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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent OassiUcation ^ 

A61K 37/12, A61F2/02 
a)7K 13/00 



Al 



(11) Inteinational Publication Number: WO 92/15323 

(43) International Publication Date: 17 September 1992 (17.09.92) 



(21) International Application Number: PCT/US92/01968 

(22) International Filing Date: 1 1 March 1992 (11.03.92) 



(30) Priority data: 
667^74 



11 March 1991 (11.03.91) US 



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

US]; 35 South Street, Hopkinton. MA 01748 (US). 

(72) Inventors: COHEN, Charles, M. ; 98 Wnthrop Street, 

Medway, MA 02053 (US). KUBERASAMPATH, Than- 
gavel ; 6 Spring Street, Medway, MA 02053 (US). 
PANG. Roy, H., L. ; 16 Kimberly Drive, Medway, MA 
02053 (US). OPPERMANN, Hermann ; 25 Summer Hill 
Road, Medway, MA 02053 (US). RUEGER, David. C. ; 
19 Downey Street, Hopkinton, MA 01748 (US). 



(74) Agent: PITCHER, Edmund, R.; Testa. Hurwitz & Thi- 
beault. Exchange Place, 53 State Street, Boston, MA 
02109-2809 (US). 

n 

(81) Designated States: AT (European patent), AU, BE (EuroC9 
pean patent), CA, CH (European patent), DE (Euro"T 
pean patent), DK (European patent), ES (European pa^^ 
tent), FR (European patent), GB (European patent), GRj^ 
(European patent), IT (European patent), JP, LU (Euro-^ J 
pean patent), MC (European patent), NL (European pa-J^ 
tent), SE (European patent). 

Published ^ 

With international search report 



(54) Title: PROTEIN-INDUCED MORPHOGENESIS 



(57) Abstract 



Disclosed are 1) amino acid sequence data, structural features, homologies and various other data characterizing morphog- 
enic proteins, 2) methods of producing these proteins from natural and recombinant sources and from synthetic constructs, 3) 
morphogenic devices comprising these morphogenic proteins and a suitably modified tissue-specific matrix, and 4) methods of 
inducing non-chondrogenic tissue growth in a mammal. 



FOR THE PURPOSES OF INFORMATION ONLY 



CcMle^ used lo itleniify Sialte pany to the PCI* on ihe fioni pages of pamphlets publishing internaitonal 
applications under Ihe PCI'. 



AT 


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(Juhun 


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Mauritania 


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Belgium 


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Uuilcil Kiiigiluiti 


MW 


Malawi 


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fiurkiiui haw 


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Guinea 


Nt. 


Neihcrlamb 


BG 


Bulgaria 


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(ircccc 


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Norway 


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Benin 


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Hungary 


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Poland 


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Bra/M 


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Ireland 


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Rumania 


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t ufuida 


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lluly 


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Ruuian Fcdcralion 


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Soviet Union 


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United Siate:i of America 


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wo 92/15323 



PCr/US92/01968 



PROTEIN- INDUCED MORPHOGENESIS 

Background of the Invention 

This invention relates to morphogenic proteins 
which can induce tissue morphogenesis in mammals; to 
5 methods of identifying these proteins and obtaining 
them from natural sources or producing synthetic forms 
of these proteins by expressing recombinant DNA 
encoding the proteins; to the fabrication of tissue- 
specific acellular matrices; and to methods for 
10 promoting tissue stasis, repair and regeneration, and 
methods for increasing progenitor cell populations 
using these proteins. 

Cell differentiation is the central 
15 characteristic of morphogenesis which initiates in the 
embryo, and continues to various degrees throughout the 
life of an organism in adult tissue repair and 
regeneration mechanisms. The degree of morphogenesis 
in adult tissue varies among different tissues and is 
20 related, among other things, to the degree of cell 

turnover in a given tissue. On this basis, tissues can 
be divided into three broad categories: (1) tissues 
with static cell populations such as nerve and skeletal 
muscle where there is no cell division and most of the 



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cells formed during early development persist 
throughout adult life; (2) tissues containing 
conditionally renewing populations such as liver where 
there is generally little cell division but, in 
5 response to an appropriate stimulus, cells can divide 
to produce daughters of the same differentially defined 
type; and (3) tissues with permanently renewing 
populations including blood, testes and stratified 
squamous epithelia which are characterized by rapid and 
10 continuous cell turnover in the adult. Here, the 

terminally differentiated cells have a relatively short 
life span and are replaced through proliferation of a 
distinct subpdpulation of cells, known as stem or 
progenitor cells. 

15 

The cellular and molecular events which govern 
the stimulus for differentiation of these cells is an 
area of intensive research. In the medical field, it 
is anticipated that the discovery of factor(s) which 

20 control cell differentiation and tissue morphogenesis 
will significantly advance medicine's ability to repair 
and regenerate diseased or damaged mammalian tissues 
and organs. Particularly useful areas include 
reconstructive surgery and in the treatment of tissue 

25 degenerative diseases including arthritis, emphysema, 
osteoporosis, cardiomyopathy, cirrhosis, and 
degenerative nerve diseases. 

A number of different factors have been 
30 isolated in recent years which appear to play a ^-ole in 
cell differentiation. Some of these factors are gene 
transcription activators such as the NOTCH gene, 
identified in Drosophila and the related XOTCH gene 
identified in Xenopus, as well as a number of 
35 transcription activators identified in Caenorhabditis 
eleqans . 



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The hemopoietic system^ because of its 
continually renewing cell population, is an area of 
concentrated study. Factors identified in this system 
which may be involved in cell renewal include 
5 interleukin 3 (IL-3)/ erythropoietin, the CSFs (GM-CSF, 
G-CSF, M-CSF et al.) and various stem cell growth 
factors • 

Other proteins thought to play a role in cell 
10 differentiation include proteins that are members of 
the family of insulin-like growth factors (IGF), 
members of the family of heparin-binding growth 
factors, (e.g., FGF - acidic and basic fibroblast 
growth factors, and ECDGF - embryonal carcinoma-derived 
15 growth factor) as well as several transforming 

oncogenes (hst and int-2, see for example. Heath et 
al., (1988), J. Cell Sci, Suppl. 10 :256-256.) DIF 
(Differentiation Inducing Factor), identified in 
Dictyostelium discoideum , is another bioregulatory 
20 protein, directing prestock cell differentiation in 
that organism. 

The structurally related proteins of the TGF-p 
superfamily of proteins also have been identified as 

25 involved in a variety of developmental events. For 
example, TGF-p and the polypeptides of the 
inhibin/activin group appear to play a role in the 
regulation of cell growth and differentiation. MIS 
(Mullerian Inhibiting Substance) causes regression of 

30 the Mullerian duct in development of the mammalian male 
embryo, and DPP, the gene product of the Drosophila 
decapentaplegic complex is required for appropriate 
dorsal-ventral specification. Similarly, Vg-1 is 
involved in mesoderm induction in Xenopus, and Vgr-1 

35 has been identified in a variety of developing murine 
tissues. 



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Another source that has revealed a wealth of 
infoimation is in the area of bone morphogenesis • The 
development and study of a bone model system has 
5 identified the developmental cascade of bone 
differentiation as consisting of chemotaxis of 
mesenchymal cells, proliferation of these progenitor 
cells, differentiation of these cells into 
chrondroblasts , cartilage calcification, vascular 
10 invasion, bone formation, remodeling, and finally, 

marrow differentiation (Reddi (1981) Collagen Rel> Res . 
1^:209-206). Proteins capable of inducing endochondral 
bone formation in a mammal when implanted in 
association with a matrix now have been identified in a 

15 number of different mammalian species, as have the 

genes encoding these proteins, (see, for example, U.S. 
Patent No. 4,968,590 and U.S. Patent No. 5,011,691, 
Ozkaynak, et al., (1990) EMBO J 9; 2085-2093, and 
Ozkaynak et al., (1991) Biochem. Biophys. Res. Commn . 

20 179:116-123 and USSN 07/841,646, filed February 21, 
1992.) These proteins, which share significant amino 
acid sequence homology with one another as well as 
structural similarities with various members of the 
TGF-p super family of proteins, have been shown to 

25 induce endochondral bone formation and/or cartilage 
formation when implanted in a mammal in association 
with a suitably modified matrix. Proteins capable of 
inducing a similar developmental cascade of tissue 
morphogenesis of other tissues have not been 

30 identified. 



It is an object of this invention to provide 
morphogenic proteins ( "morphogens " ) , and methods for 
identifying these proteins, which are capable of 
35 inducing the developmental cascade of tissue 



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morphogenesis for a variety of tissues in mammals 
different from bone or cartilage v This morphogenic 
activity includes the ability to induce proliferation 
and differentiation of progenitor cells, and the 
5 ability to support and maintain the differentiated 
phenotype through the progression of events that 
results in the formation of adult tissue* Another 
object is to provide genes encoding these proteins as 
well as methods for the expression and isolation of 
10 these proteins, from either natural sources or 

biosynthetic sources, using recombinant DNA techniques. 
Still another object is to provide tissue-specific 
acellular matrices that may be used in combination with 
these proteins, and methods for their production. 
15 Other objects include providing methods for increasing 
a progenitor cell population in a mammal, methods for 
stimulating progenitor cells to differentiate in vivo 
or in vitro and maintain their differentiated 
phenotype, methods for inducing tissue-specific growth 
20 in vivo and methods for the replacement of diseased or 
damaged tissue in vivo . These and other objects and 
features of the invention will be apparent from the 
description, drawings, and claims which follow. 



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Siimmary of the Invention 

This invention provides morphogenic proteins 
("morphogens") capable of inducing the developmental 
5 cascade of tissue morphogenesis in a mammal. In 

particular, these proteins are capable of inducing the 
proliferation of uncommitted progenitor cells, and 
inducing the differentiation of these stimulated 
progenitor cells in a tissue-specific manner under 

10 appropriate environmental conditions. In addition/ the 
morphogens are capable of supporting the growth and 
maintenance of these differentiated cells. These 
morphogenic activities allow the proteins of this 
invention to initiate and maintain the developmental 

15 cascade of tissue morphogenesis in an appropriate, 
morphogenically permissive environment, stimulating 
stem cells to proliferate and differentiate in a 
tissue-specific maimer, and inducing the progression of 
events that culminate in new tissue formation. These 

20 morphogenic activities also allow the proteins to 

stimulate the "redif ferentiation" of cells previously 
induced to stray from their differentiation path. 
Under appropriate environmental conditions it is 
anticipated that these morphogens also may stimulate 

25 the "dedif ferentiation" of committed cells (see infra.) 

In one aspect of the invention, the proteins 
and compositions of this invention are useful in the 
replacement of diseased or damaged tissue in a mammal, 

30 particularly when the damaged tissue interferes with 
normal tissue or organ function. Accordingly, it is 
anticipated that the proteins of this invention will be 
useful in the repair of damaged tissue such as, for 
example, damaged lung tissue resulting from emphysema, 

35 cirrhotic kidney or liver tissue, damaged heart or 



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blood vessel tissue, as may result from 
cardiomyopathies and/or atherothrombotic or 
cardioembolic strokes, damaged stomach tissue resulting 
from ulceric perforations or their repair, damaged 
5 neural tissue as may result from physical injury, 
degenerative diseases such as Alzheimer's disease or 
multiple sclerosis or strokes, damaged dentin tissue as 
may result from disease or mechanical injury. When the 
proteins of this invention are provided to, or their 
10 expression stimulated at, a tissue-specific locus, the 
developmental cascade of tissue morphogenesis is 
induced (see infra). Cells stimulated ex vivo by 
contact with the proteins or agents capable of 
stimulating morphogen expression in these cells also 
15 may be provided to the tissue locus. In these cases 
the existing tissue provides the necessary matrix 
requirements, providing a suitable substratum for the 
proliferating and differentiating cells in a 
morphogenically permissive environment, as well as 
20 providing the necessary signals for directing the 
tissue-specificity of the developing tissue. 
Alternatively, the proteins or stimulated cells may be 
combined with a formulated matrix and implanted as a 
device at a locus in vivo . The formulated matrix 
25 should be a biocompatible, preferably biodegradable, 

appropriately modified tissue-specific acellular matrix 
having the characteristics described below. 

In many instances, the loss of tissue function 
30 results from scar tissue, 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 type of injury. Thus, in another 



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aspect, the invention includes morphogens that may be 
used to prevent or substantially inhibit the formation 
of scar tissue by providing the morphogens, or 
raorphogen-stimulated cells, to a newly injured tissue 
5 loci ( see infra ) . 

The morphogens of this invention also may be 
used to increase or regenerate a progenitor or stem 
cell population in a mammal. For example, progenitor 
10 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 

15 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 

20 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. 

