isolated cells may be stimulated in vitro by 

15 morphogen exposure, essentially as described in Example 
3. Stimulation is performed under sterile conditions, 
using an appropriate morphogen concentration and 
incubation period to stimulate the cells. Preferred 
times and concentration for a given procedure may be 

20 determined empirically by the clinician without undue 
experimentation. In general, a period of from about 10 
minutes to 72 hours should be sufficient • Cells may be 
attached to a matrix by incubating the cells in the 
presence of matrix for at least a number of hours, 

25 e.g., 3-5 hours, or, preferably overnight. An 

efficient technique for attaching cells to a matrix 
surface! is to place a concentrated suspension of cells 
on the surface of the matrix material and allow the 
cells to infiltrate and adsorb to the material. Cells 

30 typically attach individually or in small groups. In 
the absence of added morphogen cells begin rearranging 
into clusters within 24 hours and within 3 days cells 
have almost completely infiltrated the support and have 
organized into large clusters. 

35 



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In a particularly preferred embodiment, the 
morphogen first is adsorbed to the matrix surface and 
cells subsequently attached thereto. The cell-matrix 
structure may be maintained in vitro and to allow the 
5 cells to proliferate (preferably by exposure to a 
morphogen or morphogen- stimul ting agent) or, 
alternatively/ the complex may be implanted in the 
animal and the cells allowed to proliferate (and 
differentiate) in vivo. 

10 

As with morphogen administrations, where implanted 
cells are to replace damaged or lost tissue at a liver- 
specific locus, the cells preferably are provided to a 
surgically prepared locus where from which necrotic or 

15 cirrhotic tissue has been removed, e.g., by surgical, 
chemical, ablating, or other means known in the 
medical art. The cells then are provided to the 
prepared site, preferably attached to a matrix and 
associated with a morphogen or morphogen- stimulating 

20 agent. 

The cells may be provided to a morphogenically 
permissive site in a liver-specific locus, e.g., 
following removal of necrotic and/or cirrhotic tissue, 

25 or following excision of sufficient tissue to provide a 
morphogenically permissive site. Alternatively, the 
cell-matrix structure may be implanted together with a 
morphogen or morphogen- stimulating agent at a suitable, 
vascularized liver-associated locus, such as within the 

30 folds of the mesentery. 

As described above, implanting cells together with 
a morphogen or morphogen- stimulating agent enhances 
their proliferation and their viability in vivo, such 
35 that the new tissue is formed without the significant 

1 



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associated cell loss or delay which characterizes 
existing protocols and which currently require the use 
of substantial initial seed cell populations. In 
addition, hepatic tissue growth can be stimulated using 
5 the methods described herein without the need of a 
partial hepatectomy as described in the art. Finally, 
the morphogens described herein functionally inhibit 
the tissue damage associated with the body's immune 
response, reducing the need for associated treatments 
10 with immunosuppressive drugs. 

IV. Bioassy Considerations 

The following sets forth various procedures for 
15 evaluating the in vivo morphogenic utility of the 
morphogens and morphogenic compositions of this 
invention. The proteins and compositions may be 
injected or surgically implanted in a mammal, following 
any of a number of procedures well known in the art. 

20 

Histological Evaluation 

Histological sectioning and staining is preferred 
to determine the extent of morphogenesis in vivo, 

25 particularly in tissue repair procedures. Excised 
implants are fixed in Bouins Solution, embedded in 
paraffin, and cut into 6-8 /i/m sections. Staining with 
toluidine blue or hemotoxylin/eosin demonstrates 
clearly the ultimate development of the new tissue. 

30 Twelve day implants are usually sufficient to determine 
whether the implants contain newly induced tissue. 



35 



Successful implants exhibit a controlled 
progression through the stages of induced tissue 
development allowing one to identify and follow the 



- 72 - 



tissue-specific events that occur. For example, in 
endochondral bone formation the stages include: 
(1) leukocytes on day one; (2) mesenchymal cell 
migration and proliferation on days two and three; 

(3) chondrocyte appearance on days five and six; 

(4) cartilage matrix formation on day seven; 

(5) cartilage calcification on day eight; (6) vascular 
invasion, appearance of osteoblasts, and formation of 
new bone on days nine and ten; (7) appearance of 
osteoclasts and bone remodeling and dissolution of the 
implanted matrix on days twelve to eighteen; and 

(8) hematopoietic bone marrow differentiation in the 
ossicle on day twenty-one. Similarly, in hepatic 
tissue formation the stages include leukocytes on day 
one, mesenchymal cell migration and proliferation on 
days two and three, hepatocyte appearance on days five 
and six, followed by matrix formation and 
vascularization . 

Biological Markers 

In addition to histological evaluation, biological 
markers may be used as a marker for tissue 
morphogenesis. Useful markers include tissue-specific 
enzymes whose activities may be assayed (e.g., 
spectrophotometrically ) after homogenization of the 
implant. These assays may be useful for quantitation 
and for obtaining an estimate of tissue foannation 
quickly after the implants are removed from the animal. 
For example, alkaline phosphatase activity may be used 
as a marker for osteogenesis. 

Incorporation of systemically provided morphogens 
may be followed using tagged morphogens (e.g., 
radioactively labelled) and determining their 
localization in new tissue, and/or by monitoring their 



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disappearance from the circulatory system using a 
standard pulse-chase labeling protocol. The morphogen 
also may be provided with a tissue-specific molecular 
tag^ whose uptake may be monitored and correlated with 
5 the concentration of morphogen provided. 

V. Formulations and Methods for Parenteral 
Administration of Therapeutic Agents 

10 The morphogens of this invention may be used to 

repair diseased or damaged mammalian tissue. The 
tissue to be repaired is preferably assessed, and 
excess necrotic or interfering scar tissue removed as 
needed, by surgical, chemical, ablating or other 

15 methods known in the medical arts. 

The morphogen then may be provided directly to the 
tissue locus as part of a sterile, biocompatible 
composition, either by surgical implantation or 

20 injection. Alternatively, a sterile, biocompatible 

composition containing morphogen-stimulated progenitor 
cells may be provided to the tissue locus. The 
existing tissue at the locus, whether diseased or 
damaged, provides the appropriate matrix to allow the 

25 proliferation and tissue-specific differentiation of 
progenitor cells. In addition, a damaged or diseased 
tissue locus, particularly one that has been further 
assaulted by surgical means, provides a morphogenically 
permissive environment. For some tissues, it is 

30 envisioned that systemic provision of the morphogen 
will be sufficient. 

In some circumstances, particularly where tissue 
damage is extensive, the tissue may not be capable of 
35 providing a sufficient matrix for cell influx and 

proliferation. In these instances, it may be necessary 
to provide the morphogen or morphogen-stimulated 



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progenitor cells to the tissue locus in association 
with a suitable, biocompatible formulated matrix, 
prepared by any of the means described below. The 
matrix preferably is tissue-specific, in vivo 
5 biodegradable, and comprises particles having 
dimensions within the range of 70-850/im, most 
preferably 150-420pm. 

The morphogens may be provided to an individual by 

10 any suitable means. Preferably, the morphogen or 
morphogen-stimulating agent (collectively described 
herein below as the "therapeutic agent") is provided 
directly to the liver tissue (e.g., locally, as by 
injection to the tissue locus or by periodic release 

15 from a locally implanted osmotic pump). While not 

currently preferred for most liver tissue regenerative 
applications, oral administration or systemic injection 
also may be viable administration routes for certain 
applications, such as part of a protocol to enhance 

20 viabilty of a tissue to be transplanted, or as part of 
a protocol to maintain liver function during a surgical 
or other therapeutic procedure, or for maintaining 
liver function in aged or immuno-suppressed 
individuals, or others at risk for hepatic tissue 

25 damage. A detailed description of considerations for 
systemic administration, including oral and parenteral 
administration, is disclosed, for example, in copending 
[Atty. Docket CRP-059CP], incorporated hereinabove by 
reference. It should be noted that morphogenically 

30 active protein is present in milk, including mammary 
gland extract, colostrum and 57-day milk, and also is 
present in human senun, indicating that systemic and, 
in particular, oral administration are viable 
administrative routes for morphogens. 

35 



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Where the morphogen or morphogen-stimulatig agent 
is provided by local injection, the morphogen 
preferably comprises part of an aqueous solution* The 
solution is physiologically acceptable so that in 
5 addition to delivery of the desired morphogen to the 
patient, the solution does not otherwise adversely 
affect the patient's electrolyte and volume balance. 
The aqueous medium for the morphogen thus may comprise 
normal physiologic saline { 0*85-0. 9% NaCl, 0.15M), pH 

10 7-7.4. The aqueous solution containing the morphogen 
can be made, for example, by dissolving the protein. in 
50% ethanol containing acetonitrile in 0.1% 
trif luoroacetic acid (TFA) or 0.1% HCl, or equivalent 
solvents. One volume of the resultant solution then is 

15 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, a given morphogen may be made more soluble by 

20 association with a suitable molecule. For example, the 
pro form of the morphogenic protein comprises a species 
that is soluble in physiologically buffered solutions. 
In fact, the endogenous protein is thought to be 
transported in this form. This soluble form of the 

25 protein may be obtained from the culture medium of 

morphogen-secreting mammalian cells. Alternatively, a 
soluble species may be formulated by complexing the 
mature dimer (or an active fragment thereof) with part 
or all of a pro domain. Another molecule capable of 

30 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-1 by 80%. Other components found in milk 
and/or various serum proteins also may be useful. 



35 



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Useful solutions for parenteral administration may 
be prepared by any of the methods well known in the 
pharmaceutical art, described, for example, in 
Remington's Pharmaceutical Sciences (Gennaro, A., ed.)/ 
5 Mack Pub*, 1990. Formulations may include, for 
example, polyalkylene glycols such as polyethylene 
glycol, oils of vegetable origin, hydrogenated 
naphthalenes, and the like. Formulations for direct 
administration, in particular, may include glycerol and 

10 other compositions of high viscosity. Biocompatible, 
preferably bioresorbable, polymers, including, for 
example, hyaluronic acid, collagen, polybutyrate, 
tricalcium phosphate, lactide and lactide/glycolide 
copolymers, may be useful excipients to control the 

15 release of the morphogen in vivo. Other potentially 
useful parenteral delivery systems for these morphogens 
include ethylene-vinyl acetate copolymer particles, 
osmotic pumps, implantable infusion systems, and 
liposomes. 

20 

In addition, while the mature forms of certain 
morphogens described herein typically are sparingly 
soluble, the morphogen form found in milk (and mammary 
gland extract and colostrum) is readily soluble, 

25 probably by noncovalent association of the mature, 

morphogenically active form with part or all of the pro 
domain of the intact sequence as described below, (see 
Section V.l) and/or by association with one or more 
milk components. Accordingly, the compounds provided 

30 herein also may be associated with molecules capable of 
enhancing their solubility in vitro or in vivo . 

The compounds provided herein also may be 
associated with molecules capable of targeting the 
35 morphogen or morphogen- stimulating agent to liver 

tissue. For example, an antibody , antibody fragment. 



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or other binding protein that interacts specifically 
with a surface molecule on liver tissue cells, 
including hepatocytes or epithelial cells, may be used. 
Useful targeting molecules may be desigrned, for 
5 example, using the single chain binding site technology 
disclosed, for example, in U.S. Pat. No. 5,091,513. 

As described above, the morphogens provided herein 
share significant sequence homology in the C-terminal 

10 active domains. By contrast, the sequences typically 
diverge significantly in the sequences which define the 
pro domain. Accordingly, the pro domain is thought to 
be morphogen-specif ic. As described above, it is also 
known that the various morphogens identified to date 

15 are differentially expressed in the different tissues. 
Accordingly, without being limited to any given theory, 
it is likely that, under natural conditions in the 
body, selected morphogens typically act on a given 
tissue. Accordingly, part or all of the pro domains 

20 which have been identified associated with the active 
form of the morphogen in solution, may serve as 
targeting molecules for the morphogens described 
herein. For example, the pro domains may interact 
specifically with one or more molecules at the target 

25 tissue to direct the morphogen associated with the pro 
domain to that tissue. Accordingly, another useful 
targeting molecule for targeting morphogen to hepatic 
tissue may include part or all of a morphogen pro 
domain. As described above, morphogen species 

3d comprising the pro domain may be obtained from culture 
medium of morphogen-secreting cells. Alternatively, a 
tissue-targeting species may be formulated by 
complexing the mature dimer (or an active fragment 
thereof) with part or all of a pro domain. 

35 



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Finally/ the morphogens or morphogen-stimulating 
agents provided herein may be administered alone or in 
combination with other molecules ( "cof actors" ) known to 
be beneficial in maintaining liver function, 
5 particularly symptom-alleviating cof actors, such as 
other, non-steroidal anti-inflammatory agents, 
antiseptics and antibiotics. 

The compounds provided herein can be formulated 
10 into pharmaceutical compositions by admixture with 
pharmaceutically acceptable nontoxic excipients and 
carriers. As noted above, such compositions may be 
prepared for direct, or local or systemic 
administration, particularly in the form of liquid 
15 solutions or suspensions; for oral administration, 
particularly in the form of tablets or capsules; or 
intranasally, particularly in the form of powders, 
nasal drops, or aerosols. 

20 The compositions can be formulated for 

administration to humans or other mammals in 
therapeutically effective amounts, e.g., cumounts which 
provide appropriate concentrations for a time 
sufficient to substantially eliminate or reduce the 

25 patient's pathological condition, including stimulating 
regeneration of damaged or lost hepatic tissue 
following hepatocellular injury including inhibiting 
additional damage thereto, to provide therapy for the 
liver diseases and disorders described above, and 

30 amounts effective to protect hepatic tissue in 
anticipation of injury to the tissue. 



As will be appreciated by those skilled in the art, 
the concentration of the compounds described in a 
35 therapeutic composition will vary depending upon a 

number of factors, including the dosage of the drug to 



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be administered, the chemical characteristics (e.g., 
hydrophobicity ) of the compounds employed, and the 
route of administration. The preferred dosage of 
therapeutic agent to be administered also is likely to 
5 depend on such variables as the type and extent of 

progression of the hepatic disorder, the overall health 
status of the particular patient, the relative 
biological efficacy of the compound selected, the 
formulation of the compound excipients, and its route 

10 of administration. In general terms, the compounds of 
this invention may be provided in an aqueous 
physiological buffer solution containing about 0.001 to 
10% W/v compound for liquid administration. Typical 
dose ranges are from about 10 ng/kg to about 1 g/kg of 

15 body weight per day; a preferred dose range is from 
about 0.1 //g/kg to 100 mg/kg of body weight per day. 
Optimally, the morphogen dosage given is between 
0.1-100 /jg of protein per kilogram weight of the 
patient. No obvious morphogen induced pathological 

20 lesions are induced when mature morphogen (e.g., OP-1, 

20 fjg) is administered daily to normal growing rats for 

21 consecutive days. Moreover, 10 systemic 
injections of morphogen (e.g., OP-1) injected daily for 
10 days into normal newborn mice does not produce any 

25 gross abnormal ties. 

Where morphogens are administered systemically, in 
the methods of the present invention, preferably a 
large volume loading dose is used at the start of the 
30 treatment. The treatment then is continued with a 
maintenance dose. Further administration then can be 
determined by monitoring at intervals the levels of the 
morphogen in the blood. 



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Where injury to hepatic tissue is induced 
deliberately as part of, for example, a surgical or 
other medical procedure, the morphogen preferably is 
provided just prior to, or concomitant with induction 
5 of the trauma. Preferably, the morphogen is 

administered prophylactically in a surgical setting. 
Optimally, the morphogen dosage given in all cases is 
between 1-100 fjg of protein per kilogram weight of the 
patient. 

10 

As described above, as an alternative or, in 
addition, an effective amount of an agent capable of 
stimulating endogenous morphogen levels may be 
administered by any of the routes described above. For 

15 example, an agent capable of stimulating morphogen 

production and/or secretion from liver tissue cells or 
cells at a distant which then is targeted to the liver, 
may be provided to a mammal , e.g., by direct 
administration of the morphogen to glial cells 

20 associated with the nerve tissue to be treated. A 
method for identifying and testing agents capable of 
modulating the levels of endogenous morphogens in a 
given tissue is described generally herein in 
Example 9, and in detail in international application 

25 US92/07359 (WO 93/05/72). Briefly, candidate compounds 
can be identified and tested by incubating the compound 
in vitro with a test tissue or cells thereof, for a 
time sufficient to allow the compound to affect the 
production, i.e., the expression and/or secretion, of a 

30 morphogen produced by the cells of that tissue. Here, 
suitable tissue or cultured cells of a tissue 
preferably would comprise hepatic tissue cells. 

A currently preferred detection means for 
35 evaluating the level of the morphogen in culture upon 
exposure to the candidate compound comprises an 
immunoassay utilizing an antibody or other suitable 



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binding protein capable of reacting specifically with a 
morphogen and being detected as part of a complex with 
the morphogen. Immunoassays may be performed using 
standard techniques known in the art and antibodies 
5 raised against a morphogen and specific for that 
morphogen. Agents capable of stimulating endogenous 
morphogens then may formulated into pharmaceutical 
preparations and administered as described herein. 

10 V.A Soluble Morphogen Complexes 

A currently preferred form of the morphogen useful 
in therapeutic formulations ^ having improved solubility 
in aqueous solutions and consisting essentially of 

15 amino acids, is a dimeric morphogenic protein 

comprising at least the 100 amino acid peptide sequence 
having the pattern of seven or more cysteine residues 
characteristic of the morphogen family complexed with a 
peptide comprising part or all of a pro region of a 

20 member of the morphogen family, or an allelic, species 
or other sequence variant thereof. Preferably, the 
dimeric morphogenic protein is complexed with two 
peptides. Also, the dimeric morphogenic protein 
preferably is noncovalently complexed with the pro 

25 region peptide or peptides. The pro region peptides 
also preferably comprise at least the N-terminal 
eighteen amino acids that define a given morphogen 
pro region. In a most preferred embodiment, peptides 
defining substantially the full length pro region are 

30 used. 

Other soluble forms of morphogens include dimers of 
the uncleaved pro forms of these proteins, as well as 
"hemi-dimers" wherein one subunit of the dimer is an 
35 uncleaved pro form of the protein, and the other 



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subunit comprises the mature form of the protein, 
including truncated forms thereof, preferably 
noncovalently associated with a cleaved pro domain 
peptide . 

As described above, useful pro domains include the 
full length pro regions, as well as various truncated 
forms hereof, particularly truncated forms cleaved at 
proteolytic Arg-Xaa-Xaa-Arg cleavage sites. For 
example, in OP-1, possible pro sequences include 
sequences defined by residues 30-292 (full length 
form); 48-292; and 158-292. Soluble OP-1 complex 
stability is enhanced when the pro region comprises the 
full length form rather than a truncated form, such as 
the 48-292 truncated form, in that residues 30-47 show 
sequence homology to the N-terminal portions of other 
morphogens, and are believed to have particular utility 
in enhancing complex stability for all morphogens. 
Accordingly, currently preferred pro sequences are 
those encoding the full length form of the pro region 
for a given morphogen. Other pro sequences 
contemplated to have utility include biosynthetic pro 
sequences, particularly those that incorporate a 
sequence derived from the N-terminal portion of one or 
more morphogen pro sequences. 

As will be appreciated by those having ordinary 
skill in the art, useful sequences encoding the pro 
region may be obtained from genetic sequences encoding 
known morphogens. Alternatively, chimeric pro regions 
can be constructed from the sequences of one or more 
known morphogens. Still another option is to create a 
synthetic sequence variant of one or more known pro 
region sequences. 



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In another preferred aspect, useful pro region 
peptides include polypeptide chains comprising an amino 
acid sequence encoded by a nucleic acid that hybridizes 
under stringent conditions with a DNA or RNA sequence 
5 encoding at least the N-terminal eighteen amino acids 
of the pro region sequence for OPl or 0P2, e.g., 
nucleotides 136-192 and 152-211 of Seq. ID No. 16 and 
20, respectively. 

10 V.A.I Isolation of Soluble morphogen complex from 
conditioned media or body fluid 

Morphogens are expressed from mammalian cells as 
soluble complexes. Typically, however the complex is 

15 disassociated during purification, generally by 

exposure to denaturants often added to the purification 
solutions, such as detergents, alcohols, organic 
solvents, chaotropic agents and compounds added to 
reduce the pH of the solution. Provided below is a 

20 currently preferred protocol for purifying the soluble 
proteins from conditioned media (or, optionally, a body 
fluid such as serum, cerebro- spinal or peritoneal 
fluid), under non-denaturing conditions. The method is 
rapid, reproducible and yields isolated soluble 

25 morphogen complexes in substantially pure form. 

Soluble morphogen complexes can be isolated from 
conditioned media using a simple, three step 
chromatographic protocol performed in the absence of 

30 denaturants. The protocol involves running the media 
(or body fluid) over an affinity column, followed by 
ion exchange and gel filtration chromatographies. The 
affinity column described below is a Zn-IMAC column. 
The present protocol has general applicability to the 

35 purification of a variety of morphogens, all of which 



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are anticipated to be isolatable using only minor 
modifications of the protocol described below. An 
alternative protocol also envisioned to have utility an 
immunoaf f inity column, created using standard 
5 procedures and, for example, using antibody specific 
for a given morphogen pro domain (complexed, for 
example, to a protein A-conjugated Sepharose colimn. ) 
Protocols for developing immunoaf f inity columns are 
well described in the art, (see, for example. Guide to 
10 Protein Purification , M. Deutscher, ed.. Academic 

Press, San Diego, 1990, particularly sections VII and 
XI. ) 

In this experiment OP-1 was expressed in mammalian 

15 CHO (Chinese hamster ovary) cells as described in the 
art (see, for example, international application 
US90/05903 (WO91/05802).) The CHO cell conditioned 
media containing 0.5% FBS was initially purified using 
Immobilized Metal-Ion Affinity Chromatography (IMAC). 

20 The soluble OP-1 complex from conditioned media binds 
very selectively to the Zn-IMAC resin and a high 
concentration of imidazole (50 mM imidazole, pH 8.0) is 
required for the effective elution of the bound 
complex. The Zn-IMAC step separates the soluble OP-1 

25 from the bulk of the contaminating serum proteins that 
elute in the flow through and 35 mM imidazole wash 
fractions. The Zn-IMAC purified soluble OP-1 is next 
applied to an S-Sepharose cation-exchange column 
equilibrated in 20 mM NaPO^ (pH 7.0) with SO mM NaCl. 

30 This S-Sepharose step serves to further purify and 

concentrate the soluble OP-1 complex in preparation for 
the following gel filtration step. The protein was 
applied to a Sephacryl S-200HR column equilibrated in 
TBS. Using substantially the same protocol, soluble 

35 morphogens also may be isolated from one or more body 



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fluids, including serum, cerebro-spinal fluid or 
peritoneal fluid. 

IMAC was performed using Chelating-Sepharose 
5 (Pharmacia) that had been charged with three column 
volumes of 0.2 M ZnSO^. The conditioned media was 
titrated to pH 7.0 and applied directly to the ZN-IMAC 
resin equilibrated in 20 mM HEPES (pH 7.0) with 500 mM 
NaCl. The Zn-IMAC resin was loaded with 80 mL of 

10 starting conditioned media per mL of resin. After 
loading, the column was washed with equilibration 
buffer and most of the contaminating proteins were 
eluted with 35 mM imidazole (pH 7.0) in equilibration 
buffer. The soluble OP-1 complex then is eluted with 

15 50 roH imidazole (pH 8.0) in 20 mM HEPES and 500 mM 
NaCl. 

The 50 mM imidazole eluate containing the soluble 
GP-1 complex was diluted with nine volumes of 20 mM 

20 NaPO^ (pH 7.0) and applied to an S-Sepharose 

(Phannacia) column equilibrated in 20 mM NaPO^ (pH 7.0) 
with 50 mM NaCl. The S-Sepharose resin was loaded with 
an equivalent of 800 mL of starting conditioned media 
per mL of resin. After loading the S-Sepharose column 

25 was washed with equilibration buffer and eluted with 

100 mM NaCl followed by 300 mM and 500 mM NaCl in 20 mM 
NaPO^ (pH 7.0). The 300 mM NaCl pool was further 
purified using gel filtration chromatography. Fifty 
mis of the 300 mm NaCl eluate was applied to a 5.0 X 90 

30 cm Sephacryl S-200HR (Pharmacia) equilibrated in Tris 
buffered saline (TBS), 50 mM Tris, 150 mM NaCl 
(pH 7.4). The column was eluted at a flow rat« of 5 
mL/minute collecting 10 mL fractions. The apparent 
molecular of the soluble OP-1 was determined by 

35 comparison to protein molecular weight standards 



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10 



- 86 - 

(alcohol dehydrogenase {ADH, 150 kDa), bovine serum 
albumin (BSA, 68 kDa), carbonic anhydrase (CA, 30 kDa) 
and cytochrome C (cyt C, 12.5 KDa). The purity of the 
S-200 column fractions was determined by separation on 
standard 15% polyacrylamide SDS gels stained with 
coomassie blue. The identity of the mature OP-1 and 
the pro-domain was determined by N-terminal sequence 
analysis after separation of the mature OP-1 from the 
pro-domain using standard reverse phase C18 HPLC. 



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



The complex components can be verified by running 

20 the complex-containing fraction from the S-200 or S- 

200HR columns over a reverse phase C18 HPLC column and 
eluting in an acetonitrile gradient (in 0.1% TFA)/ 
using standard procedures. The complex is dissociated 
by this step, and the pro domain and mature species 

25 elute as separate species. These separate species then 
can be subjected to N-terminal sequencing using 
standard procedures (see, for example. Guide to 
Protein Purification , M. Deutscher, ed., Academic 
Press, San Diego, 1990, particularly pp. 602-613), and 

30 the identity of the isolated 36kD, 39kDa proteins 

confirmed as mature morphogen and isolated, cleaved pro 
domain, respectively. N-terminal sequencing of the 
isolated pro domain from mammalian cell produced OP-1 
revealed 2 forms of the pro region, the intact form 

35 (beginning at residue 30 of Seq. ID No. 16) and a 



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truncated fom, (beginning at residue 48 of Seq. ID No. 
16.) N-terminal sequencing of the polypeptide subunit 
of the isolated mature species reveals a range of 
N- termini for the mature sequence, beginning at 
5 residues 293, 300, 313, 315, 316, and 318, of Seq. ID 
No. 16, all of which are active as demonstrated by the 
standard bone induction assay. 

V.A.2. In Vitro Soluble Morphogen Complex Formation 

10 

As an alternative to purifying soluble complexes 
from culture media or a body fluid, soluble complexes 
may be formulated from purified pro domains and mature 
dimeric species. Successful complex formation 

15 apparently requires association of the components under 
denaturing conditions sufficient to relax the folded 
structure of these molecules, without affecting 
disulfide bonds. Preferably, the denaturing conditions 
mimic the environment of an intracellular vesicle 

20 sufficiently such that the cleaved pro domain has an 
opportunity to associate with the mature dimeric 
species under relaxed folding conditions. The 
concentration of denaturant in the solution then is 
decreased in a controlled, preferably step-wise manner, 

25 so as to allow proper refolding of the dimer and pro 
regions while maintaining the association of the pro 
domain with the dimer. Useful denaturants include 4-6M 
urea or guanidine hydrochloride (GuHCl), in buffered 
solutions of pH 4-10, preferably pH 6-8. The soluble 

30 complex then is formed by controlled dialysis or 
dilution into a solution having a final denaturant 
concentration of less than 0. 1-2M urea or GuHCl, 
preferably 1-2 M urea of GuHCl, which then preferably 
can be diluted into a physiological buffer. Protein 

35 purif ication/renaturing procedures and considerations 



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are well described in the art, and details for 
developing a suitable renaturing protocol readily can 
be determined by one having ordinary skill in the art. 
One useful text one the subject is Guide to Protein 
5 Purification , M. Deutscher, ed.^ Academic PresS/ San 
DiegO/ 1990, particularly section V. Complex formation 
also may be aided by addition of one or more chaperone 
proteins. 

10 V.A.3 Stability of Soluble Morphoqen Complexes 

The stability of the highly purified soluble 
morphogen complex in a physiological buffer, e.g., 
tris-buffered saline (TBS) and phosphate-buffered 

15 saline (PBS), can be enhanced by any of a number of 

means. Currently preferred is by means of a pro region 
that comprises at least the first 18 amino acids of the 
pro sequence (e.g., residues 30-47 of Seq. ID NO. 16 
for OP-1), and preferably is the full length pro 

20 region. Residues 30-47 show sequence homology to the 
N-terminal portion of other morphogens and are believed 
to have particular utility in enhancing complex 
stability for all morphogens. Other useful means for 
enhancing the stability of soluble morphogen complexes 

25 include three classes of additives. These additives 

include basic amino acids (e.g., L-arginine, lysine and 
betaine); nonionic detergents (e.g., Tween 80 or 
Nonldet P-120); and carrier proteins (e.g., serum 
albumin and casein). Useful concentrations of these 

30 additives include 1-100 mM, preferably 10-70 mM, 
including 50 mM, basic amino acid;, 0.01-1.0%, 
preferably 0.05-0.2%, including 0.1% (v/v) nonionic 
detergent;, and 0.01-1.0%, preferably 0.05-0.2%, 
including 0.1% (w/v) carrier protein. 

35 



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VI • Examples 

Example !• Identification of Morphogen^Expressing 
Tissue 

5 

Determining the tissue distribution of raorphogens 
may be used to identify different morphogens expressed 
in a given tissue, as well as to identify new, related 
morphogens. Tissue distribution also may be used to 

10 identify useful morphogen-producing tissue for use in 
screening and identifying candidate morphogen- 
stimulating agents. The morphogens (or their mRNA 
transcripts) readily are identified in different 
tissues using standard methodologies and minor 

15 modifications thereof in tissues where expression may 
be low. For example, protein distribution may be 
detennined using standard Western blot analysis or 
immunof lucre scent techniques, and antibodies specific 
to the morphogen or morphogens of interest. Similarly, 

20 the distribution of morphogen transcripts may be 
determined using standard Northern hybridization 
protocols and transcript-specific probes. 

Any probe capable of hybridizing specifically to a 
25 transcript, and distinguishing the transcript of 

interest from other, related transcripts may be used. 
Because the morphogens described herein share such high 
sequence homology in their active, C-terminal domains, 
the tissue distribution of a specific morphogen 
30 transcript may best be determined using a probe 

specific for the pro region of the immature protein 
and/or the N- terminal region of the mature protein. 
Another useful sequence is the 3' non-coding region 
flanking and immediately following the stop codon^ 
35 These portions of the sequence vary substantially among 



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the morphogens described herein, and accordingly, are 
specific for each protein. For example, a particularly 
useful Vgr-l-specif ic probe sequence is the PvuII-SacI 
fragment, a 265 bp fragment encoding both a portion of 
5 the untranslated pro region and the N- terminus of the 
mature sequence (see Lyons et al, (1989) PNAS 86:4554- 
4558 for a description of the cDNA sequence). 
Similarly, particularly useful mOP-1- specific probe 
sequences are the BstXl-Bgll fragment, a 0.68 Kb 

10 sequence that covers approximately two- thirds of the 
mOP-1 pro region; a StuI-StuI fragment, a 0.2 Kb 
sequence immediately upstream of the 7 -cysteine domain; 
and the Earl-Pstl fragment, an 0.3 Kb fragment 
containing a portion of the 3 'untranslated sequence 

15 (See Seq. ID No. 18, where the pro region is defined 
essentially by residues 30-291.) Similar approaches 
may be used, for example, with hOP-1 (Seq. ID No. 16) 
or human or mouse OP-2 (Seq. ID Nos. 20 and 22.) 

20 Using these morphogen- specific probes, which may be 

synthetically engineered or obtained from cloned 
sequences, morphogen transcripts can be identified in 
mammalian tissue, using standard methodologies well 
known to those having ordinairy skill in the art. 

25 Briefly, total RNA is prepared from various adult 
murine tissues (e.g., liver, kidney, testis, heart, 
brain, thymus and stomach) by a standard methodology 
such as by the method of Chomczyaski et al. ((1987) 
Anal. Biochem 162:156-159) and described below. Poly 

30 (A)+ RNA is prepared by using oligo (dT) -cellulose 
chromatography (e.g.. Type 7, from Phamacia LKB 
Biotechnology, Inc.). Poly (A)+ RNA (generally 15 fjq) 
from each tissue is fractionated on a 1% 
agarose/f ormaldehyde gel and transferred onto a Nytran 

35 membrane (Schleicher & Schuell). Following the 



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transfer^ the membrane is bated at 80*'C and the RNA is 
cross-linked under UV light (generally 30 seconds at 1 
mW/cm^ ) • Prior to hybridization, the appropriate probe 
is denatured by heating. The hybridization is carried 
5 out in a lucite cylinder rotating in a roller bottle 
apparatus at approximately 1 rev/min for approximately 
15 hours at 37**C using a hybridization mix of 40% 
formamide, 5 x Denhardts, 5 x SSPE, and 0.1% SDS. 
Following hybridization, the non-specific counts are 
10 washed off the filters in 0.1 x SSPE, 0.1% SDS at 50*^0 . 

Examples demonstrating the tissue distribution of 
various morphogens, including Vgr-1, OP-1, BMP2, BMP3, 
BMP4, BMP5, GDF-1, and OP-2 in developing and adult 

15 tissue are disclosed international application 
US92/01968 (W092/15323) , and in Ozkaynak, et al., 
(1991) Biochem. Biophys. Res. Commn . 179 x116-123, and 
Ozkaynak, et al. (1992) ( J. Biol. Chem. 267 ; 
25220-25227), the disclosures of which are incorporated 

20 herein by reference. Using the general probing 
methodology described herein, northern blot 
hybridizations using probes specific for these 
morphogens to probe brain, spleen, lung, heart, liver 
and kidney tissue indicate that kidney-related tissue 

25 appears to be the primary expression source for OP-1, 
with brain, heart and lung tissues being secondary 
sources. Lung tissue appears to be the primary tissue 
expression source for Vgr-1, BMP5, BMP4 and BMP3. Lower 
levels of Vgr-1 also are seen in kidney and heart 

30 tissue, while the liver appears to be a secondary 

expression source for BMP5, and the spleen appears to 
be a secondary expression source for BMP4. GDF-1 
appears to be expressed primarily in brain tissue. To 
date, OP-2 appears to be expressed primarily in early 

35 embryonic tissue. Specifically, northern blots of 



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murine embryos and 6-day post-natal animals shows 
abundant 0P2 expression in 8 -day embryos. Expression 
is reduced significantly in 17-day embryos and is not 
detected in post-natal animals. 

5 

Example 2. Morphogen Localization in Developing 
Hepatic Tissue 

The onset of liver formation in a developing embryo 

10 occurs at day 14. Using the hybridization protocol 
described in Example 1, morphogen expression was 
identified at the onset of liver formation during 
embryo development. Specifically, northern blots of 
mRNA isolated from murine embryo liver tissue (probed 

15 at 15 days and 20 days) and post natal mouse liver 
tissue (probed at 7, 14, 21 and 28 days past birth) 
show mOP-1 expression in developing liver tissue only 
during the time of liver formation. Specifically, as 
illustrated, in Fig. 1, mOP-1 RNA is expressed 

20 significantly in the 15 day embryo, and is present at 
much lower amounts at later times in healthy hepatic 
tissue. In the figure, lanes 2 and 3 contain RNA from 
15- and 20-day embryo tissue, respectively; lanes 4-8, 
RNA from 3, 7, 14, 21 and 28 days post natal animals, 

25 respectively; and lane 9 is a molecular weight ladder. 
Lanes 1 and 9 are markers. In the Northern blot mOP-1 
RNA appears as a discrete band running at about 4kb and 
2.2 or 2.4 kb, as well as a shorter band at 1.8kb (see, 
for example, Ozkaynak, et al. (1991) Biochem. Biophys 

30 Res. 179 ; 116-123.) 



35 



Example 3. Mitogenic Effect of Morphogen on 
Rat Hepatocytes 

The ability of a morphogen to induce proliferation 
of primary hepatocytes may be demonstrated in vitro 
using the following assay using primary hepatocytes 



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isolated from rat liver. Unless otherwise indicated, 
all chemicals referenced are standard, commercially 
available reagents, readily available from a number of 
sources, including Sigma Chemical, Co., St. Louis; 
5 Calbiochem, Corp., San Diego, and Aldrich Chemical Co., 
Milwaukee. 

Rat primary hepatocyte cultures were prepared by a 
two-step collagenase digestion essentially as described 

10 by Fausto et al. (1987) Cell Separation: Methods and 
Selected Applications 4:45-77 the disclosure of which 
is incorporated herein by reference. Briefly, the 
liver of a male rat (e.g., CD strain, Charles River 
Laboratories, Wilmington, MA) was perfused via the 

15 portal vein with Ca^^free and Mg^* free Hank's balanced 
salt solution for 10 min at a flow of 30-40 ml/min, 
followed by perfusion with 0.05% collagenase in 
Ca^ * -containing medium (Hepes buffer) for 10 min. The 
liver capsule was removed, the cells shaken loose from 

20 the tissue and filtered hepatocytes were collected by 
repeated centrifugation of the cell suspension at 50 xg 
for 25 min. Hepatocyte suspensions were virtually free 
of non-parenchymal cell contamination. Cells (2x10* 
per dish) were plated on 35-mm dishes coated with rat 

25 tail collagen in MEM (modified Eagle's Medium, 

Gibco, Long Island) containing 5% fetal bovine serum 
(FBS), ImM pyuvate, 0.2raM aspartate, ImM proline, 0.2mM 
serine, 2mM glutamine, and 0.5 fjg of hydrocotisone and 
1 pg of insulin per ml. The cells were incubated for 

30 24 hours under standard at 37 "^C, at which time the 
growth medium was replaced with serum-free MEM. 



35 



The cell culture then was divided into two groups: 
(1) wells which received morphogen within the dose 
range of 1-100 ng of morphogen per ml meditm; and (2) 



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the control group ^ which received no additional 
factors. In this example, OP-1 was the morphogen 
tested. The cells then were incubated for an 
additional 18*24 hours after which the wells were 
5 pulsed with 2/iCi/well of ^ H-thymidine and incubated for 
six more hours. The excess label then was washed off 
with a cold solution of 0.15 M NaCl. 250 ^1 of 10% 
tricholoracetic acid then was added to each well and 
the wells incubated at room temperature for 30 minutes. 

10 The cells then were washed three times with cold 

distilled water, and lysed by the addition of 250 pi of 
1% sodium dodecyl sulfate (SDS) for a period of 30 
minutes at 37 •C. The cell ly sates then were harvested 
using standard means well known in the art, and the 

15 incorporation of ^H-thymidine into cellular DNA was 

determined by liquid scintillation as an indication of 
mitogenic activity of the cells. 

Morphogen treatment of primary hepatocyte cultures 
20 significantly stimulates ^ H-thymidine incorporation 
into DNA, and thus promotes their cell proliferation. 
The mitogenesis stimulated by 20 ng of OP-1 in 1 ml 
serum-free medium was equivalent to the mitogenic 
effect of 10% fresh senim alone. By contrast, other 
25 local-acting growth factors, such as TGF-p do not 
stimulate proliferation of primary hepatocytes (see 
Fausto et al. (1991) Ciba Found Symp 157 ; 165-174 . ) 

Example 4. Morphogen-Induced Liver Regeneration 

30 

While hepatocytes have a remarkable capacity to 
undergo compensatory growth following tissue loss, the 
reparative properties of liver differ significantly 
from embryonic morphogenesis. Specifically, following 



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a partial hepatectomy wherein a liver lobe is partially 
or completely removed, the remaining intact lobes grow 
rapidly and double in weight due to the ability of the 
differentiated hepatocytes in the intact lobe to 
5 undergo limited proliferation. However, the excised 
lobe itself is not regenerated. The following example 
demonstrates the ability of morphogens to regenerate 
lost hepatic tissue following a partial hepatectomy, 
including regenerating the excised tissue lobe. The 
10 protocol described below is a variation on a standard 
partial hepatectomy protocol, described, for example, 
by Higgins et al. (1931) Arch. Pathol. 12; 136-202 and 
Braun et al. (1989) PNAS 86:1558-1562, the disclosures 
of which are incorporated herein by reference. 

15 

Morphogen, e.g., purified recombinant human OP-1, 
mature form, was solubilized (1 mg/ml) in 50% ethanol 
(or compatible solvent) containing 0.1% trif luoroacetic 
acid (or compatible acid). The injectable OP-1 
20 solution was prepared by diluting one volume of 

OP-l/solvent-acid stock solution with 9 volumes of 0.2% 
rat serum albumin in sterile PBS (phosphate-buffered 
saline). 

25 Growing rats or aged rats were anesthetized by 

using ketamine. Two of the liver lobes (left and 
right) were cut out (approximately 1/3 of the lobe) and 
the morphogen was injected locally at multiple sites 
along the cut ends. The amount of OP-1 injected was 

30 100 fig in 100 of PBS/RSA (phosphate-buffered saline/rat 
seriim albumin) injection buffer. Placebo samples were 
injection buffer without OP-1. Five rats in each group 
were used. The wound was closed using standard 
surgical procedures and the rats were allowed to eat 

35 normal food and drink tap water. 



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After 12 days, the rats were sacrificed and liver 
regeneration was observed visually. The photomigraph 
in Fig. 2 illustrates dramatically the regenerative 
effects of OP-1 on liver tissue formation. In the 
5 figure, the arrow indicates the treated lobe. The 
OP-l-injected group showed complete liver tissue 
regeneration including reformation of the excised lobe 
tissue, and showed no sign of any cut in the liver 
(animal 2). By contrast, in the control group into 
10 which only PBS was injected, the excised lobe tissue 
was not regenerated (animal 1). The original incision 
remains in this sample. 

In a related experiment, animals were partially 
15 hepatectomized or sham-operated and Northern blot 
analysis performed on RNA isolated from the liver 
tissue. None of the animals were morphogen- treated. 
As determined by Northern blot analysis (probed with 
mOP-l-specif ic labeled oligonucleotide, see Fig. 3), in 
20 the absence of morphogen treatment, the level of 
endogenous morphogen is not enhanced significantly 
following partial hepatectomy. In the figure lanes 2, 
4, 6, 8, 10, 12, and 14, are samples from partially 
hepatectomized rats and lanes 3, 5, 7, 9, 11, 13, and 
25 15 are samples from sham-operated rats, and lanes 1 and 
16 are markers. Samples were taken at 6 hour intervals 
between 12 and 96 hours post surgery. 



Example 5. Morphogen Expression in Regenerating Liver 
30 Tissue Following Toxin-Induced 

Tissue Damage 



Hepatic tissue repair following toxic agent-induced 
damaged tissue involves proliferation and 



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differentiation of hepatocyte precursor cells. This 
tissue reparation apparently mimics the tissue 
morphogenesis cascade that occurs during embryogenesis 
(FaustO/ et al.(1989) Lab > Investigation 60 :4-13) > As 
5 demonstrated in the example below, morphogen expression 
is enhanced significantly during hepatic tissue 
regeneration following galactosamine or carbon 
tetrachloride (CCl^ )- induced liver damage. Experiments 
were performed essentially as described in Kuhlmann et 
10 al., (1980) Virchows Arch 387 ; 47-57, the disclosure of 
which is incorporated herein by reference . 

In this experiment, male rats were provided with a 
single intraperitoneal injection of galactosamine-HCl 

15 0.75 g/.kg body weight on day 0, and morphogen 

expression monitored by standard Northern blot of liver 
tissue samples taken on days 1-7 and day 10. OP-1 
expression was significantly enhanced during this 
hepatic tissue regenerative period, indicating that 

20 morphogens play a significant role in tissue 

regeneration. A representation of the Northern blot is 
presented in Fig. 4. In Fig. 4, lanes 1-8 are samples 
taken on days 0-7; lane 9 is a sample taken on day 10, 
and lane 10 contains molecular weight markers. OP-1 

25 mRNA shows a significant expression spike on days 3-7. 
Similar results were seen with tissue regeneration 
stimulated following CCl^-induced tissue, wherein GCl^ 
intoxication is induced by orally administering 1.5g 
CCl^/kg body weight. Significant morphogen expression 

30 (mOP-1 mRNA, as determined by standard Northern blot) 
is identified by a hybridization spike at 12 hours and 
continuing through at least 72 hours. 