25 Similarly, a particular population of hemopoietic stem 
cells may be increased by the morphogens of this 
invention, for example by perfusing an individual's 
blood to extract the cells of interest, stimulating 
these cells ex vivo, and returning the stimulated cells 

30 to the blood. It is anticipated that the ability to 
augment an individual's progenitor cell population will 
significantly enhance existing methods for treating 
disorders resulting from a loss or reduction of a 
renewable cell population. Two particularly 

35 significant applications include the treatment of blood 



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disorders and impaired or lost inunune function* Other 
cell populations whose proliferation may be exploited 
include the stem cells of the epidermis, which may be 
used in skin tissue regeneration, and the stem cells of 
5 the gastrointestinal lining, for example, in the 
healing of ulcers. 

In still another aspect of the invention, the 
morphogens also may be used to support the growth and 

10 maintenance of differentiated cells, inducing existing 
differentiated cells to continue expressing their 
phenotype. It is anticipated that this activity will 
be particularly useful in the treatment of tissue 
disorders where loss of function* is caused by cells 

15 becoming senescent or quiescent, such as may occur in 
osteoporosis. Application of the protein directly to 
the cells to be treated, or providing it by systemic 
injection, can be used to stimulate these cells to 
continue expressing their phenotype, thereby 
20 significantly reversing the effects of the dysfunction 
(see infra). Alternatively, administration of an agent 
capable of stimulating morphogen expression in vivo 
also may be used. In addition, the morphogens of this 
invention also may be used in gene therapy protocols to 
25 stimulate the growth of quiescent cells, thereby 
potentially enhancing the ability of these^ cells to 
incorporate exogenous DNA. 

In yet another aspect of the invention, the 
30 morphogens of this invention also may be used to .induce 
"redif ferentiation" of cells that have strayed from 
their differentiation pathway, such as can occur during 
tumorgenesis. It is anticipated that this activity of 
the proteins will be particularly useful in treatments 
35 to reduce or substantially inhibit the growth of 



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neoplasms. The method also is anticipated to induce 
the de-and re-differentiation of these cells. As 
described supra, the proteins may be provided to the 
cells directly or systemicallyr or an agent capable of 
5 stimulating morphogen expression in vivo may be 
provided. 

Finally, modulations of endogenous morphogen levels 
may be monitored as part of a method for detecting 

10 tissue dysfunction. Specifically, modulations in 

endogenous morphogen levels are anticipated to reflect 
changes in tissue or organ stasis. Tissue stasis may 
be monitored by detecting changes in the levels of the 
morphogen itself. For example, tissue samples may be 

15 obtained at intervals and the concentration of the 
morphogen present in the tissue detected by standard 
protein detection means known to those skilled in the 
art. As an example, a binding protein capable of 
interacting specifically with the morphogen of 
20 interest, such as an anti-morphogen antibody, may be 
used to detect the morphogen in a standard immunoassay. 
The morphogen levels detected then may be compared, the 
changes in the detected levels being indicative of the 
status of the tissue. Modulations in endogenous 
25 morphogen levels also may be monitored by detecting 
fluctuations in the body's natural antibody titer to 
morphogens (see infra.) 

The morphogenic proteins and compositions of 
30 this invention can be isolated from a variety of 

naturally-occurring sources, or they may be constructed 
biosynthetically using conventional recombinant DNA 
technology. Similarly, the matrices may be derived 
from organ-specific tissue, or they may be formulated 
35 synthetically, as described below. 



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A key to these developments was the discovery 
and characterization of naturally-occurring osteogenic 
proteins followed by observation of their remarkable 
properties. These proteins / originally isolated from 
5 bone, are capable of inducing the full developmental 
cascade of bone formation^ including vascularization^ 
mineralization, and bone marrow differentiation, when 
implanted in a mammalian body in association with a 
suitably modified matrix. Native proteins capable of 
10 inducing this developmental cascade, as well as DNA 
sequences encoding these proteins now have been 
isolated and characterized for a number of different 
species (e.g., human and mouse OP-1, OP-2, and CBMP-2. 
See, for example, U.S. Patent Nos. 4,968,590 and 
15 5,011,691; U.S. Application Serial No. 841,646, filed 
February 21, 1992; Sampath et al. (1990) J. Bio. Chem 
265:13198-13205; Ozkaynak, et al. (1990) EMBO J 
9:2085-2 093 and Ozkaynak, et al. (1991) Biochem. 
Biophys. Res. Commn . 179 :116-123. ) The mature forms of 
20 these proteins share substantial amino acid sequence 
homology, especially in the C-terminal regions of the 
mature proteins. In particular, the proteins share a 
conserved six or seven cysteine skeleton in this region 
(e.g., the linear arrangement of these C-terminal 
25 cysteine residues is essentially conserved in the 
different proteins, in addition to other, apparently 
required amino acids (see Table II, infra)). 

Polypeptide chains not normally associated 
30 with bone or bone formation, but sharing substantial 

amino acid sequence homology with the C-terminus of the 
osteogenic proteins, including the conserved six or 
seven cysteine skeleton, also have been identified as 
competent for inducing bone in mammals. Among these 
35 are amino acid sequences identified in Drosophila and 



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Xenopus/ (e.g., DPP and Vgl; see, for example, U.S. 
Patent No. 5,011,691 and Table II, infra). In 
addition, non-native biosynthetic constructs designed 
based on extrapolation from these sequence homologies, 
5 including the conserved six or seven cysteine skeleton, 
have been shown to induce endochondral bone formation 
in mammals when implanted in association with an 
appropriate matrix (see U.S* Pat. No. 5,011,691 and 
Table III, infra) • 

10 

It has now been discovered that this "family" 
of proteins sharing substantial amino acid sequence 
homology and the conserved six or seven cysteine 
skeleton are true morphogens, capable of inducing, in 

15 addition to bone and cartilage, tissue-specific 
morphogenesis for a variety of other organs and 
tissues. The proteins apparently bind to surface 
receptors or otherwise contact and interact with 
progenitor cells, predisposing or stimulating the cells 

20 to proliferate and differentiate in a morphogenically 
permissive environment. The morphogens are capable of 
inducing the developmental cascade of cellular and 
molecular events that culminate in the formation of new 
organ-specific tissue, including any vascularization, 

25 connective tissue formation, and nerve ennervation as 
required by the naturally occurring tissue% 

It also has been discovered that the way in 
which the cells differentiate, whether, for example, 

30 they differentiate into bone-producing osteoblasts, 
hemopoietic cells, or liver cells, depends on the 
nature of their local environment (see infra). Thus, 
in addition to requiring a suitable substratum on which 
to anchor, the proliferating and differentiating cells 

35 also require appropriate signals to direct their 



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tissue-specificity* These signals may take the form of 
cell surface markers. 

< 

5 When the morphogens (or progenitor cells 

stimulated by these morphogens) are provided at a 
tissue-specific locus (e.g., by systemic injection or 
by implantation or injection at a tissue-specific 
locus, or by administration of an agent capable of 
10 stimulating morphogen expression in vivo), the existing 
tissue at that locus, whether diseased or damaged, has 
the capacity of acting as a suitable matrix. 
Alternatively, a formulated matrix may be externally 
provided together with the stimulated progenitor cells 
15 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 
acellular matrix having dimensions such that it allows 
the influx, differentiation, and proliferation of 
20 migratory progenitor cells, and is capable of providing 
a morphogenically permissive environment (see infra). 
The matrix preferably is tissue-specific, and 
biodegradable. 

25 Formulated matrices may be generated from 

dehydrated organ-specific tissue, prepared for example, 
by treating the tissue with solvents to substantially 
remove the non- structural components from the tissue. 
Alternatively, the matrix may be formulated 

30 synthetically using a biocompatible, preferably in vivo 



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biodegradable, structural polymer such as collagen in 
association with suitable tissue-specific cell 
attachment factors. Currently preferred structural 
polymers comprise tissue-specific collagens. Currently 
5 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 and micropits on its 
surfaces, so as to enhance the influx, proliferation 
10 and differentiation of migratory progenitor cells from 
the body of the mammal. 

Among the proteins useful in this invention 
are proteins originally identified as osteogenic 

15 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 (from Xenopus), Vgr-1 (from 
mouse, see Table II and Seq. ID Nos.5-14), and the 
recently identified GDF-1 protein (Seq. ID No. 14). 

20 The members of this family, which include members of 
the TGF-p super- family of proteins, share substantial 
amino acid sequence homology in their C-terminal 
regions. Table I, below, describes the various 
morphogens identified to date, including their 

25 nomenclature as used herein, and Seq. ID references. 

TABLE I 

30 "OP-1" Refers generically to the group of 

morphogenically active proteins expressed 
from part or all of a DNA sequence 
encoding OP-1 protein, including allelic 
and species variants thereof, e.g., human 

35 OP-1 ("hOP-1", Seq. ID No. 5, mature 



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protein amino acid sequence)^ or mouse 
OP-l {"mOP-1% Seq. ID No. 6, mature 
protein amino acid sequence.) The 
conserved seven cysteine skeleton is 
defined by residues 38 to 139 of Seq. 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 



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the mature, morphogenically active 
proteins are defined essentially by 
residues 18-263 (hOP2) and residues 18-260 
(mOPl) . 

5 

"CBMP2" refers generically to the morphogenically 

active proteins expressed from a DNA 
sequence encoding the CBMP2 proteins, 
including allelic and species variants 
10 thereof, e.g., human CBMP2A { "CBMP2A{fx') 

Seg ID No. 9) or human CBMP2B DNA 
("CBMP2B(fx)", Seq. ID No. 10). 

"DPP(fx)" refers to protein sequences encoded by the 
15 Drosophila DPP gene and defining the 

conserved seven cysteine skeleton (seq. ID 
No. 11). 

"Vgl(fx)" refers to protein sequences encoded by the 
20 Xenopus Vgl gene and defining the 

conserved seven cysteine skeleton (Seq. ID 
No. 12). 

"Vgr-l(fx)" refers to protein sequences encoded by the 
25 murine Vgr-1 gene and defining the 

conserved seven cysteine skeleton (Seq. ID 
No. 13). 

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

conserved seven cysteine skeleton (seq. ID 
No. 14). 



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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 
5 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 
10 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. 
15 Thus, as defined herein, a morphogen of this invention 
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 43-139 of Seq. ID No. 5, including 
20 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 
25 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 acting as a morphogen as defined herein. 
30 Specifically, the protein is capable of any of the 
following biological functions in a morphogenically 
permissive environment: stimulating proliferation of 
progenitor cells; stimulating the differentiation of 
progenitor cells; stimulating the proliferation of 
35 differentiated cells; and supporting the growth and 
maintenance of differentiated cells, including the 



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PCr/US92/01968 



"redifferentiation" of these cells. In addition^ it is 
also anticipated that the morphogens of this invention 
will be capable of inducing dedif ferentiation of 
committed cells under appropriate environmental 
5 conditions • 

In one preferred aspect/ the morphogens of 
this 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 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 
comprise the following additional sequence at their N- 
tezminus : 

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

Preferred amino acid sequences within the 
25 foregoing generic sequences include: Generic Sequence 
3 (Seq. ID No. 3) and Generic Sequence 4 (Seq. ID 
No. 4), listed below, which accommodate the homologias 
shared among the various preferred members of this 
morphogen family identified to date (see Table II), as 
30 well as the amino acid sequence variation among them. 
Generic Sequences 3 and 4 are composite amino acid 
sequences of the proteins presented in Table II and 
identified in Seq. ID Nos. 5-14. The generic sequences 
include both the amino acid identity shared by the 
35 sequences in Table II, as well as alternative residues 



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PCr/US92/01968 



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 
4, respectively, providing an appropriate cysteine 
5 skeleton where inter- or intramolecular disulfide bonds 
can form, and contain certain critical amino acids 
which influence the tertiary structure of the proteins. 