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Example 6. Morphoqen Inhibition of Cellular and 
Humoral Inflammatory Response 

The morphogens described herein may be used to 
5 alleviate tissue damage associated with immune 

response-mediated damage to liver tissue. Details of 
this damage and the use of morphogens to alleviate this 
injury as well as to provide a cytoprotective effect in 
anticipation of this injury for example, during a 

10 transplant procedure, are disclosed in international 
application US92/07358 (WO93/04672 ) . A primary source 
of such damage to hepatic tissue results, for example, 
from reduced perfusion of the hepatic blood supply 
and/or from partial or complete occlusion of the portal 

15 vein. As described in international application 

US92/07358 (WO93/04672) morphogens have been shown to 
alleviate damage to myocardial tissue following 
ischemia-reperfusion injury. The morphogens also 
alleivate analogous tissue damage to hepatic tissue. 

20 

Morphogens described herein inhibit multinucleation 
of mononuclear phagocytic cells under conditions where 
these cells normally would be activated, e.g., in 
response to a tissue injury or the presence of a 

25 foreign substance. For example, in the absence of 
morphogen, an implanted substrate material (e.g., 
implanted subcutaneously ) composed of, for example, 
mineralized bone, a ceramic such as titanium oxide or 
any other substrate that provokes multinucleated giant 

30 cell formation, rapidly becomes surrounded by 

multinucleated giant cells, e.g., activated phagocytes 
stimulated to respond and destroy the foreign object. 
In the presence of morphogen however, the recruited 
cells remain in their mononuclear precursor form and 



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the matrix material is undisturbed* Figure 5 
illustrates this effect of morphogens, in a schematic 
representation of histology results of a titanium oxide 
substrate implanted subcutaneously . In the figure^ 
5 "mg" means multinucleated giant cells and "ob" means 
osteoblasts. The substrate represented in Fig. 5B was 
implanted together with morphogen (0P*1) and newly 
formed osteoblasts are evident surrounding the 
substrate. By contrast/ the substrate represented in 

10 Fig. 5 A was implanted without morphogen and extensive 
multinucleated giant cell formation is evident 
surrounding the substrate. Accordingly, the 
morphogens' effect in inhibiting excessive bone mass 
loss in a mammal also may include inhibiting activation 

15 of these giant cells. 

In addition, the morphogens described herein also 
suppress antibody production stimulated in response to 
a foreign antigen in a mammal. Specifically, when 

20 bovine bone collagen matrix alone was implanted in a 

bony site in a rat, a standard antibody response to the 
collagen is stimulated in the rat as determined by 
standard anti-bovine collagen ELISA experiments 
performed on blood samples taken at four week intervals 

25 following implantation (e.g., between 12 and 20 weeks.) 
Serum anti-collagen antibody titers, measured by ELISA 
essentially following the procedure described by 
Nagler-Anderson et al, (1986) PNAS 83:7443-7446, the 
disclosure of which is incorporated herein by 

30 reference, increased consistently throughout the 

experiment. However, when the matrix was implanted 
together with a morphogen (e.g., OP-1, dispersed in the 
matrix and adsorbed thereto, essentially as described 
in U.S. Pat. No. 4,968,590) anti-bovine collagen 



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10 



- 100 - 

antibody production was suppressed significantly. This 
ability of morphogen to suppress the humoral response 
is further evidence of morphogen utility in alleviating 
tissue damage associated with autoimmune diseases, 
including autoantibody diseases, such as rheumatoid 
arthritis. 

Example 7. Morphogen Effect on Fibrogenesis and Scar 
Tissue Formation 



The morphogens described herein induce tissue 
morphogenesis of damaged or lost tissue. The ability 
of these proteins to regenerate new tissue also is 
enhanced by the anti-inflammatory effect of these 

15 proteins. Provided below are a series of in vitro 

experiments demonstrating the ability of morphogens to 
induce migration and accumulation of mesenchymal cells. 
In addition, the experiments demonstrate that 
morphogens, unlike TGF-p, do not stimulate 

20 fibrogenesis or scar tissue formation. Specifically, 
morphogens do not stimulate production of collagen, 
hyaluronic acid (HA) or metalloproteinases in primary 
fibroblasts, all of which are required for fibrogenesis 
or scar tissue formation. By contrast, TGF-p, a known 

25 inducer of fibrosis, but not of tissue morphogenesis as 
described herein, does stimulate production of these 
fibrosis markers. 



Chemotaxis and migration of mesenchymal progenitor 
30 cells were measured in modified Boyden chambers 

essentially as described by Fava, R.A. et al (1991) J. 
Exp. Med. 173 ; 1121-1132, the disclosure of which is 
incorporated herein by reference, using polycarbonate 
filters of 2, 3 and 8 micron ports to measure migration 



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of progenitor neutrophils, monocytes and fibroblasts. 
Chemotaxis was measured over a range of morphogen 
concentrations, e.g., 10"^°M to 10"^^M OP-1. For 
progenitor neutrophils and monocytes, 10'^®-10"^'m OP-1 
5 consistently induced maximal migration, and 10"^* to 
10" ^^M OP-1 maximally induced migration of progenitor 
fibroblasts. In all cases the chemotactic activity 
could be inhibited with anti-OP-1 antibody. Similar 
migration activities also were measured and observed 
10 with TGF-p. 

The effect of morphogen on f ibrogenesis was 
determined by evaluating fibroblast production of 
hyaluronic acid (HA), collagen, collagenese and tissue 
15 inhibitor of metalloproteinases (TIMP). 

Human fibroblasts were established from explants of 
infant foreskins and maintained in monolayer culture 
using standard culturing procedures. (See, for 

20 example, (1976) J. Exp. Med . 144: 1188-1203.) Briefly, 
fibroblasts were grown in maintenance medium consisting 
of Eagle's MEM, supplemented with nonessential amino 
acids, ascorbic acid (50 /jg/ml), NaHCO^ and HEPES 
buffers (pH 7.2), penicillin (100 U/ml), streptomycin 

25 (100 pg/ml), amphotericin B (1 /jg/ml) and 9% heat 

inactivated FCS. Fibroblasts used as target cells to 
measure chemotaxis were maintained in 150 mm diameter 
glass petri dishes. Fibroblasts used in assays to 
measure synthesis of collagen, hyaluronic acid, 

30 collagenase and tissue inhibitors of metalloproteinases 
(TIMP) were grown in 100 mm diameter plastic tissue 
culture petri dishes. 



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The effects of morphogen on fibroblast production 
of hyaluronic acid^ collagens, collagenase and TIMP 
were determined by standard assays (See^ for example, 
Posttethwaite et al. (1989) J, Clin, Invest s 83: 629- 
5 636, Posttethwaithe (1988) J>/ Cell Biol . 106 ; 311-318 
and Clark et al (1985) Arch> Bio-chem Biophys. 241 ; 36- 
44, the disclosures of which are incorporated by 
reference. } For these assays, fibroblasts were 
transferred to 24-well tissue culture plates at a 

10 density of 8 x 10^ cells per well. Fibroblasts were 
grown confluency in maintenance medi\im containing 9% 
FCS for 72 h and then grown in serum-free maintenance 
medium for 24 h. Medium was then removed from each 
well and various concentrations of OP-1 (recombinantly 

15 produced mature or soluble form) or TGF-p-1 (R&D 
Systems, Minneapolis) in 50 fjl PBS were added to 
triplicate wells containing the confluent fibroblast 
monolayers. For experiments that measured production 
of collagenase and TIMP, maintenance medium (450 pi) 

20 containing 5% FCS was added to each well, and culture 
supernatants were harvested from each well 48 h later 
and stored at -70° C until assayed. For experiments 
that assessed HA production, maintenance medium (450 
pi) containing 2.5% FCS was added to each well, and 

25 cultures grown for 48 h. For experiments that measured 
fibroblast production of collagens, serum-free 
maintenance medium (450 /il) without non-essential amino 
acids was added to each well and cultures grown for 72 
h. Fibroblast production of HA was measured by 

30 labeling newly synthesized glycosaminoglycans (GAG) 
with t^H] -acetate the last 24 h of culture and 
quantitating released radioactivity after incubation 
with hyaluronidase from Streptomyces hyalurolyticus 
(ICN Biochemicals, Cleveland, OH) which specifically 



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degrades hyaluronic acid. Production of total collagen 
by fibroblasts was measured using a collagenase- 
sensitive protein assay that reflects [^HJ-proline 
incorporation the last 24 h of culture into newly 
5 synthesized collagens. Collagenase and TIMP protein 
levels in fibroblast cultures supernatants was measured 
by specific ELISAs. 

As shown in Fig. 6, OPl does not stimulate 
10 significant collagen or HA production, as compared with 
TGF-p. In the figure, panel A shows OP-1 effect on 
collagen production, panel B shows TGF-p effect on 
collagen production, and panels C and D show OP-1 
(panel C) and TGF-^ (panel D) effect on HA production. 
15 The morphogen results were the same whether the soluble 
or mature form of OPl was used. By contrast, the 
latent form of TGF-p (e.g., pro domain-associated form 
of TGF-p) was not active. 

20 Example 8. Liver Tissue Diagnostics 

Morphogen localization in developing and 
regenerating liver tissue can be used as part of a 
method for diagnosing a liver function disorder in 

25 vivo. The method may be particularly advantageous for 
diagnosing early stages of a liver dysfunction 
associated with a hepatocellular injury. Specifically, 
a biopsy of liver tissue is performed on a patient at 
risk, using standard procedures known in the medical 

30 art. Morphogen expression associated with the biopsied 
tissue then is assessed using standard methodologies, 
as by immunolocalization, using standard 
immunofluorescence techniques in concert with 
morphogen- specif ic antisera or monoclonal antibodies. 



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Specif ically^ the biopsied tissue is thin sectioned 
using standard methodologies known in the art, and 
fluorescently labelled (or otherwise detectable) 
antibodies having specificity for the morphogen are 
5 incubated with the tissue under conditions sufficient 
to allow specific antigen-antibody complex fonaation. 
The presence and quantity of complex formed then is 
detected and compared with a predetermined standard or 
reference value. Detection of altered levels of 

10 morphogen present in the tissue then may be used as an 
indicator of tissue dysfunction. Alternatively^ 
fluctuation in morphogen levels may be assessed by 
monitoring morphogen transcription levels, either by 
standard Northern blot analysis or by in sfitu 

15 hybridization, using a labelled probe capable of 

hybridizing specifically to morphogen RNA and standard 
RNA hybridization protocols well described in the art 
and as described in Examples 1, 2, 5 and 6. 

20 Fluctuations in morphogen levels present in the 

bloodstream or peritoneal fluid also may be used to 
evaluate liver tissue viability. For example, 
morphogens are detected associated with regenerating 
liver tissue and/or may be released from dying cells 

25 into surrounding peritoneal fluid. OP-1 recently has 
been identified in human blood, which also may be a 
means of morphogen transport. 

Serum samples may be obtained by standard 
30 venipuncture and serum prepared by centrifugation at 

3,000 RPM for ten minutes. Similarly, peritoneal fluid 
samples may be obtained by a standard fluid extraction 
methodology. The presence of morphogen in the serum or 
peritoneal fluid then may be assessed by standard 



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Western blot ( immunoblot ) ^ ELISA or RIA procedures. 
Briefly/ for example, with the ELISA, samples may be 
diluted in an appropriate buffer, such as phosphate- 
buffered saline, and 50 pi aliquot s allowed to absorb 
5 to flat bottomed wells in microtitre plates pre-coated 
with morphogen-specif ic antibody, and allowed to 
incubate for 18 hours at 4'*C. Plates then may be 
washed with a standard buffer and incubated with 50 //l 
aliquots of a second morphogen-specif ic antibody 
10 conjugated with a detecting agent, e.g., biotin, in an 
appropriate buffer, for 90 minutes at room temperature. 
Morphogen-antibody complexes then may be detected using 
standard procedures. 

15 Alternatively, a morphogen-specif ic affinity column 

may be created using, for example, morphogen-specif ic 
antibodies adsorbed to a coliunn matrix, and passing the 
fluid sample through the matrix to selectively extract 
the morphogen of interest. The morphogen then is 

20 eluted. A suitable elution buffer may be determined 
empirically by determining appropriate binding and 
elution conditions first with a control (e.g., 
purified, recombinantly-produced morphogen. ) Fractions 
then are tested for the presence of the morphogen by 

25 standard immunoblot. Morphogen concentrations in serum 
or other fluid samples then may be determined using 
standard protein quantification techniques, including 
by spectrophotometric absorbance or by quantitation by 
ELISA or RIA antibody assays. Using this procedure, 

30 OP-1 has been identified in senim. 



OP-1 was detected in human serum using the 
following assay. A monoclonal antibody raised against 
mammalian, recombinant ly produced OP-1 using standard 



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immunology techniques well described in the art and 
described generally in Example 13, was immobilized by 
passing the antibody over an activated agarose gel 
{e.g., Affi-Gel^**, from Bio-Rad Laboratories , Richmond, 
5 CA, prepared following manufacturer's instructions), 
and used to purify OP-1 from serum. Human serum then 
was passed over the column and eluted with 3M 
K-thiocyanate . K-thiocyanante fractions then were 
dialyzed in 6M urea, 20mM PO^, pH 7.0, applied to a C8 

10 HPLC column, and eluted with a 20 minute, 25-50% 

acetonitrile/0. 1% TFA gradient. Mature, recombinantly 
produced OP-1 homodimers elute between 20-22 minutes. 
Accordingly, these fractions from the affinity -purified 
human seirum sample were collected and tested for the 

15 presence of OP-1 by standard immunoblot using an 
OP-l-specifc antibody, and the protein identity 
confirmed by N- terminal sequencing. 

Morphogens may be used in diagnostic applications 
20 by comparing the quantity of morphogen present in a 

body fluid sample with a predetermined reference value, 
with fluctuations in fluid morphogen levels indicating 
a change in the status of liver tissue. Alternatively, 
fluctuations in the level of endogenous morphogen 
25 antibodies may be detected by this method, most likely 
in serum, using an antibody or other binding protein 
capable of interacting specifically with the endogenous 
morphogen antibody. Detected fluctuations in the 
levels of the endogenous antibody may be used as 
30 indicators of a change in tissue status. 



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Example 9. Screening Assay for Candidate Compounds 
which Alter Endogenous Morphogen Levels 

Candidate compound(s) which may be administered to 
5 affect the level of a given morphogen may be found 

using the following screening assay, in which the level 
of morphogen production by a cell type which produces 
measurable levels of the morphogen is determined with 
and without incubating the cell in culture with the 

10 compound, in order to assess the effects of the 

compound on the cell's production of morphogen. This 
can be accomplished by detection of the morphogen 
either at the protein or RNA level. A more detailed 
description also may be found in international 

15 application US92/07359 (WO93/05172 ) . 

9.1 Growth of Cells in Culture 

Cell cultures of kidney, adrenals, urinary bladder, 
20 brain, or other organs, may be prepared as described 
widely in the literature. For example, kidneys may be 
explanted from neonatal or new born or young or adult 
rodents (mouse or rat) and used in organ culture as 
whole or sliced (1-4 ram) tissues. Primary tissue 
25 cultures and established cell lines, also derived from 
kidney, adrenals, urinary, bladder, brain, mammary, or 
other tissues may be established in multiwell plates (6 
well or 24 well) according to conventional cell culture 
techniques, and are cultured in the absence or presence 
30 of serum for a period of time (1-7 days). Cells may be 
cultured, for example, in Dulbecco's Modified Eagle 
medium (Gibco, Long Island, NY) containing serum (e.g., 
fetal calf serum at 1%-10%, Gibco) or in serum-deprived 
medixim, as desired, or in defined mediiim (e.g.. 



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containing insulin, transferrin, glucose, albumin, or 
other growth factors ) . 

Samples for testing the level of morphogen 
5 production includes culture supernatants or cell 

lysates, collected periodically and evaluated for OP-1 
production by immunoblot analysis (Sambrook et al., 
eds., 1989, Molecular Cloning, Cold Spring Harbor 
Press, Cold Spring Harbor, NY), or a portion of the 

10 cell culture itself, collected periodically and used to 
prepare polyA+ RNA for mRNA analysis. To monitor de 
novo OP-1 synthesis, some cultures are labeled 
according to conventional procedures with an 
^^S-methionine/^^S-cysteine mixture for 6-24 hours and 

15 then evaluated to OP-1 synthesis by conventional 
immunoprecipitation methods. 

9.2 Determination of Level of Morphogenic Protein 

20 In order to quant itate the production of a 

morphogenic protein by a cell type, an immunoassay may 
be performed to detect the morphogen using a polyclonal 
or monoclonal antibody specific for that protein. For 
example, OP-1 may be detected using a polyclonal 

25 antibody specific for OP-1 in an ELISA, as follows. 

1 ^/g/100 fjl of affinity-purified polyclonal rabbit 
IgG specific for OP-1 is added to each well of a 
96-well plate and incubated at 37*^0 for an hour. The 
30 wells are washed four times with 0.167M sodiiam borate 
buffer with 0.15 M NaCl (BSB), pH 8.2, containing 0.1% 
Tween 20. To minimize non-specific binding, the wells 
are blocked by filling completely with 1% bovine serum 
albumin (BSA) in BSB and incubating for 1 hour at 37**C. 



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The wells are then washed four times with BSB 
containing 0,1% Tween 20, A 100 pi aliquot of an 
appropriate dilution of each of the test samples of 
cell culture supernatant is added to each well in 
5 triplicate and incubated at 37 for 30 min. After 
incubation, 100 fjl biotinylated rabbit anti-OP-1 serum 
(stock solution is about 1 mg/ml and diluted 1:400 in 
BSB containing 1% BSA before use) is added to each well 
and incubated at 37**C for 30 min. The wells are then 

10 washed four times with BSB containing 0.1% Tween 20* 
100 fjl strepavidin-alkaline (Southern Biotechnology 
Associates, Inc. Birmingham, Alabama, diluted 1:2000 in 
BSB containing 0.1% Tween 20 before use) is added to 
each well and incubated at 37*^0 for 30 min. The plates 

15 are washed four times with 0.5M Tris buffered Saline 
(TBS), pH 7.2. 50pl substrate (ELISA Amplification 
System Kit, Life Technologies, Inc., Bethesda, MD) is 
added to each well and incxxbated at room temperature 
for 15 min. Then, 50 fjl amplifier (from the same 

20 amplification system kit) is added and incubated for 
another 15 min at room temperature. The reaction is 
stopped by the addition of 50 /il 0.3 M sulphuric acid. 
The OD at 490 nm of the solution in each well is 
recorded. To quant itate OP-1 in culture media, a OP-1 

25 standard curve is performed in parallel with the test 
samples. 

Polyclonal antibody may be prepared as follows. 
Each rabbit is given a primary immunization of 100 
30 ug/500 pi E. coli produced OP-1 monomer (amino acids 
328-431 in SEQ ID NO: 5) in 0.1% SDS mixed with 500 /il 
Complete Freund's Adjuvant. The antigen is injected 
subcutaneous ly at multiple sites on the back and flanks 
of the animal. The rabbit is boosted after a month in 



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the same manner using incomplete Freund's Adjuvant. 
Test bleeds are taken from the ear vein seven days 
later. Two additional boosts and test bleeds are 
performed at monthly intervals until antibody against 
5 OP-1 is detected in the serum using an ELISA assay. 
Then/ the rabbit is boosted monthly with 100 fjg of 
antigen and bled (15 ml per bleed) at days seven and 
ten after boosting. 

10 Monoclonal antibody specific for a given morphogen 

may be prepared as follows. A mouse is given two 
injections of E. coli produced OP-1 monomer. The first 
injection contains 100/jg of OP-1 in complete Freund's 
adjuvant and is given subcutaneously. The second 

15 injection contains 50 pg of OP-1 in incomplete adjuvant 
and is given intraperitoneally. The mouse then 
receives a total of 230 ^g of OP-1 (amino acids 307-431 
in SEQ ID NO: 5) in four intraperitoneal injections at 
various times over an eight month period. One week 

20 prior to fusion, the mouse is boosted intraperitoneally 
with 100 fjg of OP-1 (307-431) and 30 fjg of the N- 
terminal peptide ( Ser2g3*Asn2Qg-Cys) conjugated through 
the added cysteine to bovine serum albumin with SMCC 
crosslinking agent. This boost was repeated five days 

25 (IP)/ four days (IP)/ three days (IP) and one day (IV) 
prior to fusion. The mouse spleen cells are then fused 
to myeloma (e.g., 653) cells at a ratio of 1:1 using 
PEG 1500 (Boeringer Mannheim), and the cell fusion is 
plated and screened for OP-l-specific antibodies using 

30 OP-1 (307-431) as antigen. The cell fusion and 

monoclonal screening then are according to standard 
procedures well described in standard texts widely 
available in the art. 



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The invention may be embodied in other specific 
forms without departing from the spirit or essential 
characteristics thereof • The present embodiments are 
5 therefore to be considered in all respects as 

illustrative and not restrictive, the scope of the 
invention being indicated by the appended claims rather 
than by the foregoing description / and all changes 
which come within the meaning and range of equivalency 
10 of the claims are therefore intended to be embraced 
therein. 



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SEQUENCE LISTING 



(1) GENERAL INFORMATION: 

(i) APPLICANT: 

(A) NAHE: CREATIVE BIOHOLECULES, INC. 

(B) STREET: 45 SOOTH STREET 

(C) CITY: HOPKINTON 

(D) STATE: MA 

(E) COUNTRY: USA 

(F) POSTAL CODE (ZIP): 01748 

(G) TELEPHONE: 1-508-435-9001 

(H) TELEFAX: 1-508-435-0454 

(I) TELEX; 

(ii) TITLE OF INVENTION: MORPHOGEN- INDUCED LIVER REGENERATION 
(iii) NUMBER OF SEQUENCES: 33 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: CREATIVE BIOHOLECULES, INC. 

(B) STREET: 45 SOUTH STREET 

(C) CITY: HOPKINTON 

(D) STATE: MA 

(E) COUNTRY: USA 

(F) ZIP: 01748 

(V) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

(C) OPERATING SYSTEM: PC-DOS/MS-DOS 

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 

(vi) CURRENT APPLICATION DATA: 

(A) APPLICATION NUMBER: 

(B) FILING DATE: 

(C) CLASSIFICATION: 

(vii) PRIOR APPLICATION DATA: 

(A) APPLICATION NUMBER: 

(B) FILING DATE: 

(viii) ATTORNEY/AGENT INFORMATION: 

(A) NAME: KELLEY ESQ, ROBIN D. 

(B) REGISTRATION NUMBER: 34,637 

(C) REFERENCE/DOCKET NUMBER: CRP-072 

(ix) TELECOMMUNICATION INFORMATION: 

(A) TELEPHONE: 617/248-7477 

(B) TELEFAX: 617/248-7100 



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10 



40 



(2) INFORHATION FOR SEQ ID N0:1: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acid 

(C) STRANDEONESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



(ix) FEATURE: 

(A) NAHE/KEY: Protein 

(B) LOCATION: 1..97 

15 (D) OTHER INFORHATION: /label= GENERIC-SEQl 

/note= "WHEREIN EACH XAA INDEPENDENTLY INDICATES 
ONE OF THE 20 NATURALLY-OCCURING L-ISOMER, A-AMINO 
ACIDS, OR A DERIVATIVE THEREOF." 

20 

(zi) SEQUENCE DESCRIPTION: SEQ ID N0:1: 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
25 ' ' 10 15 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 
20 25 30 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
30 35 40 45 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa 
50 55 60 

35 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

65 70 75 80 



Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 
85 90 95 

Xaa 



(2) INFORHATION FOR SEQ ID NO: 2: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



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(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..97 

(D) OTHER INFORMATION: /labels GENERIC-S£q2 
5 /note= "WHEREIN EACH XAA INDEPENDENTLY INDICATES 

ONE OF THE 20 NATURALLY OCCURING L-ISOMER A-AMINO 
ACIDS, OR A DERIVATIVE THEREOF." 



10 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2: 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
15 10 15 

15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 

20 25 30 



20 



Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
35 40 45 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa 
50 55 60 



Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
25 65 70 75 80 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 
85 90 95 

30 Xaa 



(2) INFORHATION FOR SEQ ID NO: 3: 

35 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



40 



(ii) MOLECULE TYPE: protein 



(ix) FEATURE: 
45 (A) NAME/KEY: Protein 

(B) LOCATION: 1..97 

(D) OTHER INFORMATION: /labels GENERIC-SEQ3 

/note= "WHEREIN EACH XAA IS INDEPENDENTLY SELECTED 
FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS 
50 AS DEFINED IN THE SPECIFICATION." 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: 

55 Leu Tyr Val Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa Xaa Ala 

1 5 10 15 



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10 



Pro Xaa Gly Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro 
20 25 30 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Leu 
35 40 45 . 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro 
50 55 60 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
65 70 75 60 



Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Gly Cys 
15 85 90 95 

Xaa 



20 (2) INFORHATION FOR SEQ ID NO: 4: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

25 (C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



30 



(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

(D) OTHER INFORMATION: /label= GENERIC-SEQ4 
35 /note= "yHEREIN EACH XAA IS INDEPOTOENTLY SELECTED 

FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS 
AS DEFINED IN THE SPECIFICATION." 

40 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4: 

Cys Xaa Xaa Xaa Xaa Leu Tyr Val Xaa Phe Xaa Xaa Xaa Gly Trp Xaa 
15 10 15 

45 Xaa Trp Xaa Xaa Ala Pro Xaa Gly Xaa Xaa Ala Xaa Tyr Cys Xaa Gly 

20 25 30 



50 



Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Bis Ala 
35 40 45 

Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
50 55 60 



Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 
55 65 70 75 80 



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Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val 
85 90 95 

5 Xaa Xaa Cys Gly Cys Xaa 

100 

(2) INFORHATION FOR SEQ ID NO: 5: 

10 (i) SEQUEHCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



15 



35 



50 



(ii) MOLECULE TYPE: protein 



(vi) ORIGINAL SOURCE: 

(A) ORGANISM: Homo sapiens 
20 (F) TISSUE TYPE: HIPPOCAMPUS 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..139 

25 (D) OTHER INFORMATION: /label* hOPl-MATURE 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 

30 Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro Lys 

1 5 10 15 



Asn Gin Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 
20 25 30 

Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 
35 40 45 



Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly Tyr Ala Ala 
40 50 55 60 

Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 
65 70 75 80 

45 Ala Thr Asn His Ala He Val Gin Thr Leu Val His Phe He Asn Pro 

85 90 95 



Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala He 
100 105 110 

Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val He Leu Lys Lys Tyr 
H5 120 125 



Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
55 130 135 



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(2) INFORMATION FOR SEQ ID NO: 6: 

(1) SEQUENCE CHARACTERISTICS: 
5 (A) LENGTH: 139 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

10 (ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HURIDAE 
(F) TISSUE TYPE: EMBRYO 

15 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..139 

(D) OTHER INFORMATION: /label- MOPl -MATURE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 

Ser Thr Gly Gly Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro Lys 
25 1 5 10 15 

Asn Gin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn Ser Ser Ser 
20 25 30 

30 Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 

35 40 45 



20 



35 



50 



Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly Tyr Ala Ala 
50 55 60 

Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 
65 70 75 80 



Ala Thr Asn His Ala He Val Gin Thr Leu Val His Phe He Asn Pro 
40 85 90 95 

Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala He 
100 105 110 

45 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val He Leu Lys Lys Tyr 

115 120 125 



Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
130 135 

(2) INFORMATION FOR SEQ ID NO: 7: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 
55 (B) TYPE: amino acid 

(C) STRANDQ)NESS: single 

(D) TOPOLOGY: linear 



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(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 
5 (A) ORGANISM: HOMO SAPIENS 

(F) TISSUE TYPE: HIPPOCAMPUS 

(ix) FEATURE: 

(A) NAME/KEY: Protein 
10 (B) LOCATION: 1..139 

(D) OTHER INFORMATION: /labels H0P2-HATURE 



15 



3d 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 

Ala Val Arg Pro Leu Arg Arg Arg Gin Pro Lys Lys Ser Asn Glu Leu 
15 10 15 



Pro Gin Ala Asn Arg Leu Pro Gly He Phe Asp Asp Val His Gly Ser 
20 20 25 30 

His Gly Arg Gin Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Gin 
35 40 45 

25 Asp Leu Gly Trp Leu Asp Trp Val He Ala Pro Gin Gly Tyr Ser Ala 

50 55 60 



Tyr Tyr Cys Glu Gly Glu Cys Ser Phe Pro Leu Asp Ser Cys Met Asn 
65 • 70 75 80 

Ala Thr Asn His Ala He Leu Gin Ser Leu Val His Leu Met Lys Pro 
85 90 95 



Asn Ala Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr 
35 100 105 110 

Ser Val Leu T3rr Tyr Asp Ser Ser Asn Asn Val He Leu Arg Lys His 
115 120 125 

40 Arg Asn Met Val Val Lys Ala Cys Gly Cys His 

130 135 

(2) INFORMATION FOR SEQ ID NO: 8: 

45 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

50 

(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: MURIDAE 
55 (F) TISSUE TYPE: EMBRYO 



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(ix) FEATURE: 

(A) NAHE/KEY: Protein 

(B) LOCATION: 1..139 

5 (D) OTHER INFORMATION: /labels IfOP2-IlATORE 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8: 

10 Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn Glu Leu 

15 10 15 



15 



30 



Pro His Pro Asn Lys Leu Pro Gly He Phe Asp Asp Gly His Gly Ser 
20 25 30 

Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Arg 
35 40 45 



Asp Leu Gly Trp Leu Asp Trp Val He Ala Pro Gin Gly Tyr Ser Ala 
20 50 55 60 

Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys Met Asn 
65 70 75 80 

25 Ala Thr Asn His Ala He Leu Gin Ser Leu Val His Leu Met Lys Pro 

85 90 95 



Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr 
100 105 110 

Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val He Leu Arg Lys His 
115 120 125 



Arg Asn Met Val Val Lys Ala Cys Gly Cys His 
35 130 135 

(2) INFORMATION FOR SEQ ID N0;9: 

(i) SEQUENCE CHARACTERISTICS: 
40 (A) LENGTH: 101 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

45 (ii) MOLECULE TYPE: protein 

(Vi) ORIGINAL SOURCE: 

(A) ORGANISM: bovinae 

50 (ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..101 

(D) OTHER INFORMATION: /label= CBMP-2A-FX 

55 



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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 

Cys Lys Arg His Fro Leu l^r Val Asp Phe Ser Asp Val Gly Trp Asn 
15 10 15 

5 

Asp Trp He Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly 
20 25 30 

Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala 
10 35 40 45 

He Val Gin Thr Leu Val Asn Ser Val Asn Ser Lys He Pro Lys Ala 
50 55 60 

15 Cys Cys Val Pro Thr Glu Leu Ser Ala He Ser Met Leu Tyr Leu Asp 

65 70 75 80 



20 



25 



Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gin Asp Met Val Val Glu 
85 90 95 

Gly Cys Gly Cys Arg 
100 

(2) INFORMATION FOR SEQ ID NO: 10: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 101 amino acids 

(B) TTPE: amino acid 

(C) STRANDEDNESS: single 
30 (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 
35 (A) ORGANISM: HOMO SAPIENS 

(F) TISSUE TYPE: hippocampus 

(ix) FEATURE: 

(A) NAME/KEY: Protein 
40 (B) LOCATION: 1..101 

(D) OTHER INFORMATION: /labels CBMP-2B-FX 



45 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 

Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn 
15 10 15 



Asp Trp He Val Ala Pro Pro Gly Tyr Gin Ala Phe Tyr Cys His Gly 
50 20 25 30 

Asp Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala 
35 40 45 

55 He Val Gin Thr Leu Val Asn Ser Val Asn Ser Ser He Pro Lys Ala 

50 55 60 



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Cys Cys Val Pro Thr Glu Leu Ser Ala He Ser Het Leu Tyr Leu Asp 
65 70 75 80 

5 Glu Tyt Asp Lys Val Val Leu Lys Asn Tyr Gin Glu Met Val Val Glu 

85 90 95 

Gly Cys Gly Cys Arg 
100 

10 

(2) INFOmTION FOR SEQ ID NO: 11: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 
15 (B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



20 



40 



(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: DROSOPHILA MELANOGASTER 



(ix) FEATURE: 
25 (A) NAME/KEY: Protein 

(B) LOCATION: 1..101 
(D) OTHER INFORMATION: /label= DPP-FX 

30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 

Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asp 
15 10 15 

35 Asp Trp He Val Ala Pro Leu Gly Tyr Asp Ala Tyr Tyr Cys His Gly 

20 25 30 



Lys Cys Pro Phe Pro Leu Ala Asp His Phe Asn Ser Thr Asn His Ala 
35 40 45 

Val Val <;in Thr Leu Val Asn Asn Asn Asn Pro Gly Lys Val Pro Lys 
50 55 60 



Ala Cys Cys Val Pro Thr Gin Leu Asp Ser Val Ala Met Leu Tyr Leu 
45 65 70 75 80 

Asn Asp Gin Ser Thr Val Val Leu Lys Asn Tyr Gin €lu Met Thr Val 
85 90 95 

50 Val Gly Cys Gly Cys Arg 

100 

(2) INFORMATION FOR SEQ 10 N0:12; 



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(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 
5 (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 
10 (A) ORGANISM: XENOFUS 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

15 (D) OTHER INFORMATION: /labeU VGL-FX 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 

20 Cys Lys Lys Arg His Leu Tyr Val Glu Phe Lys Asp Val Gly Trp Gin 

15 10 15 



25 



40 



50 



Asn Trp Val He Ala Pro Gin Gly Tyr Met Ala Asn Tyr Cys Tyr Gly 
20 25 30 

Glu Cys Pro Tyr Pro Leu Thr Glu He Leu Asn Gly Ser Asn His Ala 
35 40 45 



He Leu Gin Thr Leu Val His Ser He Glu Pro Glu Asp He Pro Leu 

30 50 55 60 

Pro Cys Cys Val Pro Thr Lys Met Ser Pro He Ser Met Leu Phe Tyr 
65 70 75 80 

35 Asp Asn Asn Asp Asn Val Val Leu Arg His Tyr Glu Asn Met Ala Val 

85 90 95 



Asp Glu Cys Gly Cys Arg 
100 

(2) INFORMATION FOR SEQ ID NO: 13: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 
45 (B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HURIDAE 



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(ix) FEATURE; 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

(D) OTHER INFORHATION: /label= VGR-l-FX 

5 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13; 

Cys Lys Lys His Glu Leu lyr Val Ser Phe Gin Asp Val Gly Trp Gin 
10 1 5 10 15 

Asp Trp lie lie Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly 
20 25 30 

15 Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 

35 40 45 

He Val Gin Thr Leu Val His Val Met Asn Pro Glu Tyr Val Pro Lys 
50 55 60 

20 

Pro Cys Cys Ala Pro Thr Lys Val Asn Ala He Ser Val Leu Tyr Phe 
65 70 75 80 

Asp Asp Asn Ser Asn Val He Leu Lys Lys Tyr Arg Asn Met Val Val 
25 85 90 95 

Arg Ala Cys Gly Cys His 
100 

30 (2) INFORMATION FOR SEQ ID NO: 14; 

(1) SEQUENCE CHARACTERISTICS: 

(A) LENGTH; 106 amino acids 

(B) TYPE: amino acid 

35 (C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

40 (iii) HYPOTHETICAL: NO 

(iv) ANTI-SENSE: NO 

(vi) ORIGINAL SOURCE: 
45 (A) ORGANISM: Homo sapiens 

(F) TISSUE TYPE: brain 

(ix) FEATURE; 

(A) NAME/KEY: Protein 
50 (B) LOCATION: 1..106 

(D) OTHER INFORMATION: /note= "GDF-1 (fx)" 



(Xi) SEQUENCE DESCRIPTION; SEQ ID NO: 14: 

55 

Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp His 
1 5 10 15 



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Arg Trp Val lie Ala Pro Arg Gly Phe Leu Ala Asn Tyr Cys Gin Gly 
20 25 30 

5 Gin Cys Ala Leu Fro Val Ala Leu Set Gly Ser Gly Gly Pro Fro Ala 

35 40 45 

Leu Asn His Ala Val Leu Arg Ala Leu Het His Ala Ala Ala Fro Gly 
50 55 60 

10 

Ala Ala Asp Leu Pro Cys Cys Val Fro Ala Arg Leu Ser Pro lie Ser 
65 70 75 80 

Val Leu Phe Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gin Tyr Glu 
15 85 90 95 

Asp Het Val Val Asp Glu Cys Gly Cys Arg 
100 105 

20 (2) INFOBHATION FOR SEQ ID NO: 15: 

(i) SEQUENCE CHAKACTERISTICS: 

(A) LENGTH: 5 amino acids 

(B) TYPE: amino acid 

25 (C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) HOLECULE TYPE: peptide 

30 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: 

Cys Xaa Xaa Xaa Xaa 
35 1 5 

(2) INFORHATION FOR SEQ ID NO: 16: 

(i) SEQUENCE CHARACTERISTICS: 
40 (A) LENGTH: 1822 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

45 (ii) HOLECULE TYPE: cDNA 

(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 

50 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HOHO SAPIENS 
(F) TISSUE TYPE: HIPPOCAMPUS 



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(Ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 49.. 1341 

(C) IDENTIFICATION METHOD: experimental 

5 (D) OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN" 

/products "OPl" 
/evidenceic EXPERIMENTAL 
/staiidard_names "OPl" 

10 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: 

GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG 57 

Met His Val 

15 1 

CGC TCA CTG CGA GCT GCG GCG CCG CAC AGC TTC GTG GCG CTC TGG GCA 105 
Arg Ser Leu Arg Ala Ala Ala Fro His Ser Phe Val Ala Leu Trp Ala 
5 10 15 

20 

CCC CTG TTC CTG CTG CGC TCC GCC CTG GCC GAC TTC AGC CTG GAC AAC 153 
Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn 
20 25 30 35 

25 GAG GTG CAC TCG AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG 201 
Glu Val His Ser Ser Phe lie His Arg Arg Leu Arg Ser Gin Glu Arg 
40 45 50 

CGG GAG ATG CAG CGC GAG ATC CTC TCC ATT TTG GGC TTG CCC CAC CGC 249 
30 Arg Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu Pro His Arg 
55 60 65 

CCG CGC CCG CAC CTC CAG GGC AAG CAC AAC TCG GCA CCC ATG TTC ATG 297 
Pro Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro Met Phe Met 
35 70 75 80 

CTG GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG GGC GGC GGG CCC <;GC 345 
Leu Asp Leu lyr Asn Ala Met Ala Val Glu Glu Gly Gly Gly Pro Gly 
85 90 95 

40 

GGC CAG GGC TTC TCC TAC CCC TAC AAG €CC GTC TTC AGT ACC CAG GGC 393 
Gly Gin Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gin Gly 
100 105 110 115 

45 CCC CCT CTG GCC AGC CTG CAA GAT AGC CAT TTC CTC ACC GAC GCC GAC 441 
Pro Pro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp Ala Asp 
120 125 130 

ATG GTC ATG AGC TTC GTC AAC CTC GTG GAA CAT GAC AAG GAA TTC TTC 489 
50 Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe 
135 140 145 

CAC CCA CGC TAC CAC CAT CGA GAG TTC CGG TTT GAT CTT TCC AAG ATC 537 
His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys He 
55 150 155 160 



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CCA GAA GGG GAA GCT GTC ACG GCA GCC GAA TTC CGG ATC TAC AAG GAC 585 

Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He Tyr Lys Asp 
165 170 175 

5 

TAC ATC CGG GAA CGC TTC GAC AAT GAG ACG TTC CGG ATC AGC GTT TAT 633 

Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg He Ser Val Tyr 

180 185 190 195 

10 CAG GTG CTC CAG GAG CAC TTG GGC AGG GAA TCG GAT CTC TTC CTG CTC 681 

Gin Val Leu Gin Glu His Leu Gly Arg Glu Ser Asp Leu Phe Leu Leu 
200 205 210 



GAC AGC CGT ACC CTC TGG GCC TCG GAG GAG GGC TGG CTG GTG TTT GAC 
15 Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp 
215 220 225 



729 



20 



ATC ACA GCC ACC AGC AAC CAC TGG GTG GTC AAT CCG CGG CAC AAC CTG 
He Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu 
230 235 240 



777 



GGC CTG CAG CTC TCG GTG GAG ACG CTG GAT GGG CAG AGC ATC AAC CCC 
Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser He Asn Pro 
245 250 255 

25 

AAG TTG GCG GGC CTG ATT GGG CGG CAC GGG CCC CAG AAC AAG CAG CCC 
Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys Gin Pro 
260 265 270 275 

30 TTC ATG GTG GCT TTC TTC AAG GCC ACG GAG GTC CAC TTC CGC AGC ATC 
Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser He 
280 285 290 



825 



873 



921 



CGG TCC ACG GGG AGC AAA CAG GGC AGC CAG AAC CGC TCC AAG ACG CCC 
35 Arg Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro 
295 300 305 



969 



40 



AAG AAC CAG GAA GCC CTG CGG ATG GCC AAC GTG GCA GAG AAC AGC AGC 
Lys Asn Gin Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser 
310 315 320 



1017 



45 



50 



AGC GAC CAG AGG CAG GCC TGT AAG AAG CAC GAG CTG TAT GTC AGC TTC 1065 
Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe 
325 330 335 

CGA GAC CTG GGC TGG CAG GAC TGG ATC ATC GCG CCT GAA GGC TAC GCC 1113 
Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly Tyv Ala 
340 345 350 355 

GCC TAC TAC TGT GAG GGG GAG TGT GCC TTC CCT CTG AAC TCC TAC ATG 1161 
Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met 
360 365 370 



AAC GCC ACC AAC CAC GCC ATC GTG CAG ACG CTG GTC CAC TTC ATC AAC 
55 Asn Ala Thr Asn His Ala He Val Gin Thr Leu Val His Phe He Asn 
375 380 385 



1209 



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CCG GAA ACQ GTG CCC AAG CCC TGC TGT GCG CCC ACG CA6 CTC AAT GCC 1257 
Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala 
390 395 400 

5 

ATC TCC GTG CTC TAC TTC GAT GAC AGC TCC AAC GTC ATC CTG AAG AAA 1305 
lie Ser Val Leu Tjrr Phe Asp Asp Ser Ser Asn Val lie Leu Lys Lys 
405 410 415 

10 TAC AGA AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCCTCC 1351 
Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
420 425 430 

GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT CCATTGCTCG CCTTGGCCAG 1411 

15 

GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA ACTTTAAAGG 1471 
TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CAGTGGCAGC 1531 
20 ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AAAAAACAAC 1591 
GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TGCACGGACT 1651 
CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GGAGGAAGGG 1711 

25 

GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTGAC CCGGAAGTTC 1771 
CTGTAATAAA TGTCACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A 1822 

30 

(2) INFOSHATION FOR SEQ ID NO: 17: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 431 amino acids 
35 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: 

Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 
1 5 10 15 

45 Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 
20 25 30 

Leu Asp Asn Glu Val His Ser Ser Phe lie His Arg Arg Leu Arg Ser 
35 40 45 

50 

Gin Glu Arg Arg Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu 
50 55 60 

Pro His Arg Pro Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro 
55 65 70 75 80 



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Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly 
85 90 95 

5 Gly Pro Gly Gly Gin Gly Phe Ser Tyr Pro lyr Lys Ala Val Phe Ser 
100 105 110 



10 



Thr Gin Gly Pro Pro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr 
115 120 125 

Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys 
130 135 140 



Glu Phe Phe His Fro Arg Tyr His Bis Arg Glu Phe Arg Phe Asp Leu 
15 145 150 155 160 

Ser Lys He Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He 
165 170 175 

20 Tyr Lys Asp Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg He 
180 185 190 



25 



Ser Val Tyr Gin Val Leu Gin Glu His Leu Gly Arg Glu Ser Asp Leu 
195 200 205 

Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu 
210 215 220 



Val Phe Asp He Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg 
30 225 230 235 240 