Generic Sequence 3 
10 Leu Tyr Val Xaa Phe 

1 5 

Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

• 10 

Xaa Ala Pro Xaa Gly Xaa Xaa Ala 

15 15 20 

Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 

25 30 

Xaa Pro Xaa Xaa Xaa Xaa Xaa 

35 

20 Xaa Xaa Xaa Asn His Ala Xaa Xaa 

40 45 
Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 

50 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 
25 55 60 

Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
65 



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PCf/US92/01968 



Xaa Xaa Xaa Leu Xaa Xaa Xaa 

70 75 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
80 

5 Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

85 90 
Xaa Cys Gly Cys Xaa 
95 

wherein each Xaa is independently selected from a group 

10 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^ Gln^ Ser or 
Lys); Xaa at res. 7 - (Asp or Glu); Xaa at res. 8 « (Leu 
or Val); Xaa at res. 11 - (Gin, Leu, Asp, His or Asn); 

15 Xaa at res. 12 = (Asp, Arg or Asn); Xaa at res. 14 = (He 
or Val); Xaa at res. 15 = (He 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 
or Gin); Xaa at res. 23 = (Tyr, Asn or Phe); Xaa at 

20 res. 26 = (Glu, His, Tyr, Asp or Gin); Xaa at res. 28 « 
(Glu, Lys, Asp or Gin); Xaa at res. 30 - (Ala, Ser, Pro 
or Gin); Xaa at res. 31 = (Phe, Leu or Tyr); Xaa at 
res. 33 = (Leu or Val); Xaa at res. 34 = (Asn, Asp, Ala 
or Thr); Xaa at res. 35 = (Ser, Asp, Glu, Leu or Ala); 

25 Xaa at res. 36 = (Tyr, Cys, His, Ser or He); Xaa at 
res. 37 = (Met, Phe, Gly or Leu); Xaa at res. 38 = (Asn 
or Ser); Xaa at res. 39 = (Ala, Ser or Gly); Xaa at 
res. 40 = (Thr, Leu or Ser); Xaa at res. 44 - (He or 
Val); Xaa at res. 45 = (Val or Leu); Xaa at res. 46 = 

30 (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 



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PCr/US92/01968 



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 
5 res. 57 «= (Val, Ala or He); Xaa at res. 58 = (Pro or 
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 
10 res. 68 = (Asn, Ser or Asp); Xaa at res .69 - (Ala, Pro 
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 ~ 
15 (Asp, Glu, Asn or Ser); Xaa at res. 78 = (Ser, Gin, Asn 
or Tyr); Xaa at res. 79 = (Ser, Asn, Asp or Glu); 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); 
20 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 
Arg); and Generic Seq. 4: 

25 

Generic Sequence 4 

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

15 

Xaa Ala Pro Xaa Gly Xaa Xaa Ala 

20 25 
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 
35 30 35 



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PCr/US92/01968 



Xaa Pro Xaa Xaa Xaa Xaa Xaa 

40 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 
45 50 
5 Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 

55 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

60 65 
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
10 70 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 

75 80 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
85 

15 Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

90 95 
Xaa Cys Gly Cys Xaa 
100 

wherein each Xaa is independently selected from a group 

20 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 
= (His or Arg); Xaa at res. 5 = (Glu, Ser, His, Gly, Arg 
or Pro); Xaa at res. 9 = (Ser, Asp or Glu); Xaa at 

25 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, 
or Asn); Xaa at res. 19 = (lie or Val); Xaa at res. 20 = 
(lie or Val); Xaa at res. 23 = (Glu, Gin, Leu, Lys, Pro 

30 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 = (Clu, His, Tyr, Asp 
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 

35 or Tyr); Xaa at res. 38 = (Leu or Val); Xaa at res. 39 = 



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PCr/US92/01968 



(Asn, Asp/ Ala or Thr); Xaa at res. 40 = (Ser^ Asp/ Glu, 
Leu or Ala); Xaa at res.41 = (Tyr, Cys, His, Ser or 
lie); Xaa at res. 42 = (Met, Phe, Gly or Leu); Xaa at 
res. 44 « (Ala, Ser or Gly); Xaa at res. 45 = (Thr, Leu 
5 or Ser); Xaa at res. 49 = (lie 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 
Met); Xaa at res. 55 = (His or Asn); Xaa at res. 56 « 
(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res. 57 = (lie, 
10 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, 
Lys, Asp, Tyr, Ser or Ala); Xaa at res. 62 = (Val, Ala 
or lie); Xaa at res. 63 = (Pro or Asp); Xaa at res. 64 = 
15 (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 = 
(Leu, Met or Val); Xaa at res. 73 = (Asn, Ser or Asp); 
Xaa at res. 74 = (Ala, Pro or Ser); Xaa at res. 75 = 
20 (He, 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 
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, 
25 Asn, Asp or Glu); Xaa at res. 85 = (Asn, Thr or Lys); 
Xaa at res. 87 = (He or Val); Xaa at res. 89 = (Lys or 
Arg); Xaa at res. 90 = (Lys, Asn, Gin or His); Xaa at 
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 
30 = (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). 

Particularly useful sequences for use as 
35 morphogens in this invention include the C-terminal 



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PCr/US92/01%8 



domains, e.g., the C-terminal 96-102 amino acid 
residues of Vgl, Vgr-1, DPP, OP-1, OP-2, CBMP-2A, 
CBMP-2B and GDF-1 (see Table II, infra, and Seg. ID 
Nos. 5-14) which include at least the conserved six or 
5 seven cysteine skeleton. In addition, biosynthetic 
constructs designed from the generic sequences, such as 
COP-1, 3-5, 7, 16 (see Table III, infra) aso are 
useful. Other sequences include the C- terminal CBMP3 
and the inhibins/activin proteins (see, for example, 
10 U.S. Pat. Nos. 4,968,590 and 5,011,691). Accordingly, 
other useful sequences are those sharing at least 70% 
amino acid sequence homology, and preferably 80% 
homology with any of the sequences above. These are 
anticipated to include allelic and species variants and 
15 mutants, and biosynthetic muteins, as well as novel 
members of this morphogenic family of proteins. 
Particularly envisioned in the family of related 
proteins are those proteins exhibiting morphogenic 
activity and wherein the amino acid changes from the 
20 preferred sequences include conservative changes, e.g., 
those as defined by Dayoff et al.. Atlas of Protein 
Sequence and Structure ; vol. 5, Suppl. 3, pp. 345-362, 
(M.O. Dayoff, ed., Nat'l BioMed. Research Fdn., 
Washington, D.C. 1979). 

25 

The currently most preferred protein sequences 
useful as morphogens in this invention include those 
having greater than 60% identity, preferably greater 
than 65% identity, with the amino acid sequence 
30 defining the conserved six cysteine skeleton of hOPl 
(e.g., residues 43-139 of Seq. ID No. 5). These most 
preferred sequences include both allelic and species 
variants of the OPl and 0P2 proteins. 



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PCr/US92/01968 



The invention thus provides proteins 
comprising any of the polypeptide chains described 
above/ whether isolated from naturally-occurring 
sources / or produced by recombinant DNA techniques ^ and 
5 includes allelic and species variants of these 

proteins, naturally-occurring or biosynthetic mutants 
thereof/ as well as various truncated and fusion 
constructs. Deletion or addition mutants also are 
envisioned to be active (see infra), including those 

10 which may alter the conserved C-terminal cysteine 
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 

15 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 or mutated forms of 

20 native or biosynthetic proteins, produced by expression 
of recombinant DNA in host cells. 

The morphogenic proteins can be expressed from 
intact or truncated cDNA or from synthetic DNAs in 
25 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. 

30 

Thus, in view of this disclosure, skilled 
genetic engineers can isolate genes from cDNA or 
genomic libraries of various different species which 
encode appropriate amino acid sequences, or construct 
35 DNAs from oligonucleotides, and then can express them 



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PCr/US92/01968 



in various types of host cells, including both 
procaryotes and eucaryotes, to produce large quantities 
of active proteins capable of inducing tissue-specific 
cell differentiation and tissue morphogenesis in a 
5 variety of mammals including humans- 



The invention thus further comprises these 
methods of inducing tissue-specific morphogenesis using 
the morphogenic proteins of this invention and 

10 pharmaceutical and therapeutic agents comprising the 
morphogens of this invention. The invention further 
comprises the use of these morphogens in the 
manufacture of pharmaceuticals for various medical 
procedures / including procedures for inducing tissue 

15 growth, procedures for inducing progenitor cell 

proliferation, procedures to inhibit neoplasm growth 
and procedures to promote phenotypic cell expression of 
differentiated cells. 



wo 92/15323 -27- 

Brief Description of the Drawings 



PCT/US92/01968 



The foregoing and other objects and features 
of this invention, as well as the invention itself, may 
5 be more fully understood from the following 

description, when read together with the accompanying 
drawings, in which: 

FIGURE 1 is a photomicrograph of a Northern 
10 Blot identifying Vgr-1 specific transcripts in various 
adult murine tissues; 

FIGURE 2 is a photomicrograph of a Northern 
Blot identifying mOP-l-specif ic mRNA expression in 
15 various murine tissues prepared from 2 week old mice 
(panel A) and 5 week old mice (Panel B); 

FIGURE 3 is a photomicrograph of Northern 
Blots identifying mRNA expression of EF-Tu 
20 (A, control), mOP-1 (B, D), and Vgr-1 (C) in ( 1 ) 17-day 
embryos and (2) 3-day post natal mice; 

FIGURE 4A and 4B are photomicrographs showing 
the presence of OP-1 (by immunofluorescence staining) 
25 in the cerebral cortex (A) and spinal cord (B); 

FIGURE 5A and 5B are photomicrographs 
illustrating the ability of morphogen (OP-1) to induce 
undifferentiated NG108 calls (5A) to undergo 
30 differentiation of neural morphology (SB). 

FIGURE 6A-6D are photomicrographs showing the 
effect of morphogen (OP-1) on human embryo carcinoma 
cell redifferentiation; 

35 



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PCr/US92/01968 



FIGURE 7 is a photomicrograph showing the effects 
of phosphate buffered saline (PBS^ animal 1) or 
morphogen (OP-1, animal 2) on partially hepatectomized 
rats; 

5 

FIGURE 8A - 8C are photomicrographs showing 
the effect of no treatment (8A)/ carrier matrix 
treatment (8B) and morphogen treatment (0P-1/8C) on 
dentin regeneration. 



10 



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PCT/US92/01968 



Detailed Description 

Purification protocols first were developed 
which enabled isolation of the osteogenic (bone 
5 inductive) protein present in crude protein extracts 
from mammalian bone. (See PCT US 89/01453, and 
U.S. 4,968,590.) The development of the procedure, 
coupled with the availability of fresh calf bone, 
enabled isolation of substantially pure bovine 
10 osteogenic protein (BOP). BOP was characterized 
significantly; its ability to induce cartilage and 
ultimately endochondral bone growth in cat, rabbit, and 
rat were demonstrated and studied; it was shown to be 
able to induce the full developmental cascade of bone 
15 formation previously ascribed to unknown protein or 
proteins in heterogeneous bone extracts. This dose 
dependent and highly specific activity was present 
whether or not the protein was glycosylated (see U.S. 
Patent No. 4,968,958, filed 4/8/88 and Sampath et al., 
20 (1990) J. Biol. Chem. 265 : pp. 13198-13205). Sequence 
data obtained from the bovine materials suggested probe 
designs which were used to isolate genes encoding 
osteogenic proteins from different species. Human and 
murine osteogenic protein counterparts have now been 
25 identified and characterized (see, for example, U.S. 
Pat. No. 5,011,691, Ozkaynak, et al., (1990) EMBO J 
9:2085-2093, and Ozkaynak et al., (1991) Biochem. 
Biophys. Res. Commn . 179 : 116-123, and USSN 841,646, 
filed February 21, 1992, the disclosures of which are 
30 herein incorporated by reference.) 



Sequence data from the bovine materials also 
suggested substantial homology with a number of 
proteins known in the art which were not known to play 
35 a role in bone formation. Bone formation assays 



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PCr/US92/01968 



performed with these proteins showed that, when these 
proteins were implanted in a mammal in association with 
a suitable matrix, cartilage and endochondral bone 
formation was induced (see, for example, U.S. Patent 
5 No. 5,011,691.) One of these proteins is DPP, a 
Drosophila protein known to play a role in dorsal- 
ventral specification and required for the correct 
morphogenesis of the imaginal discs. Two other 
proteins are related sequences identified in Xenopus 

10 and mouse (Vgl and Vgr-1, respectively), thought to 
play a role in the control of growth and 
differentiation during embryogenesis. While DPP and 
Vgr-1 (or Vgr-l-like) transcripts have been identified 
in a variety of tissues (embryonic, neonatal and adult, 

15 Lyons et al., (1989) PNAS 86:4554-4 558, and see 

infra), Vgl transcripts, which are maternally inherited 
and specially restricted to the vegetal endoderm, 
decline dramatically after gastrulation. 

20 From these homologies a generic consensus 

sequence was derived which encompasses the active 
sequence required for inducing bone morphogenesis in a 
mammal when implanted in association with a matrix. 
The generic sequence has at least a conserved six 

25 cysteine skeleton (Generic Sequence 1, Seq. ID No. 1) 
or, optionally, a 7-cysteine skeleton (Generic 
Sequence 2, Seq. ID No. 2), which includes the 
conserved six cysteine skeleton defined by <3eneric 
Sequence 1, and an additional cysteine at residue 36, 

30 accomodating the additional cysteine residue iden,tified 
in the 0P2 proteins. Each "Xaa" in the generic 
sequences indicates that any one of the 20 naturally- 
occurring L-isomer, «-amino acids or a derivative 



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PCT/US92/01968 



thereof may be used at that position. Longer generic 
sequences which also are useful further comprise the 
following sequence at their N-termini: 



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



Biosynthetic constructs designed from this 
10 generic consensus sequence also have been shown to 
induce cartilage and/or endochondral bone formation 
(e.g., COP-1, COP-3, COP-4, COP-5, COP-7 and COP-16, 
described in U.S. Patent No. 5,011,691 and presented 
below in Table III.) Table II, set forth below, 
15 compares the amino acid sequences of the active regions 
of native proteins that have been identified as 
morphogens, including human OP-1 (hOP-1, Seq. ID Nos. 5 
and 16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and IB- 
IS), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20- 
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 (Seq. ID No. 14.) In the table, 
three dots indicates that the amino acid in that 
25 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 illustrating 
homologies. For example, amino acid residue 60 of 
CBMP-2A and CBMP-2B is "missing". Of course, both 
30 these amino acid sequences in this region comprise 

Asn-Ser (residues 58, 59), with CBMP-2A then con?)rising 
Lys and lie, whereas CBMP-2B comprises Ser and lie. 