His Asn Leu Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser 
245 250 255 

35 He Asn Pro Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn 
260 265 270 



40 



Lys Gin Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe 
275 280 285 

Arg Ser He Arg Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Ser 
290 295 300 



Lys Thr Pro Lys Asn Gin Glu Ala Leu Arg Met Ala Asn Val Ala Glu 
45 305 310 315 320 

Asn Ser Ser Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr 
325 330 335 

50 Val Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu 
340 345 350 



55 



Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn 
355 360 365 



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Ser Tyr Het Asn Ala Thr Asn His Ala He Val Gin Thr Leu Val His 
370 375 380 

Phe He Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin 
5 385 390 395 400 

Leu Asn Ala He Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val He 
405 410 415 

10 Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
420 425 430 

(2) INFOKHATION FOR SEQ ID NO: 18: 

15 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1873 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

20 

(ii) MOLECULE TYPE: cDNA 

(iii) HYPOTHETICAL: NO 

25 (iv) ANTI^SENSE: NO 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HURIDAE 
(F) TISSUE TYPE: EMBRYO 

30 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 104.. 1393 

(D) OTHER INFORMATION: /function* "OSTEOGENIC PROTEIN" 
35 /product- "MOPl" 

/note= "MOPl (CDNA)" 



40 



50 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: 
CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CCACCTGGGG 60 



CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGGC GCG ATC CAC GTG CGC 115 

Met His Val Arg 

45 1 ^ 



TCG CTG CGC GCT GCG GCG CCA CAC AGC TTC GTG GCG CTC TGG GCG CCT 163 
Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala Pro 
5 10 15 20 

CTG TTC TTG CTG CGC TCC GCC CTG GCC GAT TTC AGC CTG GAC AAC GAG 211 
Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn Glu 
25 30 35 



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GTG CAC TCC AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG CGG 
Val His Ser Set Phe lie His Arg Arg Leu Arg Ser Gin Glu Arg Arg 
40 45 50 



259 



GAG ATG CAG CGG GAG ATC CTG TCC ATC TTA GGG TTG CCC CAT CGC CCG 
Glu Het Gin Arg Glu lie Leu Ser lie Leu Gly Leu Pro His Arg Pro 
55 60 65 



307 



CGC CCG CAC CTC CAG GGA AAG CAT AAT TCG GCG CCC ATG TTC ATG TTG 
10 Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro Het Phe Het Leu 
70 75 80 



355 



GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG AGC GGG CCG GAC GGA CAG 
Asp Leu Tyr Asn Ala Het Ala Val Glu Glu Ser Gly Pro Asp Gly Gin 
15 85 90 95 100 

GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AGT ACC CAG GGC CCC CCT 
Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gin Gly Pro Pro 
105 110 115 • 



20 



TTA GCC AGC CTG CAG GAC AGC CAT TTC CTC ACT GAC GCC GAC ATG GTC 
Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp Ala Asp Het Val 
120 125 130 



403 



451 



499 



25 ATG AGC TTC GTC AAC CTA GTG GAA CAT GAC AAA GAA TTC TTC CAC CCT 
Het Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe His Pro 
135 140 145 



547 



CGA TAC CAC CAT CGG GAG TTC CGG TTT GAT CTT TCC AAG ATC CCC GAG 
30 Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys lie Pro Glu 
150 155 160 



595 



35 



GGC GAA CGG GTG ACC GCA GCC GAA TTC AGG ATC TAT AAG GAC TAC ATC 
Gly Glu Arg Val Thr Ala Ala Glu Phe Arg He Tyr Lys Asp Tyr He 
165 170 175 180 



643 



40 



CGG GAG CGA TTT GAC AAC GAG ACC TTC CAG ATC ACA GTC TAT CAG GIG 
Arg Glu Arg Phe Asp Asn Glu Thr Phe Gin He Thr Val Tyr Gin Val 
185 190 195 

CTC CAG GAG CAC TCA GGC AGG GAG TCG GAC CTC TTC TTG CTG GAC AGC 
Leu Gin Glu His Ser Gly Arg Glu Ser Asp Leu Phe Leu Leu Asp Ser 
200 205 210 



691 



739 



45 CGC ACC ATC TGG GCT TCT GAG GAG GGC TGG TTG GTG TTT GAT ATC ACA 
Arg Thr He Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp He Thr 
215 220 225 



787 



GCC ACC AGC AAC CAC TGG GTG GTC AAC CCT CGG CAC AAC CTG GGC TTA 
50 Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu Gly Leu 
230 235 240 



835 



CAG CTC TCT GTG GAG ACC CTG GAT GGG CAG AGC ATC AAC CCC AAG TTG 
Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser He Asn Pro Lys Leu 
55 245 250 255 260 



883 



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GCA GGC CTG ATT GGA CGG CAT GGA CCC CAG AAC AAG CAA CCC TTC ATG 931 

Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys Gin Pro Phe Met 
265 270 275 

5 

GTG GCC TTC TTC AAG GCC ACG GAA GTC CAT CTC CGT AGT ATC CGG TCC 979 

Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg Ser He Arg Ser 
280 285 290 

10 ACG GGG GGC AAG CAG CGC AGC CAG AAT CGC TCC AAG ACG CCA AAG AAC 1027 
Thr Gly Gly Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro Lys Asn 
295 300 305 

CAA GAG GCC CTG AGG ATG GCC AGT GTG GCA GAA AAC AGC AGC AGT GAC 1075 
15 Gin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn Ser Ser Ser Asp 
310 315 320 

CAG AGG CAG GCC TGC AAG AAA CAT GAG CTG TAC GTC AGC TTC CGA GAC 1123 
Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp 
20 325 330 335 340 

CTT GGC TGG CAG GAC TGG ATC ATT GCA CCT GAA GGC TAT GCT GCC TAC 1171 
Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly Tyr Ala Ala Tyr 
345 350 355 

25 

TAC TGT GAG GGA GAG TGC GCC TTC CCT CTG AAC TCC TAC ATG AAC GCC 1219 
lyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala 
360 365 370 

30 ACC AAC CAC GCC ATC GTC CAG ACA CTG GTT CAC TTC ATC AAC CCA GAC 1267 
Thr Asn His Ala He Val Gin Thr Leu Val His Phe He Asn Pro Asp 
375 380 385 

ACA GTA CCC AAG CCC TGC TGT GCG CCC ACC CAG CTC AAC GCC ATC TCT 1315 
35 Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala He Ser 
390 395 400 

GTC CTC TAC TTC GAC GAC AGC TCT AAT GTC GAC CTG AAG AAG TAC AGA 1363 
Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Asp Leu Lys Lys Tyr Arg 
40 405 410 415 420 

AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCTTCC TGAGACCCTG 1413 
Asn Met Val Val Arg Ala Cys Gly Cys His 
425 430 

45 

ACCTTTGCGG GGCCACACCT TTCCAAATCT TCGATGTCTC ACCATCTAAt; TCTCTCACTG 1473 
CCCACCTTGG CGAGGAGAAC AGACCAACCT CTCCTGAGCC TTCCCTCACC TCCCAACCGG 1533 
50 AAGCATGTAA GGGTTCCAGA AACCTGAGCG TGCAGCAGCT GATGAGCGCC CTTTCCTTCT 1593 
GGCACGTGAC GGACAAGATC CTACCAGCTA CCACAGCAAA CGCCTAAGAG CAGGAAAAAT 1653 
GTCTGCCAGG AAAGTGTCCA GTGTCCACAT GGCCCCTGGC GCTCTGAGTC TTTGAGGAGT 1713 

55 



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AATCGCAAGC CTCGTTCAGC TGCAGCAGAA GGAAGGGCTT AGCCAGGGTG GGCGCTGGCG 1773 

TCTGTGTTGA AGGGAAACCA AGCAGAAGCC ACTGTAAT6A TATGTCACAA TAAAACCCAT 1833 

5 GAATGAAAAA AAAAAAAAAA AAAAAAAAAA AAAAGAATTC 1873 

(2) INFORHATION FOR SEQ ID NO: 19: 

10 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 430 amino acids 

(B) TYPE; amino acid 
(D) TOPOLOGY: linear 

15 (ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: 

Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 
20 1 5 10 15 

Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 
20 25 30 

25 Leu Asp Asn Glu Val His Ser Ser Phe He His Arg Arg Leu Arg Ser 
35 40 45 

Gin Glu Arg Arg Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu 
50 55 60 

30 

Pro His Arg Pro Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro 
65 70 75 80 

Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Ser Gly 
35 85 90 95 

Pro Asp Gly Gin Gly Phe Ser Tyr Pro lyr Lys Ala Val Phe Ser Thr 
100 105 110 

40 Gin Gly Pro Pro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp 
115 120 125 

Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu 
130 135 140 

45 

Phe Phe His Pro Arg Tyx His His Arg Glu Phe Arg Phe Asp Leu Ser 
1« 150 155 160 

Lys He Pro Glu Gly Glu Arg Val Thr Ala Ala Glu Phe Arg He Tyr 
50 165 170 175 

Lys Asp Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Gin He Tht 
180 185 190 

55 Val Tyr Gin Val Leu Gin Glu His Ser Gly Arg Glu Ser Asp Leu Phe 
195 200 205 



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Leu Leu Asp Ser Arg Thr lie Trp Ala Ser Glu Glu Gly Trp Leu Val 
210 215 220 

5 Phe Asp He Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His 
225 230 235 240 

Asn Leu Gly Leu Gin Leu Ser Val Glu "Rit Leu Asp Gly Gin Ser He 
245 250 255 

10 

Asn Pro Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys 
260 265 270 

Gin Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg 
15 275 280 285 

Ser He Arg Ser Thr Gly Gly Lys Gin Arg Ser Gin Asn Arg Ser Lys 
290 295 300 

20 Thr Pro Lys Asn Gin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn 
305 310 315 320 

Ser Ser Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val 
325 330 335 

25 

Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly 
340 345 350 

Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser 
30 355 360 365 

lyr Met Asn Ala Thr Asn His Ala He Val Gin Thr Leu Val His Phe 
370 375 380 

35 He Asn Pro Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu 
385 390 395 400 

Asn Ala He Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Asp Leu 
405 410 415 

40 

Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
420 425 430 

(2) INFORMATION FOR SEQ ID N0:20: 

45 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1723 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 
50 (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

(vi) ORIGINAL SOURCE: 
55 (A) ORGANISM: Homo sapiens 

(F) TISSUE TYPE: HIPPOCAMPUS 



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(ix) FEATURE: 

(A) HAME/KEY: CDS 

(B) LOCATION: 490.. 1696 

5 (D) OTHER INFORMATION: /function- "OSTEOGENIC PROTEIN" 

/products "hOP2-PP" 
/note= "hOP2 (cDNA)" 

10 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:20: 

GGCGCCGGCA GAGCAGGAGT GGCTGGAGGA GCTGTGGTTG GAGCAGGAGG TGGCACGGCA 60 

GGGCTGGAGG GCTCCCTATG AGTGGCGGAG ACGGCCCAGG AGGCGCTGGA GCAACAGCTC 120 

15 

CCACACCGCA CCAAGCGGTG GCTGCAGGAG CTCGCCCATC GCCCCTGCGC TGCTCGGACC 180 

GCGGCCACAG CCGGACTGGC GGGTACGGCG GCGACAGAGG CATTGGCCGA GAGTCCCAGT 240 

20 CCGCAGAGTA GCCCCGGCCT CGAGGCGGTG GCGTCCCGGT CCTCTCCGTC CAGGAGCCAG 300 

GACAGGTGTC GCGCGGCGGG GCTCCAGGGA CCGCGCCTGA GGCCGGCTGC CCGCCCGTCC 360 

CGCCCCGCCC CGCCGCCCGC CGCCCGCCGA GCCCAGCCTC CTTGCCGTCG GGGCGTCCCC 420 

AGGCCCTGGG TCGGCCGCGG AGCCGATGCG CGCCCGCTGA GCGCCCCAGC TGAGCGCCCC 480 



25 



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 
30 1 5 10 

GCG CTA TGC GCG CTG GGC GGG €GC GGC CCC GGC CTG CGA CCC CCG CCC 576 
Ala Leu Cys Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pro Pro Pro 
15 20 25 

35 

GGC TGT CCC CAG CGA CGT CTG GGC GCG CGC GAG CGC CGG GAC GTG CAG 624 
Gly Cys Pro Gin Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Val Gin 
30 35 40 45 

40 CGC GAG ATC CTG GCG GTG CTC GGG CTG CCT GGG CGG CCC CGG CCC CGC 672 
Arg Glu He Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg 
50 55 60 

GCG CCA CCC GCC GCC TCC CGG CTG CCC GCG TCC HCG CCG CTC TTC ATG 720 
45 Ala Pro Pro Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro Leu Phe Met 
65 70 75 

CTG GAC CTG TAC CAC GCC ATG GCC GGC GAC GAC GAC GAG ^C GGC GCG 768 
Leu Asp Leu Tyr His Ala Met Ala Gly Asp Asp Asp Glu Asp Gly Ala 
50 80 85 90 

CCC GCG GAG CGG CGC CTG GGC CGC GCC GAC CTG GTC ATG AGC TTC GTT 816 
Pro Ala Glu Arg Arg Leu Gly Arg Ala Asp Leu Val Met Ser Phe Val 
95 100 105 

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AAC ATG GTG GAG CGA GAC CGT GCC CTG GGC CAC CAG GAG CCC CAT TGG 
Asn Het Val Glu Arg Asp Arg Ala Leu Gly His Gin Glu Pro His Trp 
110 115 120 125 



864 



AAG GAG TTC CGC TTT GAC CTG ACC CAG ATC CCG GCT GGG GAG GCG GTC 
Lys Glu Phe Arg Phe Asp Leu Thr Gin He Pro Ala Gly Glu Ala Val 
130 135 140 



912 



ACA GCT GCG GAG TTC CGG ATT TAC AAG GTG CCC AGC ATC CAC CTG CTC 
10 Thr Ala Ala Glu Phe Arg He Tyr Lys Val Pro Ser He His Leu Leu 
145 150 155 



960 



15 



AAC AGG ACC CTC CAC GTC AGC ATG TTC CAG GTG GTC CAG GAG CAG TCC 
Asn Arg Thr Leu His Val Ser Het Phe Gin Val Val Gin Glu Gin Ser 
160 165 170 



1008 



20 



AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT CTT CAG ACG CTC CGA GCT 1056 
Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gin Thr Leu Arg Ala 
175 180 185 

GGA GAC GAG GGC TGG CTG GTG CTG GAT GTC ACA GCA GCC AGT GAC TGC 1104 
Gly Asp Glu Gly Trp Leu Val Leu Asp Val Thr Ala Ala Ser Asp Cys 
190 195 200 205 



25 TGG TTG CTG AAG CGT CAC AAG GAC CTG GGA CTC CGC CTC TAT GTG GAG 
Trp Leu Leu Lys Arg His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu 
210 215 220 



1152 



ACT GAG GAC GGG CAC AGC GTG GAT CCT GGC CTG GCC GGC CTG CTG GGT 
30 Thr Glu Asp Gly His Ser Val Asp Pro Gly Leu Ala Gly Leu Leu Gly 
225 230 235 



1200 



35 



CAA CGG GCC CCA CGC TCC CAA CAG CCT TTC GTG GTC ACT TTC TTC AGG 
Gin Arg Ala Pro Arg Ser Gin Gin Fro Phe Val Val Thr Phe Phe Arg 
240 245 250 



1248 



40 



GCC AGT CCG AGT CCC ATC CGC ACC CCT CGG GCA GTG AGG CCA CTG AGG 1296 
Ala Ser Pro Ser Pro He Arg Thr Pro Arg Ala Val Arg Pro Leu Arg 
255 260 265 

AGG AGG CAG CCG AAG AAA AGC AAC GAG CTG CCG CAG GCC AAC CGA CTC 1344 
Arg Arg Gin Pro Lys Lys Ser Asn Glu Leu Pro Gin Ala Asn Arg Leu 
270 275 280 285 



45 CCA GGG ATC TTT GAT GAC GTC CAC GGC TCC CAC GGC CGG CAG GTC TGC 
Pro Gly He Phe Asp Asp Val His Gly Ser His Gly Arg Gin Val Cys 
290 295 300 



1392 



CGT CGG CAC GAG CTC TAC GTC AGC TTC CAG GAC CTC GGC TGG CTG GAC 
50 Arg Arg His Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Leu Asp 
305 310 315 



1440 



55 



TGG GTC ATC GCT CCC CAA GGC TAC TCG GCC TAT TAC TGT GAG GGG GAG 
Trp Val He Ala Pro Gin Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu 
320 325 330 



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TCC TCC TTC CCA CTG GAC TCC TGC ATG AAT GCC ACC AAC CAC GCC ATC 1536 
Cys Ser Phe Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His Ala He 
335 340 345 

5 

CTG CAG TCC CTG GTG CAC CTG ATG AAG CCA AAC GCA GTC CCC AAG GCG 1584 
Leu Gin Ser Leu Val His Leu Met Lys Pro Asn Ala Val Pro Lys Ala 
350 355 360 365 

10 TGC TGT GCA CCC ACC AAG CTG AGC GCC ACC TCT GTG CTC TAC TAT GAC 1632 
Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu lyr Tyr Asp 
' 370 375 380 

AGC AGC AAC AAC GTC ATC CTG CGC AAA GCC CGC AAC ATG GTG GTC AAG 1680 
15 Ser Ser Asn Asn Val He Leu Arg Lys Ala Arg Asn Met Val Val Lys 
385 390 395 

GCC TGC GGC TGC CAC T GAGTCAGCCC GCCCAGCCCT ACTGCAG 1723 
Ala Cys Gly Cys His 
20 400 

(2) INFOBMATIOH FOR SEQ ID NO: 21: 

25 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 402 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGy: linear 

30 (ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21: 

Met Thr Ala Leu Pro Gly Pro Leu Trp Uu Leu Gly Leu Ala Leu Cys 
35 1 5 10 15 

Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pro Pro Pro Gly Cys Pro 
20 25 30 

40 Gin Are Arg Leu Gly Ala Arg Glu Arg Arg Asp Val Gin Arg Glu He 
35 40 45 



45 



Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg Ala Pro Pro 
50 55 60 

Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro Leu Phe Met Leu Asp Leu 
65 70 75 80 



Tyr His Ala Met Ala Gly Asp Asp Asp Glu Asp Gly Ala Pro Ala Glu 
50 85 90 95 

Are ArE Leu Gly Arg Ala Asp Leu Val Met Ser Phe Val Asn Met Val 
100 105 110 

55 Glu Are Asp Arg Ala Leu Gly His Gin Glu Pro His Trp Lys Glu Phe 
115 120 125 



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Arg Phe Asp Leu Thr Gin He Pro Ala Gly Glu Ala Val Thr Ala Ala 
130 135 140 



5 Glu Phe Arg He TJrr Lys Val Pro Ser He His Leu Leu Asn Arg Ihr 
145 150 155 160 



Leu His Val Ser Het Phe Gin Val Val Gin Glu Gin Ser Asn Arg Glu 
165 170 175 

Ser Asp Leu Phe Phe Leu Asp Leu Gin Thr Leu Arg Ala Gly Asp Glu 
180 185 190 

Gly Trp Leu Val Leu Asp Val Thr Ala Ala Ser Asp Cys Trp Leu Leu 
195 200 205 

Lys Arg His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Glu Asp 
210 215 220 

20 Gly His Ser Val Asp Pro Gly Leu Ala Gly Leu Leu Gly Gin Arg Ala 
225 230 235 240 



10 



15 



Pro Arg Ser Gin Gin Pro Phe Val Val Thr Phe Phe Arg Ala Ser Pro 
245 250 255 

Ser Pro He Arg Thr Pro Arg Ala Val Arg Pro Leu Arg Arg Arg Gin 
260 265 270 

Pro Lys Lys Ser Asn Glu Leu Pro Gin Ala Asn Arg Leu Pro Gly He 
275 280 285 

Phe Asp Asp Val His Gly Ser His €ly Arg Gin Val Cys Arg Arg His 
290 295 300 

35 Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Leu Asp Trp Val He 
305 310 315 320 



25 



30 



Ala Pro Gin Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ser Phe 
325 330 335 

Pro Leu Asp Ser Cys Het Asn Ala Thr Asn His Ala He Leu Gin Ser 
340 345 350 

Leu Val His Leu Het Lys Pro Asn Ala Val Pro Lys Ala Cys Cys Ala 
355 360 365 

Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn 
370 375 380 

50 Asn Val He Leu Arg Lys Ala Arg Asn Het Val Val Lys Ala Cys Gly 
385 390 395 400 



40 



45 



Cys His 



55 



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(2) INFOmTION FOR SEQ ID NO: 22: 

(i) SEQUENCE CHARACTERISTICS; 

(A) LENGTH: 1926 base pairs 
5 (B) TIFE; nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(vi) ORIGINAL SOURCE: 
10 (A) ORGANISM: HURIDAE 

(F) TISSUE TYPE: EMBRYO 

(ix) FEATURE: 

(A) NAME/KEY: CDS 
15 (B) LOCATION: 93.. 1289 

(D) OTHER INFORMATION: /function^ "OSTEOGENIC PROTEIN" 
/products "mOP2-PP" 
/notes '•inOP2 cDNA" 

20 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: 

GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC CCGACCAGCT 60 

25 ACCAGTGGAT GCGCGCCGGC TGAAAGTCCG AG ATG GCT ATG CGT CCC GGG CCA 113 

Met Ala Met Arg Pro Gly Pro 
1 5 

CTC TGG CTA TTG GGC CTT GCT CTG TGC GCG CTG GGA GGC GGC CAC GGT 161 
30 Leu Trp Leu Leu Gly Leu Ala Leu Cys Ala Leu Gly Gly Gly His Gly 
10 15 20 

CCG CGT CCC CCG CAC ACC TGT CCC CAG CGT CGC CTG GGA GCG CGC GAG 209 
Pro Arg Pro Pro His Thr Cys Pro Gin Arg Arg Leu Gly Ala Arg Glu 
35 25 30 35 

CGC CGC GAC ATG CAG CGT GAA ATC CTG GCG GTG CTC GGG CTA CCG GGA 257 
Arg Arg Asp Met Gin Arg Glu lie Leu Ala Val Leu Gly Leu Pro Gly 
40 45 50 55 

40 

CGG CCC CGA CCC CGT GCA CAA CCC GCC GCT GCC CGG CAG CCA GCG TCC 305 
Arg Pro Arg Pro Arg Ala Gin Pro Ala Ala Ala Arg Gin Pro Ala Ser 
60 65 70 

45 GCG CCC CTC TTC ATG TTG GAC CTA TAC CAC GCC ATG ACC GAT GAC GAC 353 
Ala Pro Leu Phe Met Leu Asp Leu Tyr His Ala Met Thr Asp Asp Asp 
75 80 85 

GAC GGC GGG CCA CCA CAG GCT CAC TTA GGC CGT €CC GAC CTG GTC ATG 401 
50 Asp Gly Gly Pro Pro Gin Ala His Leu Gly Arg Ala Asp Leu Val Met 
90 95 100 

AGC TTC GTC AAC ATG GTG GAA CGC GAC CGT ACC CTG GGC TAC CAG GAG 449 
Ser Phe Val Asn Met Val Glu Arg Asp Arg Thr Leu Gly Tyr Gin Glu 
55 105 110 115 



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CCA CAC TGG AAG GAA TTC CAC TTT GAC CTA ACC CAG ATC CCT GCT GGG 
Pro His Trp Lys Glu Phe His Phe Asp Leu Thr Gin He Pro Ala Gly 
120 125 130 135 



497 



GAG GCT GTC ACA GCT GCT GAG TTC CGG ATC TAC AAA GAA CCC AGC ACC 
Glu Ala Val Thr Ala Ala Glu Phe Arg He Tyr Lys Glu Pro Ser Thr 
140 145 150 



545 



10 CAC CCG CTC AAC ACA ACC CTC CAC ATC AGC ATG TTC GAA GTG GTC CAA 
His Pro Leu Asn Thr Thr Leu His He Ser Net Phe Glu Val Val Gin 
155 160 165 



593 



GAG CAC TCC AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT CTT CAG ACG 
15 Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gin Thr 
170 175 180 



641 



20 



CTC CGA TCT GGG GAC GAG GGC TGG CTG GTG CTG GAC ATC ACA GCA GCC 
Leu Arg Ser Gly Asp Glu Gly Trp Leu Val Leu Asp He Thr Ala Ala 
185 190 195 



689 



25 



AGT GAC CGA TGG CTG CTG AAC CAT CAC AAG GAC CTG GGA CTC CGC CTC 737 
Ser Asp Arg Trp Leu Leu Asn His His Lys Asp Leu Gly Leu Arg Leu 
200 205 210 215 

TAT GTG GAA ACC GCG GAT GGG CAC AGC ATG GAT CCT GGC CTG GCT GGT 785 
Tyr Val Glu Thr Ala Asp Gly His Ser Het Asp Pro Gly Leu Ala Gly 
220 225 230 



30 CTG CTT GGA CGA CAA GCA CCA CGC TCC ACA CAG CCT TTC ATG GTA ACC 833 
Leu Leu Gly Arg Gin Ala Pro Arg Ser Arg Gin Pro Phe Het Val Thr 
235 240 245 

TTC TTC AGG GCC AGC CAG AGT CCT GTG CGG GCC CCT CGG GCA GCG AGA 881 
35 Phe Phe Arg Ala Ser Gin Ser Pro Val Arg Ala Pro Arg Ala Ala Arg 
250 255 260 



40 



CCA CTG AAG AGG AGG CAG CCA AAG AAA ACG AAC GAG CTT CCG CAC CCC 
Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn Glu Leu Pro His Pro 
265 270 275 



929 



AAC AAA CTC CCA GGG ATC TTT GAT GAT GGC CAC «;T TCC CGC GGC AGA 977 

Asn Lys Leu Pro Gly He Phe Asp Asp Gly His Gly Ser Arg Gly Arg 
280 285 290 295 

45 

GAG GTT TGC CGC AGG CAT GAG CTC TAC GTC AGC TTC GGT GAC CTT GGC 1025 

Glu Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly 

300 305 310 

50 TGG CTG GAC TGG GTC ATC GCC CCC CAG GGC TAC TCT GCC TAT TAC TGT 1073 

Trp Leu Asp Trp Val He Ala Pro Gin Gly Tyr Ser Ala Tyr Tyr Cys 
315 320 325 



GAG GGG €AG TGT GCT TTC CCA CTG GAC TCC TGT ATG AAC GCC ACC AAC 
55 Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys Het Asn Ala Thr Asn 
330 335 340 



1121 



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CAT GCC ATC TTG CAG TCT CTG GTG CAC CTG ATG AAG CCA GAT GTT GTC 1169 

His Ala lie Leu Gin Ser Leu Val His Leu Met Lys Pro Asp Val Val 

345 350 355 

5 

CCC AAG GCA TGC TGT GCA CCC ACC AAA CTG AGT GCC ACC TCT GTG CTG 1217 

Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu 
360 365 370 375 

10 TAC TAT GAC AGC AGC AAC AAT GTC ATC CTG CGT AAA CAC CGT AAC ATG 1265 
Tyr Tyr Asp Ser Ser Asn Asn Val He Leu Arg Lys His Arg Asn Met 
380 385 390 

GTG GTC AAG GCC TGT GGC TGC CAC TGAGGCCCCG CCCAGCATCC TGCTTCTACT 1319 
15 Val Val Lys Ala Cys Gly Cys His 
395 

ACCTTACCAT CTGGCCGGGC CCCTCTCCAG AGGCAGAAAC CCTTCTATGT TATCATAGCT 1379 

20 CAGACAGGGG CAATGGGAGG CCCTTCACTT CCCCTGGCCA CTTCCTGCTA AAATTCTGGT 1439 

CTTTCCCAGT TCCTCTGTCC TTCATGGGGT TTCGGGGCTA TCACCCCGCC CTCTCCATCC 1499 

TCCTACCCCA AGCATAGACT GAATGCACAC AGCATCCCAG AGCTATGCTA ACTGAGAGGT 1559 

25 

CTGGGGTCAG CACTGAAGGC CCACATGAGG AAGACTGATC CTTGGCCATC CTCAGCCCAC 1619 

AATGGCAAAT TCTGGATGGT CTAAGAAGGC CCTGGAATTC TAAACTAGAT GATCTGGGCT 1679 

30 CTCTGCACCA TTCATTGTGG CAGTTGGGAC ATTTTTAGGT ATAACAGACA CATACACTTA 1739 

GATCAATGCA TCGCTGTACT CCTTGAAATC AGAGCTAGCT TGTTAGAAAA AGAATCAGAG 1799 

CCAGGTATAG CGGTGCATGT CATTAATCCC AGCGCTAAAG AGACAGAGAC AGGAGAATCT 1859 

35 

CTGTGAGTTC AAGGCCACAT AGAAAGAGCC TGTCTCGGGA GCAGGAAAAA AAAAAAAAAC 1919 

GGAATTC 1926 

40 

(2) INFOBMATION FOR SEQ ID NO: 23: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 399 amino acids 
45 (B) TYPE; amino acid 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

50 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: 

Met Ala Met Arg Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys 
15 10 15 

55 Ala Leu Gly Gly Gly His Gly Pro Arg Pro Pro His Thr Cys Pro Gin 
20 25 30 



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Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Het Gin Arg Glu lie Leu 
35 40 45 

5 Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg Ala Gin Pro Ala 
50 55 60 



10 



Ala Ala Arg Gin Pro Ala Ser Ala Pro Leu Phe Met Leu Asp Leu lyr 
65 70 75 80 

His Ala Het Thr Asp Asp Asp Asp Gly Gly Pro Pro Gin Ala His Leu 
85 90 95 



Gly Arg Ala Asp Leu Val Het Ser Phe Val Asn Het Val Glu Arg Asp 
15 100 105 110 

Arg Ihr Leu Gly Tyr Gin Glu Pro His Trp Lys Glu Phe His Phe Asp 
115 120 125 

20 Leu Thr Gin He Pro Ala Gly Glu Ala Val Thr Ala Ala Glu Phe Arg 
130 135 140 



25 



Zle lyr Lys Glu Pro Ser Thr His Pro Leu Asn Thr Thr Leu His He 
145 150 155 160 

Ser Het Phe Glu Val Val Gin Glu His Ser Asn Arg Glu Ser Asp Leu 
165 170 175 



Phe Phe Leu Asp Leu Gin Thr Leu Arg Ser Gly Asp Glu Gly Trp Leu 
30 180 185 190 

Val Leu Asp He Thr Ala Ala Ser Asp Arg Trp Leu Leu Asn His His 
195 200 205 

35 Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Ala Asp Gly His Ser 
210 215 220 



40 



Het Asp Pro Gly Leu Ala Gly Leu Leu Gly Arg Gin Ala Pro Arg Ser 
225 230 235 240 

Arg Gin Pro Phe Het Val Thr Phe Phe Arg Ala Ser Gin Ser Pro Val 
245 250 255 



Arg Ala Pro Arg Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys 
45 260 265 270 

Thr Asn Glu Leu Pro His Pro Asn Lys Leu Pro Gly He Phe Asp Asp 
275 280 285 

50 Gly His Gly Ser Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr 
290 295 300 



55 



Val Ser Phe Arg Asp Leu Gly Trp Leu Asp Trp Val He Ala Pro Gin 
305 310 315 320 



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Gly Tyr Ser Ala Tyr lyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp 
325 330 335 

Ser Cys Met Asn Ala Thr Asn His Ala lie Leu Gin Ser Leu Val His 
5 340 345 350 

Leu Met Lys Pro Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys 
355 360 365 

10 Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val He 
370 375 380 



15 



Leu Arg Lys His Arg Asn Met Val Val Lys Ala Cys Gly Cys His 
385 390 395 

(2) INFORMATION FOR SEQ ID N0:24: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1368 base pairs 
20 (B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



25 



30 



(ii) MOLECULE TYPE: cONA 



(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 1..1368 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24: 



ATG TCG GGA CTG CGA AAC ACC TCG GAG GCC GTT GCA GIG CTC GCC TCC 48 
35 Met Ser Gly Leu Arg Asn Thr Ser Glu Ala Val Ala Val Leu Ala Ser 
1 5 10 15 

CTG GGA CTC GGA ATG GTT CTG CTC ATG TTC GTG GCG ACC ACG CCG CCG 96 
Leu Gly Leu Gly Met Val Leu Leu Met Phe Val Ala Thr Thr Pro Pro 
40 20 25 30 

GCC GTT GAG GCC ACC CAG TCG GGG ATT TAC ATA GAC AAC GGC AAG GAC 144 

Ala Val Glu Ala Thr Gin Ser Gly He Tyr He Asp Asn Gly Lys Asp 

35 40 45 

45 

CAG ACG ATC ATG CAC AGA GTG CTG AGC GAG GAC GAC AAG CTG GAC GTC 192 

Gin Thr He Met His Arg Val Leu Ser Glu Asp Asp Lys Leu Asn Val 

50 55 60 

50 TCG TAC GAG ATC CTC GAG TTC CTG GGC ATC GCC GAA CGG CCG ACG CAC 240 
Ser Tyr Glu He Leu Glu Phe Leu Gly He Ala Glu Arg Pro Thr His 
65 70 75 80 

CTG AGC AGC CAC CAG TTG TCG CTG AGG AAG TCG GCT CCC AAG TTC CTG 288 
55 Leu Ser Ser His Gin Leu Ser Leu Arg Lys Ser Ala Pro Lys Phe Leu 

85 90 95 



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CTG GAC GTC TAC CAC CGC ATC ACG GCG GAG GAG GGT CTC AGC GAT CAG 
Leu Asp Val Tyr His Arg lie Tbr Ala Glu Glu Gly Leu Ser Asp Gin 
100 105 110 

GAT GAG GAC GAC GAC TAC GAA CGC GGC CAT CGG TCC AGG AGG AGC GCC 
Asp Glu Asp Asp Asp Tyr Glu Arg Gly His Arg Ser Arg Arg Ser Ala 
115 120 125 



336 



384 



10 GAC CTC GAG GAG GAT GAG GGC GAG CAG CAG AAG AAC TTC ATC ACC GAC 
Asp Leu Glu Glu Asp Glu Gly Glu Gin Gin Lys Asn Fhe He Thr Asp 
130 135 140 



432 



CTG GAC AAG CGG GCC ATC GAC GAG AGC GAC ATC ATC ATG ACC TTC CTG 
15 Leu Asp Lys Arg Ala He Asp Glu Ser Asp He He Het Thr Fhe Leu 
145 150 155 160 



480 



20 



AAC AAG CGC CAC CAC AAT GTG GAC GAA CTG CGT CAC GAG CAC GGC CGT 
Asn Lys Arg His His Asn Val Asp Glu Leu Arg His Glu His Gly Arg 
165 170 175 



528 



25 



CGC CTG TGG TTC GAC GTC TCC AAC GTG CCC AAC GAC AAC TAC CTG GTG 576 
Arg Leu Trp Fhe Asp Val Ser Asn Val Fro Asn Asp Asn Tyr Leu Val 
180 185 190 

ATG GCC GAG CTG CGC ATC TAT CAG AAC GCC AAC GAG GGC AAG TGG CTG 624 
Met Ala Glu Leu Arg He Tyr Gin Asn Ala Asn Glu Gly Lys Trp Leu 
195 200 205 



30 ACC GCC AAC AGG GAG TTC ACC ATC ACG GTA TAC GCC ATT GGC ACC GGC 
Thr Ala Asn Arg Glu Fhe Thr He Thr Val Tyr Ala He Gly Thr Gly 
210 215 220 



672 



ACG CTG GGC CAG CAC ACC ATG GAG CCG CTG TCC TCG GTG AAC ACC ACC 
35 Thr Leu Gly Gin His Thr Met Glu Fro Leu Ser Ser Val Asn Thr Thr 
225 230 235 240 



720 



40 



GGG GAC TAC GTG GGC TGG TTG GAG CTC AAC GTG ACC GAG GGC CTG CAC 
Gly Asp Tyr Val Gly Trp Leu Glu Leu Asn Val Thr Glu Gly Leu His 
245 250 255 



768 



45 



GAG TGG CTG GTC AAG TCG AAG GAC AAT CAT GGC ATC TAC ATT GGA GCA 816 
Glu Trp Leu Val Lys Ser Lys Asp Asn His Gly He Tyr He Gly Ala 
260 265 270 

CAC GCT GTC AAC CGA CCC GAC CGC GAG GTG AAG CTG GAC GAC ATT £GA 864 
His Ala Val Asn Arg Pro Asp Arg Glu Val Lys Leu Asp Asp He Gly 
275 280 285 



50 CTG ATC CAC CGC AAG GTG GAC GAC GAG TTC CAG CCC TTC ATG ATC GGC 
Leu He His Arg Lys Val Asp Asp Glu Fhe Gin Fro Fhe Het He Gly 
290 295 300 



912 



TTC TTC CGC GGA CCG GAG CTG ATC AAG GCG ACG GCC CAC AGC AGC CAC 
55 Fhe Fhe Arg Gly Fro Glu Leu He Lys Ala Thr Ala His Ser Ser His 
305 310 315 320 



960 



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CAC AGG AGC AAG CGA AGC GCC AGC CAT CCA CGC AAG CGC AAG AAG TCG 1008 

His Arg Ser Lys Arg Ser Ala Ser His Pro Arg Lys Arg Lys Lys Ser 
325 330 335 

5 

GTG TCG CCC AAC AAC GTG CCG CTG CTG GAA CCG ATG GAG AGC ACG CGC 1056 

Val Ser Pro Asn Asn Val Pro Leu Leu Glu Pro Met Glu Ser Thr Arg 
340 345 350 

10 AGC TGC CAG ATG CAG ACC CTG TAC ATA GAC TTC AAG GAT CTG GGC TGG 1104 
Ser Cys Gin Met Gin Thr Leu Tyr lie Asp Phe Lys Asp Leu Gly Trp 
355 360 365 

CAT GAC TGG ATC ATC GCA CCA GAG GGC TAT GGC GCC TTC TAC TGC AGC 1152 
15 His Asp Trp lie He Ala Pro Glu Gly Tyr Gly Ala Phe Tyr Cys Ser 
370 375 380 

GGC GAG TGC AAT TTC CCG CTC AAT GCG CAC ATG AAC GCC ACG AAC CAT 1200 
Gly Glu Cys Asn Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 
20 385 390 395 400 

GCG ATC GTC CAG ACC CTG GTC CAC CTG CTG GAG CCC AAG AAG GTG CCC 1248 
Ala He Val Gin Thr Leu Val His Leu Leu Glu Pro Lys Lys Val Pro 
405 410 415 

25 

AAG CCC TGC TGC GCT CCG ACC AGG CTG GGA GCA CTA CCC GTT CTG TAC 1296 
Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Ala Leu Pro Val Leu Tyr 
420 425 430 

30 CAC CTG AAC GAC GAG AAT GTG AAC CTG AAA AAG TAT AGA AAC ATG ATT 1344 
His Leu Asn Asp Glu Asn Val Asn Leu Lys Lys Tyr Arg Asn Met He 
435 440 445 

GTG AAA TCC TGC GGG TGC CAT TGA 1368 
35 Val Lys Ser Cys Gly Cys His 
450 455 



(2) INFORMATION FOR SEQ ID N0:2S: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 455 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: 

50 Met Ser Gly Leu Arg Asn Thr Ser Glu Ala Val Ala Val Leu Ala Ser 
1 5 10 15 



40 



45 



Leu Gly Leu Gly Met Val Leu Leu Met Phe Val Ala Thr Thr Pro Pro 
20 25 30 



55 



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Ala Val Glu Ala Thr Gin Ser Gly He Tyr He Asp Asn Gly Lys Asp 
35 40 45 

Gin Thr He Met His Arg Val Leu Ser Glu Asp Asp Lys Leu Asp Val 
5 50 55 60 

Ser lyr Glu He Leu Glu Phe Leu Gly He Ala Glu Arg Pro Thr His 
65 70 75 80 

10 Leu Ser Ser His Gin Leu Ser Leu Arg Lys Ser Ala Pro Lys Phe Leu 

85 90 95 



15 



Leu Asp Val Tyr His Arg He Thr Ala Glu Glu Gly Leu Ser Asp Gin 
100 105 110 

Asp Glu Asp Asp Asp Tyr Glu Arg Gly His Arg Ser Arg Arg Ser Ala 
115 120 125 



20 



Asp Leu Glu Glu Asp Glu Gly Glu Gin Gin Lys Asn Phe He Thr Asp 
130 135 140 



Leu Asp Lys Arg Ala He Asp Glu Ser Asp He He Met Thr Phe Leu 
145 150 155 160 

25 Asn Lys Arg His His Asn Val Asp Glu Leu Arg His Glu His Gly Arg 

165 170 175 



30 



Arg Leu Trp Phe Asp Val Ser Asn Val Pro Asn Asp Asn Tyr Leu Val 
180 185 190 

Met Ala Glu Leu Arg He Tyr Gin Asn Ala Asn Glu Gly Lys Trp Leu 
195 200 205 



35 



Thr Ala Asn Arg Glu Phe Thr He Thr Val Tyr Ala He Gly Thr Gly 
210 215 220 



Thr Leu Gly Gin His Thr Met Glu Pro Leu Ser Ser Val Asn Thr Thr 
225 230 235 240 

40 Gly Asp Tyr Val Gly Trp Leu Glu Leu Asn Val Thr Glu Gly Leu His 

245 250 255 



45 



Glu Trp Leu Val Lys Ser Lys Asp Asn His Gly He Tyr He Gly Ala 
260 265 270 

His Ala Val Asn Arg Pro Asp Arg Glu Val Lys Leu Asp Asp He Gly 
275 280 285 



Leu He His Arg Lys Val Asp Asp Glu Phe Gin Pro Phe Met He Gly 
50 290 295 300 

Phe Phe Arg Gly Pro Glu Leu He Lys Ala Thr Ala His Ser Ser His 
305 310 315 320 

55 His Arg Ser Lys Arg Ser Ala Ser His Pro Arg Lys Arg Lys Lys Ser 

325 330 335 



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Val Ser Pro Asn Asn Val Pro Leu Leu Glu Pro Met Glu Ser Thr Arg 
340 345 350 

5 Ser Cys Gin Met Gin Thr Leu Tyr He Asp Phe Lys Asp Leu Gly Trp 
355 360 365 

His Asp Trp He He Ala Pro Glu Gly Tyr Gly Ala Phe Tyr Cys Ser 
370 375 380 

10 

Gly Glu Cys Asn Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 
385 390 395 400 

Ala He Val Gin Thr Leu Val His Leu Leu Glu Pro Lys Lys Val Pro 
15 405 410 415 

Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Ala Leu Pro Val Leu Tyr 
420 425 430 

20 His Leu Asn Asp Glu Asn Val Asn Leu Lys Lys Tyr Arg Asn Met He 
435 440 445 

Val Lys Ser Cys Gly Cys His 
450 455 

25 

(2) INFORMATION FOR SEQ ID NO: 26: 

(i) SEQUENCE CHARACTERISTICS: 
30 (A) LENGTH: 104 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

35 (ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME/KEY: Protein 
40 (B) LOCATION: 1..104 

(D) OTHER INFORMATION: /note== "BMP3" 



45 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: 

Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp He Gly Trp Ser 
1 5 10 15 



Glu Trp He He Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ser Gly 
50 20 25 30 

Ala Cys Gin Phe Pro Met Pro Lys Ser Leu Lys Pro Ser Asn His Ala 
35 40 45 

55 Thr lie Gin Ser He Val Ala Arg Ala Val Gly Val Val Pro €ly He 

50 55 60 



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Pro Glu Pro Cys Cys Val Pro Glu Lys Met Ser Ser Leu Ser lie Leu 
65 70 75 80 

5 Phe Phe Asp Glu Asn Lys Asn Val Val Leu Lys Val Tyr Pro Asn Met 

85 90 95 

Thr Val Glu Ser Cys Ala Cys Arg 
100 

10 

(2) INFORMATION FOR SEQ ID NO: 27: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 
15 (B) TYPE: amino acid 

(C) STRAMDEDNESS: single 

(D) TOPOLOGY: linear 



20 



40 



(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HOMO SAPIENS 



(ix) FEATURE: 
25 (A) NAME/KEY: Protein 

(B) LOCATION: 1..102 
(D) OTHER INFORMATION: /note= "BMP5" 

30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: 

Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gin 
15 10 15 

35 Asp Trp He He Ala Pro Glu Gly Tyr Ala Ala Phe Tyr Cys Asp Gly 

20 25 30 



Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 
35 40 45 

He Val Gin Thr Leu Val His Leu Met Phe Pro Asp His Val Pro Lys 
50 55 60 



Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala He Ser Val Leu Tyr Phe 
45 65 70 75 80 

Asp Asp Ser Ser Asn Val He Leu Lys Lys Tyr Arg Asn Met Val Val 
85 90 95 

50 Arg Ser Cys Gly Cys His 

100 



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10 



(2) INFORMATION FOR SEQ ID NO: 28: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HOMO SAPIENS 



(ix) FEATURE: 
15 (A) NAME/KEY: Protein 

(B) LOCATION: 1..102 
(D) OTHER INFORMATION: /note= "BMP6" 

20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: 

Cys Arg Lys His Glu Leu lyr Val Ser Phe Gin Asp Leu Gly Trp Gin 
15 10 15 

25 Asp Trp He He Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly 

20 25 30 



30 



Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 
35 AO 45 

He Val Gin Thr Leu Val His Leu Met Asn Pro Glu T3rr Val Pro Lys 
50 55 60 



Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala He Ser Val Leu Tyr Phe 
35 65 70 75 80 

Asp Asp Asn Ser Asn Val He Leu Lys Lys Tyr Arg Trp Met Val Val 
85 90 95 

40 Arg Ala Cys Gly Cys His 

100 

(2) INFORMATION FOR SEQ ID NO: 29: 

45 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

50 (ii) MOLECULE TYPE: protein 



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(ix) FEATDRE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

(D) OTHER INFORMATION; /labels OPX 
5 /note» "VHEREIN EACH lAA IS INDEPENDENTLY SELECTED 

FROH A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS 
AS DEFINED IN THE SPECIFICATION (SECTION 11.6.2.)" 