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PCr/US92/01968 



TABLE II 



10 



hOP-1 

mOF-1 

hOP-2 

inOP-2 

DPP 

Vgl 

Vgr-1 

CBHP-2A 

CBHF-2B 

GDF-1 



Cys Lys Lys His Glu Leu Tyr Val 



Arg Arg 
Arg Arg 



Arg Arg 



Ser 



Lys Arg His 

*«* Gl^^ 

Arg . . . Pro 

Arg Arg ... Ser 

Arg Ala Arg Arg 



15 



20 



25 



hOP-1 

mOP-1 

hOP-2 

inOP-2 

DPP 

Vgl 

Vgr-l 

CBHP-2A 

CBHP-2B 

GDF-1 



Ser Phe Arg Asp Leu Gly Trp Gin Asp 



Ser 
Asp 
Glu 

• • • 

Asp 
Asp 



Gin 

Ser 
Lys 
Gin 
Ser 
Ser 



Glu 



Val 
Val 
Val 
Val 
Val 
Val 



Leu 
Leu 
Asp 



Asn 



Asn 

Asn ... 

His Arg 



10 



15 



30 



hOP-1 

nOP-l 

hOP-2 

mOP-2 

DPP 

Vgl 

Vgr-1 



Trp He He Ala Pro Glu Gly Tyr Ala 



Val 
Val 

Val 



Val 



Gin 
Gin 
Leu 
Gin 
Lys 



Ser 
Ser 
Asp 
Met 



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PCT/US92/01968 



CBHP-2A 
CBHF-2B 



Val 



Val 
Val 

• * ♦ 

20 



Pro 
Pro 
Arg 



Phe 
25 



His 
Gin 
Leu 



10 



15 



hOP-1 

mOF-l 

hOP-2 

raOF-2 

i)PP 

Vgl 

Vgr-1 

CBMP-2A 

CBlfP-2B 

GDF-1 



Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 



Asn 
Asn 
Phe 
Fhe 
Asn 



His 
Tyr 
Asp 
His 
His 
Gin 



Lys 



Glu 
Asp 
Gin 



30 



Ser 

Pro 
Pro 
Ser 
Pro 
Pro 

• • • 

35 



20 



25 



30 



35 



hOP-1 

nOF-1 

bOP-2 

inOP-2 

DPP 

Vgl 

Vgr-1 

CBMF-2A 

CBHF-2B 

GDF-1 



hOP-1 
mOP-l 
hOP-2 
bOP-2 
DPP 



Phe Pro Leu Asn Ser Tyr Met Asn Ala 



Tyr 



Leu 



Asp 
Asp 



Cys 
Cys 



Phe 
Leu 



Ala Asp His 
Thr Glu He 
Ala His 
Ala Asp His Leu 
Leu 



Ser 
Gly 
• • ■ 
Ser 
Ser 



Ala Asp His 
Val Ala Leu Ser Gly Ser** 
40 



Thr Asn His Ala He Val Gin Thr Leu 



Leu 
Leu 



Ser 
Ser 



Val 



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PCT/US92/01968 



Vgl 

Vgr-1 

CBHP-2A 

CBHP-2B 

GDF-1 



Ser 



Leu 
45 



Leu 



Val Leu Arg Ala 
50 



10 



15 



20 



25 



30 



hOP-1 

mOP-l 

hOP-2 

inOP-2 

DPP 

Vgl 

Vgr-l 

CBHP-2A 

CB1IP-2B 

GDF-1 



Val 



hOP-1 

mOP-1 . 

hOP-2 

inOP-2 

DPP 

Vgl 

Vgr-1 

CB1IP-2A 

CBHP-2B 

GDF-1 



Het 



His 


rne 


lie 


Asn 


rro 


GIU 


Thr 


Val 












Asp 






His 


Leu 


Het 


Lys 




Asn 


Ala 




His 


Leu 


Het 


Lys 


... 


Asp 


Val 


• • • 


Asn 


Asn 


Asn 






oiy 


i*ys 




... 


ber 




rill 

ulU 


a . • 


• • . 


Asp 


lie 


« « • 


Vox 






* • • 




lyr 


• • • 


Asn 


Ser 


Val 




Ser 




Lys 


lie 


Asn 


Ser 


Val 


• • * 


Ser 




Ser 


lie 


• « • 


Ala 


Ala 


Ala 


. • • 


Gly 


Ala 


Ala 


55 










60 






Lys 


Pro 


Cys 


Cys 


Ala 


Pro 


Thr 


Gin 




Ala 


• • « 

• • • 


• • • 


. • • 


• . * 

* « • 


• • • 
« « • 


Lys 


• • « 


Ala 








• * ■ 




Lys 


• • • 


Ala 






Val 


• « • 


. • • 





Leu 



Asp Leu 



Ala 
Ala 

65 



Val 

Val 
Val 
Val 



Lys 
Lys 
Glu 
Glu 

Ala Arg 
70 



35 



hOP-1 
nOP-l 



Leu Asn Ala lie Ser Val Leu lyr Phe 



WOW/15323 -35- PCT/l)S92/0I968 





hOP-2 


• • • 


Ser 


• • • 


Thr 




« • • 


• • » 


• • • 


Tyr 




nOP-2 


. • • 


Ser 


• • • 


Thr 


• • • 


• • » 




• • • 


Tyr 




Vgl 


Met 


Ser 


Pro 


• • • 




Met 


... 


Phe 


lyr 




Vgr-l 


Val 


• • • 


• « « 


• « • 


• • * 


• • • 


• • • 


« • « 


• • • 


5 


DPP 


• * • 


Asp 


Ser 


Val 


Ala 


Met 


• • • 


• • • 


Leu 




CfillP-2A 




Ser 


• • * 


• • • 


• • • 


Met 


« • • 


• • • 


Leu 




CBMP-2B 


• * • 


Ser 


• • ■ 


• • « 


• • • 


Met 


• • * 


• • • 


Leu 




GDF-1 


• • • 


Ser 


Pro 


• • • 


• • • 


« « • 


• « • 


Phe 


• • • 












75 










80 


10 


hOP-1 


Asp 


Asp 


Ser 


Ser 


Asn 


Val 


lie 


Leu 


Lys 




mOP-1 


• • • 


• • * 


• • • 


• • • 


• • • 


• • • 


• • • 


• • • 


• • • 




hOP-2 


• • • 


Ser 




Asn 


• • • 


• • • 


• • • 


• « • 


Arg 




mOP-2 


• • • 


Ser 


• • • 


Asn 


• • • 


• • • 


• • • 


• • • 


Arg 




DPP 


Asn 




Gin 


• • • 


Thr 


• • • 


Val 


• • • 


• « • 


15 


Vgl 


• • • 


Asn 


Asn 


Asp 


• • • 


• • • 


Val 


• • • 


Arg 




Vgr-l 


• • • 


• • • 


Asn 


• « • 


• • « 


• • • 


• • » 


• • « 


* • • 




CBHP-2A 


• « a 


Glu 


Asn 


Glu 


Lys 


• • • 


Val 








CBHP-2B 


• « • 


Glu 


Tyr 


Asp 


Lys 


... 


Val 


• • • 


• • • 




GDF-1 




Asn 




Asp 


• • • 




Val 


« * * 


Arg 


20 












85 












hOP-1 


Lys 


Tyr 


Arg 


Asn 


Met 


Val 


Val 


Arg 






mOP-1 




• • • 


• • • 






• « • 




• • ■ 




25 


hOP-2 


• • • 


His 


• • • 


• • • 


• • • 






Lys 






iaOP-2 


. * • 


His 


• • « 




• • • 


• • • 


• • . 


Lys 






DPP 


Asn 




Gin 


Glu 


« • • 


Thr 


• * * 


Val 






Vgl 


His 




Glu 


• • • 




Ala 




Asp 






Vgr-l 


• • • 


• • • 


* • ■ 


• • • 


• w • 


• • • 


• • • 


« • « 




30 


CBHF-2A 


Asn 




Gin 


Asp 


• • • 


• • • 




Glu 






CBHP-2B 


Asn 




Gin 


Glu 


« • • 


• • • 




Glu 






GDF-1 


Gin 




Glu 


Asp 


« • • 


• • » 




Asp 








90 










95 









35 



wo 92/15323 



-36- 



PCT/US92/01968 







Ala 


Cvs Glv Cys His 




mOP-l 








hOP-2 








iUw* ^ 








DPP 


Glv 


Arg 




Vgl 


rill 


AXg 




Vgr-1 


• • • 


••• 




CBMP-2A 


GI7 


Arg 




CB!fP-2B 


Gly 


Arg 


10 


GDF-1 


Glu 


Arg 



100 

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

15 Table III/ set forth below, compares the amino 

acid sequence data for six related biosynthetic 
constructs designated COPs 1, 3, 4, 5, 7, and 16. 
These sequences also are presented in U.S. Fat. No. 
5,011/691. As with Table II, the dots mean that in that 

20 position there is an identical amino acid to that of 
COP-1, and dashes mean that the COF-1 amino acid is 
missing at that position. 



25 



TABLE III 



30 



COP-1 
COP-3 
COP-4 
COP-5 
COP-7 
COP-16 



Leu Tyr Val Asp Phe Gin Arg Asp Val 



Ser 
Ser 
Ser 
Ser 



1 



5 



wo 92/15323 



-37- 



PCr/US92/01968 





COP-1 


Gly 


Trp 


Asp 


Asp 


Trp 


He 


He 


Ala 




COP-3 


... 


• • • 


• • • 


• • • 




• • • 


Val 


• • • 




COP-4 


... 


... 


• « • 


• • • 


• • • 


« • • 


Val 


« « « 






... 






• • • 




• • • 


VcLX 


• • • 


5 


COP-7 


• . . 


... 


Asn 




« • • 


• • • 


Val 


• • » 




COP-16 


... 


... 


Asn 


• • • 


• • • 


• • • 


Val 


« * • 






10 










15 








COP-1 


Pro 


Val 


Asp 


Fhe 


Asp 


Ala 


Tyr 


Tyr 


10 


COP-3 


• • • 


Pro 


Gly 


Tjrr 


Gin 


... 


Fhe 


• • * 




COP-4 


• « • 


Pro 


Gly 


lyr 


Gin 


... 


Phe 


... 




COP-5 




Pro 


Gly 


Tyr 


Gin 




Phe 






COP-7 




Pro 


Gly 


Tyv 


His 




Phe 


« • « 




COP-16 


• • • 


Pro 


Gly 


Tyr 


Gin 


• • • 


Phe 




15 








20 










25 




COP-1 


Cys 


Ser 


Gly 


Ala 


Cys 


Gin 


Phe 


Pro 




COP-3 


... 


• • • 


• • * 


• • • 


• • • 


• • • 




• • • 


20 


COP-4 


• • • 


« • • 


• • « 


• • . 


• • • 


• • • 




• • . 




COP-5 


• • • 


His 


• « • 


Glu 


• • • 


Pro 




• • • 




COP-7 




His 


• • • 


Glu 


. • • 


Pro 




• . • 




COP-16 




His 


• • • 


Glu 


• • • 


Pro 




• • • 












30 










25 






















COP-1 


Ser 


Ala 


Asp 


His 


Phe 


Asn 


Ser 


Thr 




COP-3 


• • • 


• • • 




• • • 


* * • 






« • . 