10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: 

Cys Zaa Xaa His Glu Leu Tyr Val Xaa Phe Xaa Asp Leu Gly Trp Xaa 
1 5 10 15 

15 Asp Trp Xaa He Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly 

20 25 30 



20 



40 



Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala 
35 AO 45 

He Xaa Gin Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys 
50 55 60 



Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 
25 65 70 75 80 

Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xaa Arg Asn Met Val Val 
85 90 95 

30 Xaa Ala Cys Gly Cys His 

100 

(2) INFORMATION FOR SEQ ID NO: 30: 

35 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



(ix) FEATURE: 
45 (A) NAME/KEY: Protein 

(B) LOCATION: 1..97 

(D) OTHER INFORMATION: /label- GENERIC-SEQ5 

/note>= "VHEREIN EACH XAA IS INDEPENDENTLY SELECTED 
FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS 
50 AS DEFINED IN THE SPECIFICATION." 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: 

55 Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa Xaa Xaa 

15 10 15 



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Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro 
20 25 30 

5 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa 

35 AO 45 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro 
50 55 60 

10 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
65 70 75 80 

Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys 
15 85 90 95 

Xaa 



20 (2) INFORMATION FOR SEQ ID NO: 31: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

25 (C) iSTRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

30 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

(D) OTHER INFORMATION: /label= GENERIC-SEQ6 
35 /note= "WHEREIN EACH XAA IS INDEPENDENTLY SELECTED 

FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS 
AS DEFINED IN THE SPECIFICATION. " 



40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: 

Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa 
1 5 10 15 

45 Xaa Trp Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly 

20 25 30 



50 



Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala 
35 AO 45 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
50 55 60 



Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 
55 65 70 75 80 



- 151 - 



Zaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val 
85 90 95 

Xaa Xaa Cys Xaa Cys Xaa 
100 

(2) INFOSHATION FOR SEQ ID NO: 32: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1247 base pairs 

(B) TYPE; nucleic acid 

(C) STRANDEONESS: single 

(D) TOPOLOGY: linear 

(ii) HOLECULE TYPE: cDNA 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HOMO SAPIENS 
(F) TISSUE TYPE: BRAIN 

(iz) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 84.. 1199 

(D) OTHER INFORMATION: /product^ "GDF-1" 
/note- "GDF-1 CDNA" 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: 

GGGGACACCG GCCCCGCCCT CAGCCCACTG GTCCCGGGCC GCCGCGGACC CTGCGCACTC 

TCTGGTCATC GCCTGGGAGG AAG ATG CCA CCG CCG CAG CAA GGT CCC TGC 

Met Pro Pro Pro Gin Gin Gly Pro Cys 
1 5 

GGC CAC CAC CTC CTC CTC CTC CTG GCC CTG CTG CTG CCC TC€ CTG CCC 
Gly His His Leu Leu Leu Leu Leu Ala Leu Leu Leu Pro Ser Leu Pro 
10 15 20 25 

CTG ACC CGC GCC CCC GTG CCC CCA GGC CCA GCC GCC GCC CTG CTC CAG 
Leu Thr Arg Ala Pro Val Pro Pro Gly Pro Ala Ala Ala Leu Leu Gin 
30 35 40 

GCT CTA GGA CTG CGC GAT GAG CCC CAG GGT GCC CCC AGG CTC CGG CCG 
Ala Leu Gly Leu Arg Asp Glu Pro Gin Gly Ala Pro Arg Leu Arg Pro 
45 50 55 

GTT CCC CCG GTC ATG TGG CGC CTG TTT CGA CGC CGG GAC CCC CAG GAG 
Val Pro Pro Val Met Trp Arg Leu Phe Arg Arg Arg Asp Pro Gin Glu 
60 65 70 

ACC AGG TCT GGC TCG CGG CGG ACG TCC CCA GG6 GTC ACC CTG CAA CCG 
Thr Arg Ser Gly Ser Arg Arg Thr Ser Pro Gly Val Thr Leu Gin Pro 
75 80 85 



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TGC CAC GTG GAG GAG CTG GGG GTC GCC GGA AAC ATC GTG CGC CAC ATC 
Cys His Val Glu Glu Leu Gly Val Ala Gly Asn lie Val Arg His He 
90 95 100 105 

5 

CCG GAC CGC GGT GCG CCC ACC CGG GCC TCG GAG CCT GTC TCG GCC GCG 
Pro Asp Arg Gly Ala Pro Thr Arg Ala Ser Glu Pro Val Ser Ala Ala 
110 115 120 

10 GGG CAT TGC CCT GAG TGG ACA GTC GTC TTC GAC CTG TCG GCT GTG GAA 
Gly His Cys Pro Glu Trp Thr Val Val Phe Asp Leu Ser Ala Val Glu 
125 130 135 



15 



20 



25 



CCC GCT GAG CGC CCG AGC CGG GCC CGC CTG GAG CTG CGT TTC GCG €CG 
Pro Ala Glu Arg Pro Ser Afg Ala Arg Leu Glu Leu Arg Phe Ala Ala 
140 145 150 

GCG GCG GCG GCA GCC CCG GAG GGC GGC TGG GAG CTG AGC GTG GCG CAA 
Ala Ala Ala Ala Ala Pro Glu Gly Gly Trp Glu Leu Ser Val Ala Gin 
155 160 165 

GCG GGC CAG GGC GCG GGC GCG GAC CCC GGG CCG GTG CTG CTC CGC CAG 
Ala Gly Gin Gly Ala Gly Ala Asp Pro Gly Pro Val Leu Leu Arg Gin 
170 175 180 185 

TTG GTG CCC GCC CTG GGG CCG CCA GTG CGC GCG GAG CTG CTG GGC GCC 
Leu Val Pro Ala Leu Gly Pro Pro Val Arg Ala Glu Leu Leu Gly Ala 
190 195 200 



398 



446 



494 



542 



590 



638 



686 



30 GCT TGG GCT CGC AAC GCC TCA TGG CCG CGC AGC CTC CGC CTG GCG CTG 
Ala Trp Ala Arg Asn Ala Ser Trp Pro Arg Ser Leu Arg Leu Ala Leu 
205 210 215 

GCG CTA CGC CCC CGG GCC CCT GCC GCC TGC GCG CGC CTG GCC GAG GCC 
35 Ala Leu Arg Pro Arg Ala Pro Ala Ala Cys Ala Arg Leu Ala Glu Ala 
220 225 230 

TCG CTG CTG CTG GTG ACC CTC GAC CCG CGC CTG TGC CAC CCC CTG GCC 
Ser Leu Leu Leu Val Thr Leu Asp Pro Arg Leu Cys His Pro Leu Ala 
40 235 240 245 

CGG CCG CGG CGC GAC GCC GAA CCC GTG TTG GGC GGC GGC CCC GGG GGC 
Arg Pro Arg Arg Asp Ala Glu Pro Val Leu Gly Gly Gly Pro Gly Gly 
250 255 260 265 

GCT TGT CGC GCG CGG CGG CTG TAC CTG AGC TTC CGC GAG GTG GGC TGG 
Ala Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp 
270 275 280 

50 CAC CGC TGG GTC ATC GCG CCG CGC GGC TTC CTG GCC AAC TAC TGC CAG 
His Arg Trp Val He Ala Pro Arg Gly Phe Leu Ala Asn Tyr Cys Gin 
285 290 295 

GGT CAG TGC GCG CTG CCC GTC GCG CTG TCG GGG TCC GGG €GG CCG CCG 
55 Gly Gin Cys Ala Leu Pro Val Ala Leu Ser Cly Ser Gly Gly Pro Pro 
300 305 310 



45 



734 



782 



830 



878 



926 



974 



1022 



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GCG CTC AAC CAC GCT GTG CTG CGC GCG CTC ATG CAC GCG GCC GCC CCG 1070 

Ala Leu Asn His Ala Val Leu Arg Ala Leu Met His Ala Ala Ala Pro 
315 320 325 

5 

GGA GCC GCC GAC CTG CCC TGC TGC GTG CCC GCG CGC CTG TCG CCC ATC 1118 

Gly Ala Ala Asp Leu Pro Cys Cys Val Pro Ala Arg Leu Ser Pro lie 
330 335 340 345 

10 TCC GTG CTC TTC TTT GAC AAC AGC GAC AAC GTG GTG CTG CGG CAG TAT 1166 
Ser Val Leu Phe Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gin Tyr 
350 355 360 

GAG GAC ATG GTG GTG GAC GAG TGC GGC TGC CGC TAACCCGGGG CGGGCAGGGA 1219 
15 Glu Asp Met Val Val Asp Glu Cys Gly Cys Arg 
365 370 



20 



CCCGGGCCCA ACAATAAATG CCGCGTGG 1247 
(2) INFORMATION FOR SEQ ID NO: 33: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 372 amino acids 
25 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(il) MOLECULE TYPE: protein 

30 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:33: 

Met Pro Pro Pro Gin Gin Gly Pro Cys Gly His His Leu Leu Leu Leu 
1 5 10 15 

35 Leu Ala Leu Leu Leu Pro Ser Leu Pro Leu Thr Arg Ala Pro Val Pro 
20 25 30 

Pro Gly Pro Ala Ala Ala Leu Leu Gin Ala Leu Gly Leu Arg Asp Glu 
35 40 45 

40 

Pro Gin Gly Ala Pro Arg Leu Arg Pro Val Pro Pro Val Met Trp Arg 
50 55 60 

Leu Phe Arg Arg Arg Asp Pro Gin Glu Thr Arg Ser Gly Ser Arg Arg 
45 65 70 75 80 

Thr Ser Pro Gly Val Thr Leu Gin Pro Cys His Val Glu Glu Leu Gly 
85 90 95 

50 Val Ala Gly Asn lie Val Arg His lie Pro Asp Arg Gly Ala Pro Thr 
100 105 110 

Arg Ala Ser Glu Pro Val Ser Ala Ala Gly His Cys Pro Glu Trp Thr 
115 120 125 

55 



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Val Val Phe Asp Leu Ser Ala Val Glu Pro Ala Glu Arg Pro Ser Arg 
130 135 140 

Ala Are Leu Glu Leu Arg Phe Ala Ala Ala Ala Ala Ala Ala Pro Glu 
5 145 150 155 160 

Gly Gly Trp Glu Leu Ser Val Ala Gin Ala Gly Gin Gly Ala Gly Ala 
165 170 175 

10 Asp Pro Gly Pro Val Leu Leu Arg Gin Leu Val Pro Ala Leu Gly Pro 
180 185 190 



15 



Pro Val Arg Ala Glu Leu Leu Gly Ala Ala Trp Ala Arg Asn Ala Ser 
195 200 205 

Trp Pro Arg Ser Leu Arg Leu Ala Leu Ala Leu Arg Pro Arg Ala Pro 
210 215 220 



Ala Ala Cys Ala Arg Leu Ala Glu Ala Ser Leu Leu Leu Val Thr Leu 
20 225 230 235 240 

Asp Pro Arg Leu Cys His Pro Leu Ala Arg Pro Arg Arg Asp Ala Glu 
245 250 255 

25 Pro Val Leu Gly Gly Gly Pro Gly Gly Ala Cys Arg Ala Arg Arg Leu 
260 265 270 



30 



Tyr Val Ser Phe Arg Glu Val Gly Trp His Arg Trp Val He Ala Pro 
275 280 285 

Arg Gly Phe Leu Ala Asn Tyr Cys Gin Gly Gin Cys Ala Leu Pro Val 
290 295 300 



Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala Leu Asn His Ala Val Leu 
35 305 310 315 320 

Arg Ala Leu Met His Ala Ala Ala Pro Gly Ala Ala Asp Leu Pro Cys 
325 330 335 

40 Cys Val Pro Ala Arg Leu Ser Pro He Ser Val Leu Phe Phe Asp Asn 
340 345 350 



45 



Ser Asp Asn Val Val Leu Arg Gin Tyr Glu Asp Met Val Val Asp Glu 
355 360 365 

Cys Gly Cys Arg 
370 



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What is claimed is: 

1 1. A method for maintaining normal liver function 

2 following hepatic tissue injury in a mammal or in 

3 anticipation of such injury, the method comprising 

4 the step of providing to said liver a 

5 therapeutically effective concentration o£ a 

6 morphogen. 

1 2. A method for enhancing the level of a depressed 

2 liver function in a mammal/ said liver function 

3 being depressed due to a tissue injury or disease, 

4 the method comprising the step of providing to said 

5 liver a therapeutically effective concentration of 

6 a morphogen. 

1 3. The method of claim 1 or 2 wherein said step of 

2 providing a therapeutically effective morphogen 

3 concentration comprises the step of administering a 

4 therapeutically effective concentration of a 

5 morphogen to said mammal. 

1 4. The method of claim 1 or 2 wherein said step of 

2 providing a therapeutically effective morphogen 

3 concentration comprises the step of administering 

4 to said mammal an agent that stimulates in vivo a 

5 therapeutically effective concentration of an 

6 endogenous morphogen. 

1 5. The method of claim 1 or 2 wherein said liver 

2 function is reduced due to a hepatocellular injury. 

1 6. The method of claim 5 wherein the etiology of said 

2 hepatocellular injury is metabolic, infectious, 

3 toxic, autoimmune, ischemic or nutritional. 



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1 7. The method of claim 6 wherein said hepatocellular 

2 injury comprises hyperbilirubinemia , viral 

3 hepatitis, alcoholic liver disease, portal 

4 hypertension, neonatal hepatitis or hepatic 

5 encephalopathy • 

1 8, The method of claim 1 or 2 wherein said liver 

2 function is reduced due to liver cirrhosis. 

1 9. The method of claim 1 or 2 wherein said liver 

2 function is reduced due to a neoplasm. 

1 10. The method of claim 9 wherein said neoplasm 

2 comprises hepatocytes. 

1 11. The method of claim 10 wherein said neoplasm 

2 comprises a hepatic adenoma, nodular hyperplasia, 

3 hepatocellular carcinoma or a hemagio sarcoma. 

1 12. The method of claim 9 wherein said neoplasm 

2 comprises cells of a metastatic cancer. 

1 13. The method of claim 12 wherein said metastatic 

2 cancer originated in tissue of the gastrointestinal 

3 tract, breast, lung or skin. 

1 14. The method of claim 1 or 2 wherein said liver is at 

2 risk of hepatic failure. 

1 15. The method of claim 5 wherein said tissue injury 

2 results from toxic concentrations of ammonia, 

3 phenol, ethanol, infectious agent byproduct, carbon 

4 tetrachloride or a metal. 



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1 16. The method of claim 5 wherein said tissue injury 

2 results from a toxic concentration of a 

3 pharmaceutical agent or its metabolite. 

1 17. The method of claim 1 or 2 wherein said tissue 

2 injury is induced in a clinical procedure. 

1 18. The method of claim 17 wherein said tissue injury 

2 is induced in a surgical procedure. 

1 19. A method for inducing regeneration of lost or 

2 damaged hepatic tissue in a mammal, the method 

3 comprising the step of: 

4 providing to the locus of said damaged or lost 

5 tissue, a therapeutically effective concentration 

6 of a morphogen. 

1 20. The method of claim 19 wherein said morphogen is 

2 provided to said locus in association with a 

3 biocompatible, acellular matrix. 

1 21. The method of claim 20 wherein said matrix has 

2 components specific for said tissue. 

1 22. The method of claim 20 wherein said matrix is 

2 biodegradable. 

1 23. The method of claim 20 wherein said matrix is 

2 derived from organ-specific tissue. 

1 24. The method of claim 20 wherein said matrix 

2 comprises collagen and cell attachment factors 

3 specific for said tissue. 



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- 158 - 



1 25. The method of claim 20 wherein said matrix 

2 comprises a synthetic polymeric material. 

1 26. The method of claim 20 wherein said matrix defines 

2 pores of a dimension sufficient to permit the 

3 influx, differentiation and proliferation of 

4 migratory progenitor cells from the body of said 

5 mammal • 

1 27. The method of claim 25 wherein said polymeric 

2 material comprises poly lactic acid, polybutyric 

3 acid, polyglycolic acid, polyanydr ide , or 

4 copolymers thereof. 

1 28. A method for inducing hepatic tissue formation in a 

2 mammal, said method comprising the steps of: 

3 a) stimulating progenitor cells by exposure 

4 to a therapeutically effective morphogen 

5 concentration, 

6 b) implanting said stimulated cells at a 

7 liver-specific locus in vivo, such that said 

8 stimulated cells are capable of proliferation and 

9 differentiation at said locus. 

1 29. The method of claim 28 wherein said progenitor 

2 cells are of mesenchymal origin. 

1 30. The method of claim 28 wherein said stimulated 

2 cells are implanted at said locus, in association 

3 with a biocompatible, acellular matrix. 



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1 31. A method for enhancing integration of a liver 

2 tissue transplant, the method comprising the step 

3 of providing a therapeutically effective 

4 concentration of a morphogen to the liver tissue 

5 transplant locus. 

1 32. The method of claim 31 wherein said morphogen is 

2 provided to said locus prior to transplantation. 

1 33. The method of claim 31 wherein said morphogen is 

2 provided to said locus concurrent with 

3 transplantation . 

1 34. A method for enhancing integration of a liver 

2 tissue transplant, the method comprising the step 

3 of providing a therapeutically effective 

4 concentration of a morphogen to the transplant 

5 tissue. 

1 35. The method of claim 34 wherein said morphogen is 

2 provided to said tissue prior to transplantation. 

1 36. The method of claim 34 wherein said morphogen is 

2 provided to said transplant tissue prior to removal 

3 of said tissue from the donor. 

1 37. The method of claim 35 wherein said tissue is a 

2 synthetic tissue. 



1 
2 
3 



38. 



The method of claim 37 wherein said synthetic 
tissue comprises proliferating hepatocytes disposed 
on a biocompatible acellular matrix. 



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1 39. The method of claims 31 or 34 wherein said step of 

2 providing a therapeutically effective morphogen 

3 concentration is performed by administering a 

4 morphogen to said tissue or transplant locus* 

1 40. The method of claims 31 or 34 wherein said step of 

2 providing a therapeutically effective morphogen 

3 concentration is performed by administering a 

4 morphogen- stimulating agent to said tissue or 

5 transplant locus. 

1 41. The method of claim 1, 2, 19, 28, 31 or 34 wherein 

2 said morphogen comprises an amino acid sequence 

3 sharing at least 70% homology with one of the 

4 sequences selected from the group consisting of: 

5 OP-1, OP-2, CBMP2, Vgl(fx), Vgr(fx), DPP(fx), 

6 GDF-l(fx) and 60A(fx). 

1 42. The method of claim 41 wherein said morphogen 

2 comprises an amino acid sequence sharing at least 

3 80% homology with one of the sequences selected 

4 from the group consisting of: OP-1, OP-2, CBMP2, 

5 Vgl(fx), Vgr(fx), DPP(fx), GDF-l(fx), and 60A(fx). 

1 43. The method of claim 42 wherein said morphogen 

2 comprises an amino acid sequence having greater 

3 than 60% amino acid identity with the sequence 

4 defined by residues 43-139 of Seq. ID No. 5 (hOPl.) 

1 44. The method of claim 43 wherein said morphogen 

2 comprises an amino acid sequence having greater 

3 than 65% amino acid identity with the sequence 

4 defined by residues 43-139 of Seq. ID No. 5 (hOPl.) 



w6 94/06449 



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- 161 - 

1 45. The method of claim 44 wherein said morphogen 

2 comprises an amino acid sequence defined by 

3 residues 43-139 of Seq. ID No. 5 (hOPl), including 

4 allelic and species variants thereof. 

1 46. The method of claim 1, 2, 19, 28, 31 or 34 wherein 

2 said morphogen is provided in its pro form. 

1 47. The method of claim 45 wherein the morphogen is 

2 provided in its pro form. 

1 48. The method of claim 47 wherein said morphogen 

2 comprises an amino acid sequence defined by 

3 residues 30-431 of Seq. ID No. 16. 

1 49. A method for correcting a liver function deficiency 

2 in a mammal, the method comprising the step of : 
3 

4 a) attaching cells to a biocompatible, acellular 

5 matrix to create a cell-matrix structure, the 

6 matrix being suitable for cellular attachment, 

7 proliferation and ingrowth, and said cells being 

8 capable of expressing one or more proteins in vivo 

9 to correct said liver function deficiency; and 
10 

11 b) implanting said cell-matrix structure, 

12 together with a therapeutically effective 

13 concentration of a morphogen, in said mammal. 