COP-4 


• • • 


• • • 


* • • 




• • • 






• * • 




COP-5 


Leu 


• • • 


• • * 


• « • 


• • « 


• • • 




• « • 


30 


COP-7 


Leu 








Leu 









COP-16 Leu 



35 



40 



wo 92/15323 



-38- 



PCr/US92/01968 



COP-1 
COP-3 
COP-4 
COP-5 
COP-7 
COP- 16 



Asn His Ala Val Val Gin Thr 



10 


COP-1 
COP-3 
COP-4 


Asn Asn 

« • * • • • 
• • • • • • 


Met 

• • • 

• • • 


Asn Pro 

• • • • • « 

• • • • • • 


Gly 
• • • 


Lys 

• • « 

• • • 




COP-5 


Ser 


Val 


Ser 


Lys 


He 




COP-7 


Ser 


Val 


Ser 


Lys 


He 


15 


COP- 16 


Ser 


Val 


Ser 
55 


Lys 


He 



20 



25 



COP-1 
COP-3 
COP-4 
COP-5 
COP-7 
COP-16 



Pro Lys Pro Cys Cys Val Pro 



Ala 
Ala 
Ala 



60 



65 



30 



COP-1 
COP-3 
COP-4 
COP-5 
COP-7 
COP-16 



Glu Leu Ser Alia lie Ser Het 



70 



Leu Val 



50 



Val 



Thr 



Leu 



35 



wo 92/15323 



-39- 



PCr/US92/01968 



COP-1 


Tyr 


Leu Asp Glue Asn Ser 


Thr Val 


COP-3 


• • • 


••• ••• GXu 


Lys 


COP-4 


* • • 


••• ••• G3>u 


Lys 


COP-5 


« • • 


• 


Lys 


COP-7 


* • • 


• ••• GXix 


Lys 


COP-16 


• • • 


Glu 


Lys 




75 


80 





10 



15 



COP-1 
COP-3 
COP-4 
COP-5 
COP-7 
COP-16 



Val Leu Lys Asn Tyr Gin Glu Met 



85 



I • • • • • 



) • • • < 



• • • • • 



90 



20 



25 



COP-1 
COP-3 
COP-4 
COP-5 
COP-7 
COP-16 



Thr 
Val 
Val 
Val 
Val 
Val 



Val 



Val 
Glu 
Glu 
Glu 
Glu 
Glu 



Gly Cys Gly Cys Arg 



95 



30 As is apparent from the foregoing eunino 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 

35 only about 50% amino acid identity with the hOPl 



wo 92/15323 



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PCr/US92/01968 



sequence described therein, the GDF-1 sequence shares 
greater than 70% amino acid sequence homology with the 
hOPl sequence, where homology is defined by allowed 
conservative amino acid changes within the sequence as 
5 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.) 

It now has been discovered that the family of 
10 proteins described by these sequences also is capable 
of initiating and maintaining the tissue-specific 
developmental cascade in tissues other than bone and 
cartilage. When combined with naive progenitor cells 
as disclosed herein, these proteins, termed morphogens, 
15 are capable of inducing the proliferation and 
differentiation of the progenitor cells. In the 
presence of appropriate tissue-specific signals to 
direct the differentiation of these cells, and a 
morphogenically permissive environment, these 
20 morphogens are capable of reproducing the cascade of 
cellular and molecular events that occur during 
embryonic development to yield functional tissue. 

A key to these developments was the creation 
25 of a mammalian tissue model system, namely a model 
system for endochondral bone formation, and 
investigation of the cascade of events, important for 
bone tissue morphogenesis. Work on this system has 
enabled discovery not only of bone inductive 
30 morphogens, but also of tissue inductive morphogens and 
their activities. The methods used to develop the bone 
model system, now well known in the art, along with the 
proteins of this invention, can be used to create model 
systems for other tissues, such as liver (see infra). 



wo 92/15323 



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PCr/US92/01968 



Using the model system for endochondral bone 
formation, it also has been discovered that the local 
environment in which the morphogenic material is placed 
5 is important for tissue morphogenesis. As used herein, 
"local environment" is understood to include the tissue 
structural matrix and the environment surrounding the 
tissue. For example, in addition to needing an 
appropriate anchoring sxibstratum for their 
10 proliferation, the morphogen-stimulated cells need 
signals to direct the tissue-specificity of their 
differentiation. These signals vary for the different 
tissues and may include cell surface markers. In 
addition, vascularization of new tissue requires a 

15 local environment which supports vascularization. 

Using the bone model system as an example, it is known 
that, under standard assay conditions, implanting 
osteoinductive morphogens into loose mesenchyme in the 
absence of a tissue-specifying matrix generally does 

20 not result in endochondral bone formation unless very 
high concentrations of the protein are implanted. By 
contrast, implanting relatively low concentrations of 
the morphogen in association with a suitably modified 
bone-derived matrix results in the formation of fully 

25 functional endochondral bone (see, for example, Sampath 
et al. (1981) PNAS 78:7599-7 603 and U.S. Patent 
No. 4,975,526). In addition, a synthetic matrix 
comprised of a structural polymer such as tissue- 
specific collagen and tissue-specific cell attachment 

30 factors such as tissue-specific glycosylaminoglycans, 
will allow endochondral bone formation (see, for 
example, PCT publication US9 1/03603, published December 
12, 1991 (WO 91/18558), incorporated herein by 
reference). Finally, if the morphogen and a suitable 

35 bone or cartilage-specific matrix (e.g., comprising 
Type I cartilage) are implanted together in loose 
mesenchyme, cartilage and endochondral bone formation 
will result, including the formation of bone marrow and 



wo 92/15323 



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PCr/US92/01968 



a vascular system. However, if the same composition is 
provided to a nonvascular environment, such as to 
cultured cells in vitro or at an cartilage-specific 
locus, tissue development does not continue beyond 
5 cartilage formation (see infra). Similarly, a 

morphogenic composition containing a cartilage-specific 
matrix composed of Type 2 collagen is expected to 
induce formation of non-cartilage tissue in vivo (e.g., 
hyaline). However, if the composition is provided to a 
10 vascular- supporting environment, such as loose 

mesenchyme, the composition is capable of inducing the 
differentiation of proliferating progenitor cells into 
chondrocytes and osteoblasts, resulting in bone 
formation. 

15 

It also has been discovered that tissue 
morphogenesis requires a morphogenically permissive 
environment. Clearly, in fully- functioning healthy 
tissue that is not composed of a permanently renewing 

20 cell population, there must exist signals to prevent 
continued tissue growth. Thus, it is postulated that 
there exists a control mechanism, such as a feedback 
control mechanism, which regulates the control of cell 
growth and differentiation. In fact, it is known that 

25 both TGF-p, and MIS are capable of inhibiting cell 

growth when present at appropriate concentrations. In 
addition, using the bone model system it can be shown 
that osteogenic devices comprising a bone-derived 
carrier (matrix) that has been demineralized and 

30 guanidine-extracted to substantially remove the 

noncollagenous proteins does allow endochondral bone 
formation when implanted in association with an 



wo 92/15323 



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PCT/US92/01968 



osteoinductive morphogen. If# however, the bone- 
derived carrier is not demineralized but rather is 
washed only in low salt, for example, induction of 
endochondral bone formation is inhibited, suggesting 
5 the presence of one or more inhibiting factors within 
the carrier. 



Another key to these developments was 
determination of the broad distribution of these 

10 morphogens in developing and adult tissue. For 
example, DPP is expressed in both embryonic and 
developing Drosophila tissue. Vgl has been identified 
in Xenopus embryonic tissue. Vgr-1 transcripts have 
been identified in a variety of murine tissues, 

15 including embryonic and developing brain, lung, liver ^ 
kidney and calvaria (dermal bone) tissue. Recently, 
Vgr-1 transcripts also have been identified in adult 
murine lung, kidney, heart, and brain tissue, with 
especially high abundance in the lung (see infra). 

20 

OP-1 and the CBMP2 proteins, both first 
identified as bone morphogens, have been identified in 
mouse and human placenta, hippocampus, calvaria and 
osteosarcoma tissue as determined by identification of 

25 OP-1 and CMBP2-specif ic sequences in cDNA libraries 
constructed from these tissues (see Ozkaynak, et al., 
(1990) EMBO_J 9:2085-2093, and Ozkaynak et al., (1991) 
Biochem. Biophys. Res. Commn . 179 ; 116-123) > 
Additionally, the OP-1 protein is present in a variety 

30 of embryonic and developing tissues including kidney, 
liver, heart, adrenal tissue and brain as determined by 
Western blot analysis and immunolocalization (see 
infra). OP-l-specif ic transcripts also have been 
identified in both embryonic and developing tissues, 

35 most abundantly in developing kidney, bladder and brain 



wo 92/15323 



PCr/US92/01968 



(see infra) • OP-1 also has been identified as a 
mesoderm inducing factor present during embryogenesis 

(see infra). Moreover/ OP-1 has been shown to be 
associated with in satellite muscle cells and 
5 associated with pluripotential stem cells in bone 
marrow following damage to adult murine endochondral 
bone, indicating its morphogenic role in tissue repair 
and regeneration. In addition, the recently identified 
protein GDF-1 (see Table II) has been identified in 
10 neural tissue (Lee, (1991) PNAS 88 4250-4254). 

Exemplification 

IDENTIFICATION AND ISOLATION OF MORPHOGENS 

15 

Among the proteins useful in this invention 
are proteins originally identified as bone inductive 
proteins, such as the OP-1, OP-2 and the CBMP proteins, 
as well as amino acid sequence-related proteins such as 

20 DPP (from Drosophila), Vgl (from Xenopus) and Vgr-1 
(from mouse, see Table II and Sequence Listing). The 
members of this family, which include particular 
members of the TGF-p super family of structurally 
related proteins, share substantial amino acid sequence 

25 homology in their C-terminal regions. The OP-2 

proteins have an extra cysteine residue in this region 
(position 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 proteins are 

30 inactive when reduced, but are active as oxidized 
homodimeric species as well as when oxidized in 
combination with other morphogens. 

Accordingly, the morphogens of this invention 
35 can be described by either of the following two species 



wo 92/15323 



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PCr/US92/01968 



of generic amino acid sequences: Generic Sequence 1 or 
Generic Sequence 2, (Seq. ID Nos* 1 and 2), where each 
Xaa indicates one of the 20 naturally-occurring 
L-isomer/ a-amino acids or a derivative thereof, 
5 Particularly useful sequences that fall within this 
family of proteins include the 96-102 C-terminal 
residues of Vgl, Vgr-1, DPP, OP-1, OP-2, CBMP-2A, 
CBMP-2B, and GDF-1, as well as their intact mature 
amino acid sequences. In addition, biosynthetic 
10 constructs designed from the generic sequences, such as 
COP-1, COP-3-5, COP-7, and COP-16 also are useful (see, 
for example, U.S. Pat. No. 5,011,691. ) 

Generic sequences showing preferred amino 
15 acids compiled from sequences identified to date and 
useful as morphogens (e.g.. Tables II and III) are. 
described by Generic Sequence 3 (Seq. ID No. 3) and 
Generic Sequence 4 (Seq. ID No. 4). Note that these 
generic sequences have a 7 or 8-cysteine skeleton where 
20 inter- or intramolecular disulfide bonds can form, and 
contain certain critical amino acids which influence 
the tertiary structure of the proteins. It is also 
contemplated that the differing N-termini of the 
naturally occurring proteins provide a tissue-specific 
25 or other, important modulating activity of these 
proteins . 

Given the foregoing amino acid and DNA 
sequence information, the level of skill in the art, 

30 and the disclosures of U.S. Patent Nos. 4,968,590 and 
5,011,691, PCT application US 89/01469, published 
October 19, 1989 (WO89/09788 ) , and Ozkaynak, et al., 
(1990) EMBO J 9:2085-2093, and Ozkaynak et al., (1991) 
Biochem. Biophys. Res. Commn . 179 :116-123 the 

35 disclosures of which are incorporated herein by 

reference, various DNAs can be constructed which encode 



W092/IS323 



-46- 



PCr/US92/01968 



at least the active region of a morphogen of this 
invention, and various analogs thereof (including 
allelic variants and those containing genetically 
engineered mutations)/ as well as fusion proteins, 
5 truncated forms of the mature proteins, deletion and 
insertion mutants, and similar constructs* Moreover, 
DNA hybridization probes can be constructed from 
fragments of the genes encoding any of these proteins, 
including seqeunces encoding the active regions or the 
10 pro regions of the proteins (see infra), or designed de 
novo from the generic sequence. These probes then can 
be used to screen different genomic and cDNA libraries 
to identify additional morphogenic proteins from 
different tissues. 

15 

The DNAs can be produced by those skilled in 
the art using well known DNA manipulation techniques 
involving genomic and cDNA isolation, construction of 
synthetic DNA from synthesized oligonucleotides, and 

20 cassette mutagenesis techniques. 15-lOOmer 

oligonucleotides may be synthesized on a Biosearch DNA 
Model 8600 Synthesizer, and purified by polyacrylamide 
gel electrophoresis (PAGE) in Tris-Borate-EDTA buffer. 
The DNA then may be electroeluted from the gel. 

25 Overlapping oligomers may be phosphorylated by T4 

polynucleotide kinase and ligated into larger blocks 
which also may be purified by PAGE. 