1 50. A gene therapy treatment method for correcting a 

2 protein deficiency in a mammal, the method 

3 comprising the step of: 
4 



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5 a) attaching cells to a biocompatible, acellular 

6 matrix to create a cell-matrix structure, the 

7 matrix being suitable for cellular attachment, 

8 infiltration, proliferation and differentiation, 

9 and said cells being capable of expressing one or 

10 more proteins in vivo to correct said protein 

11 deficiency; and 
12 

13 b) implanting said cell-matrix structure, 

14 together with a therapeutically effective 

15 concentration of a morphogen, in said mammal. 
16 

1 51. The method of claim 49 or 50 wherein said morphogen 

2 is adsorbed to a surface of said matrix. 

1 52. The method of claim 49 or 50 comprising the 

2 additional step of stimulating proliferation of 

3 said cells prior to implantation. 

1 53. The method of claim 52 wherein said cells are 

2 stimulated by exposure to a morphogen. 

1 54. The method of claim 49 or 50 wherein said cells 

2 comprise foreign genetic material. 

1 55. The method of claim 49 or 50 wherein said cells are 

2 allogenic. 

1 56. the method of claim 49 or 50 wherein said matrix is 

2 in vivo biodegradable. 



1 
2 



57. 



The method of claim 49 or 50 wherein said matrix is 
derived from organ-specific tissue. 



wo 94/06449 



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- 163 ^ 

1 58. The method of claim 49 or 50 wherein said matrix 

2 comprises a synthetic polymeric material. 

1 59. The method of claim 58 wherein said polymeric 

2 material comprises polylactic acid^ polybutyric 

3 acid, polyglycolic acid, polyanhydride , or 
,4 copolymers thereof. 

1 60. The method of claim 49 or 50 wherein said matrix 

2 comprises one or more tissue-derived structural 

3 molecules • 

1 61. The method of claim 60 wherein said matrix 

2 comprises hyalurinc acid, laminin or collagen. 

1 62. The method of claim 49 or 50 wherein said matrix 

2 further comprises cell attachment factors. 

1 63. The method of claim 62 wherein said cell attachment 

2 factors include glycosaminoglycans, proteoglycans. 

1 64. The method of claim 49 or 50 wherein said cell- 

2 matrix structure is implanted at a liver-specific 

3 tissue locus. 

1 65. The method of claim 49 or 50 wherein said cell- 

2 matrix structure is implanted at an extra-hepatic 

3 tissue locus. 

1 66. A composition for correcting a liver function 

2 deficiency in a mammal, the composition comprising: 
3 

4 a) cells capable of expressing ona or more 

5 protein in vivo to correct said liver function 

6 deficiency; 



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7 

8 b) a biocompatible/ acellular matrix having a 

9 three-dimensional structure suitable for the 

10 attachment, infiltration, differentiation and 

11 proliferation of said hepatocytic cells; and 
12 

13 c) a morphogeny such that said cells, when 

14 attached to said matrix and stimulated by said 

15 morphogen, are capable of correcting said liver 

16 function deficiency when implanted in said mammal • 
17 

1 67. A composition useful in a gene therapy protocol to 

2 correct a protein deficiency in a manunal, the 

3 composition comprising: 
4 

5 a) cells capable of expressing one or more 

6 protein in vivo to correct said protein deficiency; 
7 

8 b) a biocompatible, acellular matrix having a 

9 three-dimensional structure suitable for the 

10 attachment, infiltration, differentiation and 

11 proliferation of said cells; and 
12 

13 c) a morphogen, such that said cells, when 

14 attached to said matrix and stimulated by said 

15 morphogen, are capable of expressing one or more 

16 proteins to correct said protein deficiency when 

17 implanted in said mammal. 

1 68. The composition of claim 66 or 67 wherein said 

2 cells comprise foreign genetic material. 



1 

2 



69. The composition of claim 67 wherein said foreign 
genetic material encodes said correcting proteins. 



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1 70. The composition of claim 66 or 67 wherein said 

2 cells are allogenic. 

1 71. The composition of claim 66 or 67 wherein said 

2 matrix is in vivo biodegradable. 

1 72. The composition of claim 66 or 67 wherein said 

2 matrix is derived from organ-specific tissue. 

1 73. The composition of claim 72 wherein said matrix is 

2 derived from hepatic t^issue. 
3 

1 74. The composition of claim 66 or 67 wherein said 

2 matrix comprises a synthetic polymeric material . 

1 75. The composition of claim 74 wherein said polymeric 

2 material comprises poly lactic acid, polybutyric 

3 acid, polyglycolic acid, polyanhydride, or 

4 copolymers thereof. 

1 76. The composition of claim 66 or 67 wherein said 

2 matrix comprises a tissue-derived structural 

3 molecule. 

1 77. The composition of claim 76 wherein said structural 

2 molecule includes collagen, laminin or hyaluronic 

3 acid. 

1 78. The composition of claim 66 or 67 wherein said 

2 matrix further comprises cell attachment factors. 



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1 79 • The composition of claim 78 wherein said cell 

2 attachment factors include glycosaminoglycans or 

3 proteoglycans . 

1 80. The invention of claim 49^ 50, 66 or 67 wherein 

2 said morphogen comprises an amino acid sequence 

3 sharing at least 70% homology with one of the 

4 sequences selected from the group consisting of: 

5 OP-1, OP-2, CBMP2, Vgl(fx), Vgr(fx), DPP(fx), 

6 GDF-l(fx) and 60A(fx). 

1 81. The invention of claim 80 wherein said morphogen 

2 comprises an amino acid sequence sharing at least 

3 80% homology with one of the sequences selected 

4 from the group consisting of: OP-1, OP-2, CBMP2, 

5 Vgl(fx), Vgr(fx), DPP(fx), GDF-l(fx), and 60A(fx). 

1 82. The invention of claim 81 wherein said morphogen 

2 comprises an amino acid sequence having greater 

3 than 60% amino acid identity with the sequence 

4 defined by residues 43-139 of Seq. ID No. 5 (hOPl.) 

1 83. The invention of claim 82 wherein said morphogen 

2 comprises an amino acid sequence having greater 

3 than 65% amino acid identity with the sequence 

4 defined by residues 43-139 of Seq. ID No. 5 (hOPl.) 

1 84. The invention of claim 83 wherein said morphogen 

2 comprises an amino acid sequence defined by 

3 residues 43-139 of Seq. ID No. 5 (hOPl), including 

4 allelic and species variants thereof. 

1 85. The invention of claim 49, 50, 66 or 67 wherein 

2 said morphogen is provided in its pro form. 



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1 86. The invention of claim 84 wherein the morphogen is 

2 provided in its pro form. 

« 1 87. The invention of claim 86 wherein said morphogen 

2 comprises an amino acid sequence defined by 

3 residues 30-431 of Seq. ID No. 16. 

1 88. The use of a morphogen in the manufacture of a 

2 pharmaceutical for enhancing the level of depressed 

3 liver function or for maintaining normal liver 

4 function following tissue injury or disease. 

1 89. The use of a morphogen in the manufacture of a 

2 pharmaceutical to regenerate lost or damaged 

3 hepatic tissue or to enhance integration of a liver 

4 transplant. 

1 90. The use of a morphogen in the manufacture of an 

2 implantable, proliferating cellular device to 

3 correct a liver function deficiency or protein 

4 deficiency in a mammal. 

1 91. The use according to claim 88, 89 or 90 wherein 

2 said morphogen comprises an amino acid sequence 

3 sharing at least 70% homology with one of the 

4 sequences selected from the group consisting of: 

5 OP-1, OP-2, CBMP2, Vgl(fx), Vgr(fx), DPP(fx), 

6 GDF-l(fx) and 60A(fx). 

1 92. The use according to claim 88, 89 or 90 wherein 

2 said morphogen comprises an amino acid sequence 

3 having greater than 60% amino acid identity with 

4 the sequence defined by residues 43-139 of Seq. ID 

5 No. 5 (hOPl.) 



wo 94/06449 



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1 93. The use according to claim 88, 89 or 90 wherein 

2 said morphogen comprises an amino acid sequence 

3 defined by residues 43-139 of Seq. ID No. 5 (hOPl), 

4 including allelic and species variants thereof. 

1 94. A kit for detecting a reduced liver function or 

2 hepatocellular injury in a mammal, or for 

3 evaluating the efficacy of a therapy for treating a 

4 malady associated with reduced liver function or 

5 hepatocellular injury in a mammal, the kit 

6 comprising: 

7 c) means for capturing a cell or body fluid 

8 sample obtained from a mammal; 

9 b) a binding protein that interacts specifically 

10 with a morphogen in said sample so as to form a 

11 binding protein-morphogen complex; 

12 c) means for detecting said complex. 

1 95. The kit of claim 94 which said binding protein has 

2 specificity for an epitope defined by part or all 

3 of the pro region of a morphogen. 

1 96. A method for detecting a reduced liver function or 

2 hepatocellular injury in a mammal, or for 

3 evaluating the efficacy of a therapy for treating a 

4 malady associated with reduced liver function or 

5 hepatocellular injury in a mammal, the method 

6 comprising the step of: 

7 detecting fluctuations in the physiological 

8 concentration of a morphogen or a morphogen 

9 antibody titer present in the serum or peritoneal 

10 fluid of said mammal, said fluctuations being 

11 indicative of an increase in hepatic cell death. 



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- 169 - 

1 97, The invention of claim 1, 2, 28, 31, A9, 50, 66, 

2 67, 88, 89 or 90 wherein said morphogen comprises 

3 dimeric protein species complexed with a peptide 

4 comprising a pro region of a member of the 

5 morphogen family, or an allelic, species or other 

6 sequence variant thereof. 

1 98. The invention of claim 97 wherein said dimeric 

2 morphogen species is noncovalently complexed with 

3 said peptide. 

1 99. The invention of claim 97 wherein said dimeric 

2 morphogen species is complexed with two said 

3 peptides. 

1 100. The invention of claim 97 wherein said peptide 

2 comprises at least the first 18 amino acids of a 

3 sequence defining said pro region. 

1 101. The invention of claim 100 wherein said peptide 

2 comprises the full length form of said pro region. 

1 102. The invention of claim 97 wherein said peptide 

2 comprises a nucleic acid that hybridizes under 

3 stringent conditions with a DNA defined by nucleotides 

4 136-192 of Seq. ID No. 16, or nucleotides 157-211 of 

5 Seq. ID Ko. 20. 

1 103. The invention of claim 97 wherein said complex is 

2 further stabilized by exposure to a basic amino acid, 

3 detergent or a carrier protein. 



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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Oassification ^ : 
A61K 37/02 



A3 



(1 1) International Publication Number: WO 94/06449 

(43) International Publication Date : 3 1 March ] 994 (3 1 .03.94) 



(21) International Application Number: PCT/US93/08808 

(22) International Filing Date : 1 6 September 1 993 ( 1 6.09.93) 



(30) Priority data: 

946,238 
029,335 
040,510 



16 September 1992 (16.09.92) US 
4 March 1993(04.03.93) US 
31 March 1993(31.03.93) US 



(71) Applicant: CREATIVE BIOMOLECULES, INC. [US/ 

US]; 45 South Street, Hopkinton, MA 01748 (US). 

(72) Inventors: KUBERASAMPATH, Thangavel ; Six Spring 

Street, Medway, MA 02053 (US). RUEGER, David, C. ; 
19 Downey Street, Hopkinton, MA 01748 (US). OPPER- 
MANN, Hermann ; 25 Summer Hill Road, Medway, 
MA 02053 (US). PANG, Roy, H., L. ; 15 Partridge 
Road. Etna, NH 03750 (US). COHEN, Charles, M. ; 98 
Wnthrop Street, Medway, MA 02053 (US). 02KAY- 
NAK, Engin ; 44 Purdue Drive, Milford, MA 01757 
(US). SMART, John, E. ; 50 Meadow Brook Road, Wes- 
ton, MA 02193 (US). 



(74) Agent: KELLEY, Robin, D.; Testa, Hurwitz & Thibeault, 
Exchange Place, 53 State Street, Boston, MA 02109 
(US). 



(81) Designated States: AT, AU, BB, BG, BR, CA, CH, CZ, 
DE, DK, ES, FI, GB, HU, JP, KP, KR, LK, LU, MG, 
MN, MW, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SK. 
UA, European patent (AT, BE, CH, DE, DK, ES, PR, 
GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent 
(BP, BJ, CP, CG, CI, CM, GA, GN, ML, MR, NE, SN, 
TD, TG). 



Published 

Wiih iniernauonal search report. 

Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 
amendments. 

(88) Date of publication of the international search report: 

1st September 1994 (01.09.94) 



(54) Title: MORPHOGEN-INDUCED LIVER REGENERATION 



(57) Abstract 

Disclosed are therapeutic treatment methods, compositions and devices for maintaining liver function in a mammal, in- 
cluding means for regenerating lost or damaged hepatic tissue, means for enhancing viability and integration of hepatic tissue 
and organ transplants, and means for correcting liver function deficiencies, including means for enhancing diminished liver func- 
tion due to tissue injury or disease. The methods, compositions and devices on this invention all provide a therapeutically effec- 
tive morphogen concentration to the hepatic cells to be treated. Also disclosed are methods and compositions useful in a ^ene 
therapy or drug delivery protocol for correcting a protein deficiency in a mammal. 



FOR THE PURPOSES OF INFORMATION ONLY 

Codes used to identi^ States party to the PCX on the front pages of pamphlets publishing international 
applications under the PCT. 



AT 


AusuU 


GB 


United Kingdom 


MR 


Mauritania 


AU 


Australia 


ce 


Georgia 


MW 


Malawi 


BB 


Bartiados 


CN 


Guinea 


NE 


Niger 


BE 


Belgium 


GR 


Greece 


NL 


Netherlands 


BF 


Burkina Faso 


HU 


Hungary 


NO 


Norway 


BC 


Bulgaria 


IE 


Ireland 


NZ 


New Zealand 


BJ 


Benin 


IT 


lUly 


PL 


Poland 


BR 


Brazil 


JP 


Japan 


PT 


Portugal 


BY 


Belarus 


KE 


Kenya 


RO 


Romania 


CA 


Canada 


KG 


Kyrgystan 


RU 


Russian Federation 


CF 


Central African Republic 


KP 


Democratic People's Republic 


SD 


Sudan 


CC 


Congo 




of Korea 


SE 


Sweden 


CH 


Swiueerland 


KR 


Republic of Korea 


SI 


Slovenia 


CI 


Cdtc d*lvoirc 


KZ 


Kazakhstan 


5K 


Slovakia 


CM 


Cameroon 


Li 


Uechlcnstein 


SN 


Senegal 


CN 


China 


LK 


Sri Lanka 


TD 


Chad 


cs 


Ceechoslovakia 


LU 


Luxembourg 


TC 


Togo 


cz 


Oecch Republic 


LV 


Latvia 


TJ 


Tajikistan 


DE 


Germany 


MC 


Monaco 


TT 


Trinidad and Tobago 


DK 


Denmark 


MD 


Republic of Moldova 


UA 


Ukraine 


ES 


Spain 


MC 


Madagascar 


US 


United States of America 


Fl 


Finland 


ML 


Mali 


uz 


Uzbekistan 


FR 


France 


MN 


Mongolia 


VN 


Vict Nam 


CA 


Gabon 









INTERNATIONAL SEARCH REPORT 



Intel jnal AppUcatiofli No 

PCT/US 93/08808 



A. CLASSinCATION OF SUBJECT MATTER 

IPC 5 A61K37/02 



Acconlipg to International Patent Qasnfication gPQ or to both national dagiecation and IPC 



B. FIELDS SEARCHED 



Minimum documentation seardted (classification system followed by dassificalion symfatds) 

li^C 5 A61K 



Documentation searched other than nmurnum documentatioD to the extent that luch documents arc included in the fields searched 



Electronic dau base consulted dunng the imcmaiicnal search (name of dau base and. wh« |»t»«»i^i, ^r^t. 



C DOCUMENTS CONSIDERED TO BE RELEVANT 



Category* 


Otation of document, with indication, where appropiiate, of the relevant pasagcs 


Relevant to claim No. j 


P.X 


W0,A.92 15323 (CREATIVE BIOMOLECULES) 17 

September 1992 

cited in the application 

see page 6, line 27 - page 7, line 8 

see page 14. line 13-25; claims 


1-91 


P.X, 
Y 


W0,A,93 04692 (CREATIVE BIOMOLECULES) 18 
March 1993 
see page 1-4 

see page 6, line 26 - page 7 

see page 80, line 15 - page 83, line 4: 

claims 1,26-32,49,55 


1-91 


X 


W0.A,92 09301 (THE AMERICAN NATIONAL RED 

CROSS) 11 June 1992 

see page 9, line 8-15 

see page 15, line 15-20; claims 

1,4.8,11,15 

-/-- 


1.5.17. 
19.35 



Fmtber documents arc listed in the coniinuaiion of boi C 



jX I patent fiimily monbers are listed in 



Spedal categories of dted documents : 

A* document defining the general state of the art which is not 
considered to be of particular relevance 

'£' cartier document but publidted on or alter the intemationa] 
filing date 

L' document which may throw doubts on priority dain^s) or 
which is died to establish the pubUcabon dau of anoQicr 
dtation or other spedal reason <as specified) 

*0* document referring to an oral disdosurc, use, cxhitaition or 
other means 

P* document pubbshed prior to the international filing date but 
later than the pribhiy date claimed 



T* later document published after the intematicnal filing date 
or priority dau and not in conflict with the application but 
dted to undentand the ptindple or theory underlying the 
invcnttOD 

*X* document of particular relcvanee; the daimed invention 
carmot be considered novel or cannot be considered to 
involve an inventive step when the document is taken alone 

*Y' document of particular relevance; fte daimed invention 
cannot be c ons ider e d to involve an inventive step wtten the 
document is combined with one or more other such docu- 
nkcnts, such combtiuDon being obvious to a penon ddUed 
in the art. 

*&* document member of the same patent family 



Date of the actual completion of the international search 

19 July 1994 



Name and mailing address of the ISA 

European Patent Office, P.B. 581 8 Patcnttaan 2 
NL- 2380 HV Rijswiik 
Td. (-^ 31-70) 340.2040»T^ 31 651 eponl. 
Fax (+31-70) 340-3016 



Date of mading of the international search report 

25.07.94 



Authorized officer 



Orviz Diaz, P 



Fonn PCT/ISA/aiO (ncend sliMt) 4July 1983} 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



Intel jDAl AppliMiioD No 

PCT/US 93/08808 



C^Cootinuaoon) DOCUMENTS CONSIDERED TO BE RELEVANT 

Category * j Qtation of document, with indication, what approptiate. of the nlcvant puages" 



Relevant to daim No. 



W0,A,89 10409 (GENETICS INSTITUTE, INC.) 2 
November 1989 

see page 8, line 1-7; claims 



1-91 



Fonn PCT/ISA/aiO (eDfittniMtion of Meond sheet) <July 1993} 



page 2 cf 2 



INTXRNATIONAL SEARCH REPORT 



inici. ^onal application No. 

PCT/ US 93/ 08808 



Box! Observations where certain claims were found unsearchable (Continuation of item I of first sheet) 



This international search report has not been csublished in respect of certain claims under ArUcle 17(2Xa) for the following reasons: 

1. [X] Claims Nos.: 

because ihey reUte to subjea matter not required to be searched by this Authority, namely: 

REMARK: Although some claims are directed to a method of treatment of the 

human body the search has been carried out. It was based on the alleged 
effects of the compositions. 

2. [Y] Claims Nos.: 

^vfA!*l21."**^ ? ■ >n«™tional applicauon that do not comply with the prescribed requirements to sudi 

an extent that no meanmgful mternaUonal search can be carried out. spedfically: 

The expression "roorphogen" is not sufficient to characterize specific chemi- 
cal compounds. In view of the extremely large number of substances encom- 
passed by this term, the search had to be limited to the general concept 
and to the specific compounds mentioned in the claims ... (please see annex 

I 1 Claims Nos.: 

because ihey are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). 



Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) 



This International Searching Authority found multiple invcnuons in this intcmaiiona] application, as follows: 



^- EZl all required addiUonal search fees were limciy paid by the applicant, this international search report covers all 
searchable claims. 

Q As all searchable claims could be searches without effort justifying an additbnal fee. this Authority did not invite payment 
oi any aoaiiionai fee. * r j 



^' n *^*y some of the required additional search fees were umcly paid by the applicant, this international search report 
covers only those claims for which fees were paid, spedftcaily claims Nos.: 



•□n o required additional search fees were timely paid by the applicant. Consequently, this international search report is 
restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 



Remark on Protest [~j The additional search fees were accompanied by the applicant's protest 

1 I Pro«" accompanied the payment of additional search fees. 



Form PCT/ISA/210 (continuation of first sheet (1)) (July 1992) 



international Apprication No. PCT/US93/ 08808 

FURTHER INFORMATION CONTINUED FROM PCT^SA/210 



....and In the pharmacological examples (see PCT, art. 6; Guidelines 
for Examination In the E.P.O,, Part B, Chapter II. 7, last sentence and 
Chapter III. 3. 7), 



INTERNATIONAL SEARCH REPORT 

InfonnttioB cni patent ftaiiy muubn 



loto^ joal ApplicMion No 

PCT/US 93/08808 



Patent document 
died in cearcfa fcport 



Publicuion 
date 



Patent fiunOy 
member(s) 



Publication 
date 



WO-A-9215323 



17-09-92 



WO-A-9304692 



18-03-93 



WO-A-9209301 



WO-A-8910409 



11-06-92 



02-11-89 



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AU-A- 
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AU-A- 
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AU-B- 
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US-A- 
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1754392 
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06-10-92 
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29-12-93 



2564592 
3176293 
2104678 
2116559 
2116562 
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9305751 
2862492 
2116560 
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05-04-93 
27-04-93 
12-09-92 
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15-06-94 
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9109391 
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25-06-92 
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13-10-93 

13-01-94 
24-11-89 
23-01-91 

15- 08-91 
21-04-92 

16- 02-93 



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