The DNA from appropriately identified clones 
30 then can be isolated, subcloned (preferably into an 

expression vector), and sequenced. Plasmids containing 
sequences of interest then can be trans fected into an 
appropriate host cell for expression of the morphogen 
and further characterisation. The host may be a 



wo 92/15323 



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PCr/US92/01968 



procaryotic or eucaryotic cell since the former's 
inability to glycosylate protein will not destroy the 
protein's morphogenic activity. Useful host cells 
include coli, Saccharomyces , the insect/baculovirus 
5 cell system, myeloma cells, and various other mammalian 
cells. The vectors additionally may encode various 
sequences to promote correct expression of the 
recombinant protein, including transcription promoter 
and termination sequences, enhancer sequences, 
10 preferred ribosome binding site sequences, preferred 
mRNA leader sequences, preferred signal sequences for 
protein secretion, and the like. 

The DNA sequence encoding the gene of interest 
15 also may be manipulated to remove potentially 

inhibiting sequences or to minimize unwanted secondary 
and tertiary structure formation. The recombinant 
morphogen also may be expressed as a fusion protein. 
After being translated, the protein may be purified 

20 from the cells themselves or recovered from the culture 
medium. All biologically active protein forms comprise 
dimeric species joined by disulfide bonds or otherwise 
associated, produced by refolding and oxidizing one or 
more of the various recombinant polypeptide chains 

25 within an appropriate eucaryotic cell or in vitro after 
expression of individual subunits. A detailed 
description of morphogens expressed from recombinant 
DNA in E. coli and in numerous different mammalian 
cells is disclosed in PCT publication US90/05903, 

30 published May 2, 1991 (WO91/05802) and U.S. Serial 

No. 841,646 filed February 21, 1992, the disclosures of 
which are hereby incorporated by reference. 



Alternatively, morphogenic polypeptide chains 
35 can be synthesized chemically using conventional 

peptide synthesis techniques well known to those having 



wo 92/15323 



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PCrAJS92/01%8 



ordinary skill in the art. For example, the proteins 
may be synthesized intact or in parts on a Biosearch 
solid phase peptide synthesizer, using standard 
operating procedures. Completed chains then are 
5 deprotected and purified by HPLC (high pressure liquid 
chromatography). If the protein is synthesized in 
parts, the parts may be peptide bonded using standard 
methodologies to form the intact protein. In general, 
the manner in which the morphogens are made can be 
10 conventional and does not form a part of this 
invention. 

MORPHOGEN DISTRIBDTION 

15 The generic function of the morphogens of this 

invention throughout the life of the organism can be 
evidenced by their expression in a variety of disparate 
mammalian tissues. Determination of the tissue 
distribution of morphogens also may be used to identify 

20 different morphogens expressed in a given tissue, as 
well as to identify new, related morphogens. The 
proteins (or their mRNA transcripts) are readily 
identified in different tissues using standard 
methodologies and minor modifications thereof in 

25 tissues where expression may be low. For example, 

protein distribution may be determined using standard 
Western blot analysis or immunof Increscent techniques, 
and antibodies specific to the morphogen or morphogens 
of interest. Similarly, the distribution of morphogen 

30 transcripts may be determined using standard Northern 
hybridization protocols and transcript-specific probes. 



35 



Any probe capable of hybridizing specifically 
to a transcript, and distinguishing the transcript of 
interest from other, related transcripts may be used. 



wo 92/15323 



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PCr/US92/01968 



Because the morphogens of this invention share such 
high sequence homology in their active/ C-terminal 
domains, the tissue distribution of a specific 
morphogen transcript may best be determined using a 
5 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. These portions of the sequence vary 

10 substantially among the morphogens of this invention, 
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 the untranslated pro region 

15 and the N-terrainus 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 sequence that covers approximately two-thirds 

20 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 
(See Seq. ID No. 18, where the pro region is defined 

25 essentially by residues 30-291.) Similar approaches 
may be used, for example, with hOPl (Seq. ID No. 16) or 
human or mouse 0P2 (Seq. ID Nos. 20 and 22.) 

Using these morphogen-specif ic probes, which 
30 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 ordinary skill in the art. 
Briefly, total RNA is prepared from various adult 
35 murine tissues (e.g., liver, kidney, testis, heart. 



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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 
(A)+ RNA is prepared by using oligo (dT) -cellulose 
5 chromatography (e.g./ Type 1, from Pharmacia LKB 

Biotechnology, Inc.). Poly (A)+ RNA (generally 15 yq) 
from each tissue is fractionated on a 1% 
agarose/formaldehyde gel and transferred onto a Nytran 
membrane (Schleicher & Schuell). Following the 
10 transfer / the membrane is baked 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 
(e.g., the PvuII-SacI Vgr-1 fragment) is denatured by 
heating. The hybridization is carried out in a lucite 
15 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 washed off 
20 the filters in 0.1 x SSPE, 0.1% SDS at 50 *C. Northern 
blots performed using Vgr-1 probes specific to the 
variable N terminus of the mature sequence indicate 
that the Vgr-1 message is approximately 3.5 Kb. 

25 Figure 1 is a photomicrograph representing a 

Northern blot analysis probing a number of adult murine 
tissues with the Vgr-1 specific probes: liver, kidney, 
testis / heart/ brain, thymus and stomach, represented 
in lanes 3-10, respectively. Lanes 1 and 12 are size 

30 standards and lanes 2 and 11 are blank. Among the 
tissues tested, Vgr-1 appears to be expressed most 
abundantly in adult lung, and to a lesser extent in 
adult kidney, heart and brain. These results confirm 
and expand on earlier studies identifying Vgr-1 and 

35 Vgr-l-like transcripts in several embryonic and adult 



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murine tissue (Lyons et al. (1989) PNAS 86:4554-4558), 
as well as studies identifying OP-1 and CBMP2 in 
various human cDNA libraries (e»g., placenta, 
hippocampus, calvaria, and osteosarcoma, see Ozkaynak 
5 et al., (1990) EMBO 9:2085-2093 ) • 

Using the same general probing methodology, 
mOP-1 transcripts also have been identified in a 
variety of murine tissues, including embryo and various 

10 developing tissues, as can be seen in Figures 2 and 3* 
Details of the probing methodology are disclosed in 
Ozkaynak, et al., (1991) Biochem. Biophys. Res. Commn . 
179 :116-123, the disclosure of which is incorporated 
herein. The Northern blots represented in Figure 2 

15 probed RNA prepared from developing brain, spleen, 
lung, kidney (and adrenal gland), heart, and liver in 
13 day post natal mice (panel A) or 5 week old mice 
(panel B) . The OP-1 specific probe was a probe 
containing the 3' untranslated sequences described 

20 supra (0.34 Kb Earl-Pst I fragment). As a control for 
RNA recovery, EF-Tu ( translational elongation factor) 
mRNA expression also was measured (EF-Tu expression is 
assumed to be relatively uniform in most tissues). 

25 The arrowheads indicate the OPl-specific messages 

observed in the various tissues. As can be seen in 
Fig. 2, OP-1 expression levels vary significantly in 
the spleen, lung, kidney and adrenal tissues, while the 
EF-Tu mRNA levels are constant. Uniformly lower levels 

30 of EF-Tu mRNA levels were found in the heart, brain and 
liver. As can be seen from the photomicrograph, the 
highest levels of OP-1 mRNA appear to be in kidney and 
adrenal tissue, followed by the brain. By contrast, 
heart and liver did not give a detectable signal. Not 



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shown are additional analyses performed on bladder 
tissue, which shows significant OP-1 raRNA expression, 
at levels close to those in kidney/adrenal tissue. The 
Northern blots also indicate that, like GDF-1, OP-1 
5 mRNA expression may be bicistonic in different tissues. 
Four transcripts can be seen: 4 Kb, 2.4 Kb, 2.2 Kb, 
and 1.8 Kb transcripts can be identified in the 
different tissues, and cross probing with OP-1 specific 
probes from the proregion and N-terminal sequences of 
10 the gene indicate that these transcripts are OP-1 
specific. 

A side by side comparison of OP-1 and Vgr-1 in 
Figure 3 shows that the probes distinguish between the 
15 morphogens Vgr-1 and OP-1 transcripts in the different 
tissues, and also highlights the multiple transcription 
of OP-1 in different tissues. Specifically, Fig. 3 
compares the expression of OP-1 (Panels B and D), Vgr-1 
(Panel C) and EF-Tu (Panel A) (control) mRNA in 17 day 
20 embryos (lane 1) and 3 day post-natal mice (lane 2). 
The same filter was used for sequential hybridizations 
with labeled DNA probes specific for OP-1 (Panels B and 
D), Vgr-1 (Panel C), and EF-Tu (Panel A). Panel A: 
the EF-Tu specific probe (control) was the 0.4 Kb 
25 Hindlll-SacI fragment (part of the protein coding 
region), the Sad site used belonged to the vector; 
Panel B: the OP-1 specific probe was the 0.68 Kb 
BstXI-Bgll fragment containing pro region sequences; 
Panel D; the OP-1 specific probe was the 0.34 Kb Earl- 
30 PstI fragment containing the 3' untranslated sequence; 
Panel C: the Vgr-1 specific probe was the 0.26 Kb 
PvuII-SacI fragment used in the Vgr-1 blots described 
above • 



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The 1.8-2.5 Kb OP-1 mRNA appears approximately two 
times higher in three day post natal mice than in 17 
day embryos, perhaps reflecting phases in bone and/or 
kidney development. In addition, of the four messages 
5 found in brain, the 2.2 Kb transcript appears most 

abundant, whereas in lung and spleen the 1.8 Kb message 
predominates. Finally, careful separation of the renal 
and adrenal tissue in five week old mice reveals that 
the 2.2 Kb transcripts were derived from renal tissue 
10 and the 4 Kb mRNA is more prominent in adrenal tissue 
( see Figure 2 ) . 

Similarly, using the same general probing 
methodology, BMP3 and CBMP2B transcripts recently have 
15 been identified in abundance in lung tissue. 

Morphogen distribution in embryonic tissue can 
be determined using five or six-day old mouse embryos 
and standard immunofluorescence techniques in concert 

20 with morphogen-specif ic antisera. For example, rabbit 
anti-OP-1 antisera is readily obtained using any of a 
number of standard antibody protocols well known to 
those having ordinary skill in the art. The antibodies 
then are fluorescently labelled using standard 

25 procedures. A five or six -day old mouse embryo then is 
thin-sectioned and the various developing tissues 
probed with the labelled antibody, again following 
standard protocols. Using this technique, OP-1 protein 
has been detected in developing brain and heart. 

30 

This method also may be used to identify 
morphogens in adult tissues undergoing repair. For 
example, a fracture site can be induced in a rat long 
bone such as the femur. The fracture then is allowed 
35 to heal for 2 or 3 days. The animal then is sacrificed 



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and the fractured site sectioned and probed for the 
presence of the morphogen e.g.f OP-l, with 
fluorescently labelled rabbit anti-OP-1 antisera using 
standard inimunolocalization methodology. This 
5 technique identifies OP-1 in muscle satellite cells / 
the progenitor cells for the development of muscle, 
cartilage and endochondral bone. In addition, OP-1 is 
detected with potential pluripotential stem cells in 
the bone marrow, indicating its morphogenic role in 
10 tissue repair and regeneration. 

OP-1 protein also has been identified in rat brain 
using standard immunofluorescence staining technique. 
Specifically, adult rat brain (2-3 months old) and 

15 spinal cord is frozen and sectioned. Anti-OP-1, raised 
in rabbits and purified on an OP-1 affinity column 
prepared using standard methodologies, was added to the 
sections under standard conditions for specific 
binding. Goat anti-rabbit IgG, labelled with 

20 fluorescence, then was used to visualize OP-1 antibody 
binding to tissue sections. 

As can be seen in FIG 4A and 4B, immunofluorescence 
staining demonstrates the presence of OP-1 in adult rat 

25 central nervous system (CNS.) Similar and extensive 
staining is seen in both the brain (4A) and spinal cord 
(4B). OP-1 appears to be predominantly localized to 
the extracellular matrix of the grey matter, present in 
all areas except the neuronal cell bodies. In white 

30 matter, staining appears to be confined to astrocytes. 
A similar staining pattern also was seen in newborn rat 
(10 day old) brain sections. 

CELL DIFFERENTIATION 



35 



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The ability of morphogens of this invention to 
induce cell differentiation can be determined by 
culturing early mesenchymal cells in the presence of 
the morphogen and then studying the histology of the 
5 cultured cells by staining with toluidine blue. For 
example, it is known that rat mesenchymal cells 
destined to become mandibular bone, when separated from 
the overlying epithelial cells at stage 11 and cultured 
in vitro under standard tissue culture conditions, will 

10 not continue to differentiate. However, if these same 
cells are left in contact with the overlying endodezm 
for an additional day, at which time they become stage 
12 cells, they will continue to differentiate on their 
own in vitro to form chondrocytes. Further 

15 differentiation into obstepblasts and, ultimately, 
mandibular bone, requires an appropriate local 
environment , e.g., a vascularized environment . 

It has now been discovered that stage 11 
20 mesenchymal cells, cultured in vitro in the presence of 
a morphogen, e.g., OP-1, continue to differentiate in 
vitro to form chondrocytes. These stage 11 cells also 
continue to differentiate in vitro if they are cultured 
with the cell products harvested from the overlying 
25 endodermal cells. Moreover, OP-1 can be identified in 
the medium conditioned by endodermal cells either by 
Western blot or immunofluorescence. This experiment 
may be performed with other morphogens and with 
different mesenchymal cells to assess the cell 
30 differentiation capability of different morphogens, as 
well as their distribution in different developing 
tissues. 

As another example of morphogen- induced cell 
35 differentiation, the effect of OP-1 on the 



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differentiation of neuronal cells has been tested in 
culture. Specifically, the effect of OP-1 on the 
NG108-15 neuroblastoma x glioma hybrid clonal cell line 
has been assessed. The cell line shows a f ibroblastic- 
5 type morphology in culture. The cell line can be 
induced to differentiate chemically using 0.5 mM 
butyrate/ 1% DMSO or 500 mM For skol in, inducing the 
expression of virtually all important neuronal 
properties of cultured primary neurons. However, 
10 chemical induction of these cells also induces 
cessation of cell division. 

In the present experiment NG108-15 cells were 
subcultured on poly-L-lysine coated 6 well plates. 

15 Each well contained 40-50,000 cells in 2.5 ml of 

chemically defined medium. On the third day 2.5 pi of 
OP-1 in 60% ethanol containing 0,025% trifluoroacetic 
was added to each well. OP-1 concentrations of 0, 1, 
10, 40 and 100 ng/ml were tested. The media was 

20 changed daily with new aliquots of OP-1. After four 
days with 40 and 100 ng OP-l/ral concentrations, OP-1 
induced differentiation of the NG108-15 cells. 
Figure 5 shows the morphological changes that occur. 
The OP-1 induces clumping and rounding of the cells and 

25 the production of neurite outgrowths (processes). 
Compare FIG 5A (naive NG108-15 cells) with FIG 5B, 
showing the effects of OPI-treated cells. Thus the 
OP-1 can induce the cells to differentiate into a 
neuronal cell morphology. Some of the outgrowths 

30 appear to join in a synaptic-type junction. This 

effect was not seen in cells incubated with T<;f-B1 at 
concentrations of 1 to 100 ng/ml. 

The neuroprotective effects of OP-1 were 
35 demonstrated by comparison with chemical 



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differentiation agents on the NG108-15 cells. 50,000 
cells were plated on 6 well plates and treated with 
butyrate, DMSO, Forskolin or OP-1 for four days. Cell 
counts demonstrated that in the cultures containing the 
5 chemical agents the differentiation was accompanied by 
a cessation of cell division. In contrast, the cells 
induced to differentiate by OP-1 continued to divide, 
as determined by -thymidine uptake. The data suggest 
that OP-1 is capable of maintaining the stability of 
10 the cells in culture after differentiation. 

As yet another, related example, the ability of the 
morphogens of this invention to induce the 
"redif ferentiation" of transformed cells also has been 

15 assessed. Specifically, the effect of OP-1 on human EC 
cells (embryo carcinoma cells, NTERA-Z CL.Dl) is 
disclosed herein. In the absence of an external 
stimulant these cells can be maintained as 
undifferentiated stem cells, and can be induced to grow 

20 in serum free media (SFM). In the absence of morphogen 
treatment the cells proliferate rampantly and are 
anchorage- independent. The effect of morphogen 
treatment is seen in Figs. 6A-D. Figs 6A and 6B show 
4 days of growth in SFM in the presence of OP-1 
25 (25ng/ml, 6A) or the absence of morphogen (6B). 

Figs. 6C and 6D are 5 days growth in the presence of 
lOng/ml OP-1 (6C) or no morphogen (6D). Figs. 6C and 
6D are at lOx and 2 Ox magnification compared to FIGs 6A 
and 5B. As can readily be seen, in the presence of 
30 OP-1, EC cells grow as flattened cells, becoming 
anchorage dependent. In addition, growth rate is 
reduced approximately 10 fold. Finally, the cells are 
induced to differentiate. 

35 MAINTENANCE OF PHENOTYPE 



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The morphogens of this invention also may be 
used to maintain a cell's differentiated phenotype. 
This morphogenic capability is particularly useful for 
5 inducing the continued expression of phenotype in 
senescent or quiescent cells. 

The phenotypic maintenance capability of 
morphogens is readily assessed. A number of 

10 differentiated cells become senescent or quiescent 
after multiple passages under standard tissue culture 
conditions in vitro. However, if these cells are 
cultivated in vitro in association with a morphogen of 
this invention, the cells are induced to maintain 

15 expression of their phenotype through multiple 
passages. For example, the alkaline phosphatase 
activity of cultured osteoblasts, like cultured 
osteoscarcoma cells and calvaria cells, is 
significantly reduced after multiple passages in vitro. 

20 However, if the cells are cultivated in the presence of 
a morphogen (e.g., OP-1), alkaline phosphatase activity 
is maintained over extended periods of time. 
Similarly, phenotypic expression of myocytes also is 
maintained in the presence of the morphogen. This 
25 experiment may be performed with other morphogens and 
different cells to assess the phenotypic maintenance 
capability of different morphogens on cells of 
differing origins. 

30 Phenotypic maintenance capability alsQ may be 

assessed in vivo , using a rat model for osteoporosis, 
disclosed in co-pending USSN 752,857, filed August 30, 
1991,, incorporated herein by reference. As disclosed 
therein. Long Evans rats are ovariectomized to produce 

35 an osteoporotic condition resulting from decreased 



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estrogen production. Eight days after ovariectomy, 
rats are systemically provided with phosphate buffered 
saline (PBS) or OP-1 (21 pg or 20 pg) for 22 days. The 
rats then are sacrificed and serum alkaline phosphatase 
5 levels, serum calcium levels, and serum osteocalcin 
levels are determined, using standard methodologies. 
Three-fold higher levels of osteocalcin levels are 
found in rats provided with 1 or 20 fjg of OP-1. 
Increased alkaline phosphatase levels also were seen. 
10 Histomorphometric analysis on the tibial diaphysical 
bone shows QP-l can reduce bone mass lost due to the 
drop in estrogen levels. 

CELL STIMULATION 

15 

The ability of the morphogens of this 
invention to stimulate the proliferation of progenitor 
cells also can be assayed readily in vitro- Useful 
naive stem cells include pluripotential stem cells, 

20 which may be isolated from bone marrow or umbilical 

cord blood using conventional methodologies, {see, for 
example, Faradji et al., (1988) Vox Sang . 55 
(3):133-138 or Broxmeyer et al., (1989) PNAS 86 
(10) ;3828-3832) , as well as naive stem cells obtained 

25 from blood. Alternatively, embryonic cells (e.g., from 
a cultured mesodermal cell line) may be useful. 

Another method for obtaining progenitor cells 
and for determining the ability of morphogens to 

30 stimulate cell proliferation is to capture progenitor 
cells from an in vivo source. For example, a 
biocompatible matrix material able to allow the influx 
of migratory progenitor cells may be implanted at an in 
vivo site long enough to allow the influx of migratory 

35 progenitor cells. For example, a bone-derived. 



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guanidine-extracted matrix, formulated as disclosed for 
example in Sampath et al. ((1983) PNAS 80:6591-6595), 
or U.S. Patent No. 4,975,526, may be implanted into a 
rat at a subcutaneous site, essentially following the 
5 method of Sampath et al. (ibid). After three days the 
implant is removed, and the progenitor cells associated 
with the matrix dispersed and cultured. 

Progenitor cells, however obtained, then are 
incubated in vitro with a suspected morphogen under 
standard cell culture conditions well known to those 
having ordinary skill in the art. In the absence of 
external stimuli, the progenitor cells do not, or 
minimally proliferate on their own in culture. 
However, if the cells are cultured in the presence of a 
morphogen, such as OP-1, they are stimulated to 
proliferate. Cell growth can be determined visually or 
spectrophotometrically using standard methods well 
known in the art. 

PROLIFERATION OF PROGENITOR CELL POPULATIONS 

Progenitor cells may be stimulated to 
proliferate in vivo or ex vivo. The cells may be 
25 stimulated in vivo by injecting or otherwise providing 
a sterile preparation containing the morphogen into the 
individual. For example, the hemopoietic 
pluripotential stem cell population of an individual 
may be stimulated to proliferate by injecting or 
30 otherwise providing an appropriate concentration of the 
morphogen to the individual's bone marrow. 

Progenitor cells may be stimulated ex vivo by 
contacting progenitor cells of the population to be 
35 enhanced with a morphogen under sterile conditions at a 



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concentration and for a time sufficient to stimulate 
proliferation of the cells. In general/ a period of 
from about 10 minutes to about 24 hours should be 
sufficient. The stimulated cells then are provided to 
5 the individual as, for example, by injecting the cells 
to an appropriate in vivo locus. Suitable 
biocompatible progenitor cells may be obtained by any 
of the methods known in the art or described herein. 

10 REGENERATION OF DAMAGED OR DISEASED TISSUE 

The morphogens of this invention may be used 
to repair diseased or damaged mammalian tissue. The 
tissue to be repaired is preferably assessed, and 
15 excess necrotic or interfering scar tissue removed as 
needed, by surgical, chemical, ablating or other 
methods knovm 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 
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 
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 
envisioned that systemic provision of the morphogen 
will be sufficient. 



20 



25 



30 



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In some circumstances, particularly where 
tissue damage is extensive, the tissue may not be 
capable of providing a sufficient matrix for cell 
influx and proliferation. In these instances, it may 
5 be necessary to provide the morphogen or morphogen- 
stimulated 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 
10 biodegradable, and comprises particles having 
dimensions within the range of 70-850/im, most 
preferably 150-420^nn. 

The morphogens of this invention also may be used 
15 to prevent or substantially inhibit scar tissue 
formation following an injury. If a morphogen is 
provided to a newly injured tissue locus, it can induce 
tissue morphogenesis at the locus, preventing the 
aggregation of migrating fibroblasts into non- 
20 differentiated connective tissue. The morphogen 
preferably is provided as a sterile pharmaceutical 
preparation injected into the tissue locus within five 
hours of the injury. Several non- limiting examples 
follow, illustrating the morphogens regenerate 
25 capabilities in different issues. The proteins of this 
invention previously have been shown to be capable of 
inducing cartilage and endochondral bone formation 
(See, for example U.S. Patent No. 5,011,691). 

30 As an example, protein- induced morphogenesis of 

substantially injured liver tissue following a partial 
hepatectomy is disclosed. Variations on this general 
protocol may be used to test morphogen activity in 
other different tissues. The general method involves 

35 excising an essentially nonregenerating portion of a 



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tissue and providing the morphogen, preferably as a 
soluble pharmaceutical preparation to the excised 
tissue locus, closing the wound and examining the site 
at a future date. Like bone, liver has a potential to 
5 regenerate upon injury during post-fetal life. 

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 
10 acid (or compatible acid). The injectable OP-1 
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) . 

15 

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 OP-1 was injected locally at multiple sites along 

20 the cut ends. The amount of OP-1 injected was 100 pg 
in 100 of PBS/RSA (phosphate buffered saline/rat serum 
albumin) injection buffer. Placebo samples are 
injection buffer without OP-1. Five rats in each group 
were used. The wound was closed and the rats were 

25 allowed to eat normal food and drink tap water. 

After 12 days, the rats were sacrificed and liver 
regeneration was observed visually. The 
photomicrograph in Fig. 7 illustrates dramatically the 

30 regenerative effects of OP-1 on liver regeneration. 
The OP-l-injected group showed complete liver tissue 
regeneration and no sign remained of any cut in the 
liver (animal 2). By contrast, in the control group 
into which only PBS was injected only minimal 

35 regeneration was evidenced (animal 1). In addition, 
the incision remains in this sample. 



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AS another example^ the ability of the morphogens 
of this invention to induce dentinogenesis also was 
assessed. To date, the unpredictable response of 
5 dental pulp tissue to injury is a basic clinical 

problem in dentistry. Cynomolgus monkeys were chosen 
as primate models as monkeys are presiamed to be more 
indicative of human dental biology than models based on 
lower non-primate mammals. 

10 

Using standard dental surgical procedures, small 
areas (e.g., 2mm) of dental pulps were surgically 
exposed by removing the enamel and dentin immediately 
above the pulp (by drilling) of sample teeth, 
15 performing a partial amputation of the coronal pulp 
tissue, inducing hemostasis, application of the pulp 
treatment, and sealing and filling the cavity by 
standard procedures. 

20 Pulp treatments used were: OP-1 dispersed in a 

carrier matrix; carrier matrix alone and no treatment. 
Twelve teeth per animal (four for each treatment) were 
prepared, and two animals were used. At four weeks, 
teeth were extracted and processed histologically for 

25 analysis of dentin formation, and/or ground to analyze 
dentin mineralization. FIG. 8 illustrates dramatically 
the effect of morphogen on osteodentin reparation. 
FIG. 8A is a photomicrograph of the control treatment 
(PBS) and shows little or no reparation. FIG. 8B is a 

30 photomicrograph of treatment with carrier alone, 

showing minimal reparation. By contrast, treatment 
with morphogen (FIG. 8C) shows significant reparation. 
The results of FIG. 8 indicate that OP-l-CM (OP-1 plus 



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carrier matrix) reliably induced formation of 
reparative or osteodentin bridges on surgically exposed 
healthy dental pulps. By contrast, pulps treated with 
carrier matrix alone, or not treated failed to form 
5 reparative dentin. 

As another example, the morphogen- induced 
regenerative effects on central nervous system (CNS) 
repair may be assessed using a rat brain stab model. 

10 Briefly, male Long Evans rats are anesthesized and the 
head area prepared for surgery. The calvariae is 
exposed using standard surgical procedures and a hole 
drilled toward the center of each lobe using a 0.035K 
wire, just piercing the calvariae. 25/il solutions 

15 containing either morphogen (OP-1, 25pg) or PBS then is 
provided to each of the holes by Hamilton syringe. 
Solutions are delivered to a depth approximately 3 mm 
below the surface, into the underlying cortex, corpus 
callosum and hippocampus. The skin then is sutured and 

20 the animal allowed to recover. 

Three days post surgery, rats are sacrificed by 
decapitation and their brains processed for sectioning. 
Scar tissue formation is evaluated by immunofluoresence 

25 staining for glial fibrillary acidic protein, a marker 
protein for glial scarring, to qualitatively determine 
the degree of scar formation. Sections ^Iso are probed 
with anti-OP-1 antibodies to determine the presence of 
OP-1. Reduced levels of glial fibrillary acidic 

30 protein are anticipated in the tissue sections of . 

animals treated with morphogen, evidencing the ability 
of morphogen to inhibit glial scar formation, thereby 
stimulating nerve regeneration. 

35 MORPHOGEN ACTIVITY MODULATION 



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Antibodies to xnorphogens of this invention have 
been identified in healthy human sera. In addition, 
implanting devices comprising morphogens (e.g., OP-1) 
5 have been discovered to induce an increase in anti- 
morphogen antibodies (e.g., anti-OP-1 antibodies). It 
is anticipated that these antibodies comprise part of 
the body's regulation of morphogen activity in vivo . 
The presence of the antibodies, and fluctuations in 

10 their levels, which are readily monitored, can provide 
a useful method for monitoring tissue stasis and tissue 
viability (e.g., identification of a pathological 
state). For example, standard radioimmunoassays or 
ELISA may be used to detect and quantify endogeous 

15 anti-morphogen antibodies in sera. Antibodies or other 
binding proteins capable of detecting anti-morphogen 
antibodies may be obtained using standard 
methodologies. 

20 MATRIX PREPARATION 

The morphogens of this invention may be 
implanted surgically, dispersed in a biocompatible, 
preferably in vivo biodegradable matrix appropriately 

25 modified to provide a structure in which the morphogen 
may be dispersed and which allows the influx, 
differentiation and proliferation of migrating 
progenitor cells. The matrix also should provide 
signals capable of directing the tissue specificity of 

30 the differentiating cells, as well as a morphogenically 
permissive environment, being essentially free of 
growth inhibiting signals. 



35 



In the absence of these features the matrix 
does not appear to be suitable as part of a morphogenic 
composition. Recent studies on osteogenic devices 



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(morphogens dispersed within a formulated matrix) using 
matrices formulated from polylactic acid and/or 
polyglycolic acid biopolymers, ceramics (a-tri-calcium- 
phosphate ) / or hydroxyapatite show that these 
5 materials, by themselves, are unable to provide the 
appropriate environment for inducing de novo 
endochondral bone forination in rats by themselves. In 
addition, matrices formulated from commercially 
available highly purified, reconstituted collagens or 

10 naturally-derived non-bone, species-specific collagen 
(e.g., from rat tail tendon) also are unsuccessful in 
inducing bone when implanted in association with an 
osteogenic protein. These matrices apparently lack 
specific structurally-related features which aid in 

15 directing the tissue specificity of the morphogen- 
stimulated, differentiating progenitor cells. 

The formulated matrix may be shaped as desired 
in anticipation of surgery or may be shaped by the 

20 physician or technician during surgery. Thus, the 
material may be used in topical, subcutaneous, 
intraperitoneal, or intramuscular implants to repair 
tissue or to induce its growth de novo. The matrix 
preferably is biodegradable in vivo , being slowly 

25 absorbed by the body and replaced by new tissue growth, 
in the shape or very nearly in the shape of the 
implant. 

Details of how to make and how to use the 
30 matrices useful in this invention are disclosed below. 

TISSUE-DERIVED MATRICES 

Suitable biocompatible, in vivo biodegradable 
35 acellular matrices may be prepared from naturally- 



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occurring tissue* The tissue is treated with suitable 
agents to substantially extract the cellular, 
nonstructural components of the tissue. The agents 
also should be capable of extracting any growth 
5 inhibiting components associated with the tissue. The 
resulting material is a porous, acellular matrix, 
substantially depleted in nonstructurally-associated 
components • 

10 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 

15 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 

20 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 

25 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®C. The suspension then is filtered. 
The insoluble material that remains is collected and 

30 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 

35 alters the surface structure of the matrix material. 



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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 
and PCT publication US90/00912, published September 7, 
5 1990 (WO90/10018) . 

The currently most preferred agent is a heated 
aqueous f ibril*modifying medium such as water, to 
increase the matrix particle surface area and porosity. 

10 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% 
acetic acid, which has a pH of about 3, currently is 

15 roost preferred. 0.1 M acetic acid also may be used. 

Various amounts of delipidated, demineralized 
guanidine-extracted bone collagen are heated in the 
aqueous medium ( Ig matrix/30ml aqueous medium) under 

20 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 
to two hours appear acceptable. The temperature 

25 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 60**C. 

30 After the heat treatment, the matrix is filtered, 

washed, lyophilized and used for implant. Where an 
acidic aqueous medium is used, the matrix also is 
preferably neutralized prior to washing and 
lyophilization. A currently preferred neutralization 

35 buffer is a 200mM sodium phosphate buffer, pH 7.0. To 



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neutralize the matrix, the matrix preferably first is 
allowed to cool following thermal treatment, the acidic 
aqueous medium (e.g., 0.1% acetic acid) then is removed 
and replaced with the neutralization buffer and the 
5 matrix agitated for about 30 minutes. The 

neutralization buffer then may be removed and the 
matrix washed and lyophilized. 

Other useful fibril-modifying treatments include 
10 acid treatments (e.g., trifluoroacetic acid and 
hydrogen fluoride) and solvent treatments such as 
dichloromethane, acetonitrile, isopropanol and 
chloroform, as well as particular acid/solvent 
combinations. 

15 

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 below: 

20 

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 
(UTBS) or water and stir at room temperature (RT) for 

25 30 minutes (sufficient time to neutralize the pH); 

2. Centrifuge and repeat wash step; and 

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

SYNTHETIC TISSUE-SPECIFIC MATRICES 
35 In addition to the naturally-derived tissue- 



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specific matrices described above, useful tissue- 
specific matrices may be formulated synthetically if 
appropriately modified. These porous biocompatible, in 
vivo biodegradable synthetic matrices are disclosed in 
5 PCT publication US91/03603, published December 12, 1991 
(W091/18558) , the disclosure of which is hereby 
incorporated by reference. Briefly, the matrix 
comprises a porous crosslinked structural polymer of 
biocompatible, biodegradable collagen and appropriate, 

10 tissue-specific glycosaminoglycans as tissue-specific 
cell attachment factors. Collagen 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 

15 aqueous solutions, as well as those collagens which are 
commercially available. 

Glycosaminoglycans (GAGs) or 
mucopolysaccharides are hexosamine-containing 

20 polysaccharides of animal origin that have a tissue 
specific distribution, and therefore may be used to 
help determine the tissue specificity of the morphogen- 
stimulated differentiating cells. Reaction with the 
GAGs also provides collagen with another valuable 

25 property, i.e., inability to provoke an immune reaction 
(foreign body reaction) from an animal host. 

Chemically, GAGs are made up of residues of 
hexoseamines glycosidically bound and alternating in a 

30 more-or-less regular manner with either hexouronic acid 
or hexose moieties (see, e«g., Dodgson et al. in 
Carbohydrate Metabolism and its Disorders (Dickens et 
al., eds.) Vol. 1, Academic Press (1968)). Useful CAGs 
include hyaluronic acid, heparin, heparin sulfate, 

35 chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan 



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sulfate, and keratin sulfate. Other GAGs are suitable 
for forming the matrix described herein / and those 
skilled in the art will either know or be able to 
ascertain other suitable GAGs using no more than 
5 routine experimentation. For a more detailed 
description of mucopolysaccharides, see Aspinall, 
Polysaccharides , Pergamon Press, Oxford (1970). For 
example, as disclosed in U.S. Application Serial 
No. 529,852, chondroitin-6-sulf ate can be used where 
10 endochondral bone formation is desired. Heparin 

sulfate, on the other hand, may be used to formulate 
synthetic matrices for use in lung tissue repair. 

Collagen can be reacted with a GAG in aqueous 
15 acidic solutions, preferably in diluted acetic acid 

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

. Insolubility of the collagen-GAG products can 
be raised to the desired degree by covalently cross- 
linking these materials, which also serves to raise the 
25 resistance to resorption of these materials. In 

general, any covalent cross-linking method suitable for 
cross-linking collagen also is suitable for cross- 
linking these composite materials, although 
crosslinking by a dehydrothermal process is preferred. 

30 

When dry, the crosslinked particles are 
essentially spherical, with diameters of about 500 ym. 
Scanning electron miscroscopy shows pores of about 
20 /im on the surface and 40 pm on the interior. The 
35 interior is made up of both fibrous and sheet-like 



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staructureS/ providing surfaces for cell attachment. 
The voids interconnect, providing access to the cells 
throughout the interior of the particle. The material 
appears to be roughly 99.5% void volume, making the 
5 material very efficient in terms of the potential cell 
mass that can be grown per gram of microcarrier. 

The morphogens described herein can be . 
combined and dispersed in an appropriately modified 
10 tissue-specific matrix using any of the methods 
described below: 

1. Ethanol Precipitation 

15 Matrix is added to the morphogen dissolved in 

guanidine-HCl. Samples are vortexed and incubated at a 
low temperature. Samples are then further vortexed. 
Cold absolute ethanol is added to the mixture which is 
then stirred and incubated. After centrifugation 

20 (microfuge/ high speed) the supernatant is discarded. 
The matrix is washed with cold concentrated ethanol in 
water and then lyophilized. 

2. Acetonitrile Trifluoroacetic 
25 Acid Lyophilization 

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

3. Buffered Saline Lyophilization 

Morphogen preparations in physiological saline 
35 may also be vortexed with the matrix and lyophilized to 



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produce morphogenically active material. 

BIOASSAY 

5 

The following sets forth various procedures 
for evaluating the in vivo morphogenic utility of the 
morphogens and morphogenic compositions of this 
invention. The proteins and compositions may be 
10 injected or surgically implanted in a mammal/ following 
any of a number of procedures well known in the art. 
For example/ surgical implant bioassays may be 
performed essentially following the procedure of 
Sampath et al. (1983) PNAS 80:6591-6595. 

15 

Histological Evaluation 

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

20 vivO/ particularly in tissue repair procedures. 

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

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

Successful implants exhibit a controlled 
progression through the stages of induced tissue 

30 development allowing one to identify and follow the 
tissue-specific events that occur. For example/ in 
endochondral bone fomation the stages include: 
(1) leukocytes on day one; (2) mesenchymal cell 
migration and proliferation on days two and three; 

35 (3) chondrocyte appearance on days five and six; 



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PCr/US92/01968 



(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 

5 osteoblastic 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* 

10 Biological Markers 

In addition to histological evaluation/ 
biological markers may be used as a marker for tissue 
morphogenesis. Useful markers include tissue-specific 

15 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 formation 
quickly after the implants are removed from the animal. 

20 For example, alkaline phosphatase activity may be used 
as a marker for osteogenesis. 

Incorporation of systemically provided 
morphogens may be followed using tagged morphogens 

25 (e.g., radioactively labelled) and determining their 
localization in new tissue, and/or by monitoring their 
disappearance from the circulatory system using a 
standard pulse-chase labeling protocol. The morphogen 
also may be provided with a tissue-specific molecular 

30 tag, whose uptake may be monitored and correlated with 
the concentration of morphogen provided. As an 
example, ovary removal in female rats results in 
reduced bone alkaline phosphatase activity, rendering 
the rats predisposed to osteoporosis. If the female 

35 rats now are provided with a morphogen, e.g., OP-1, a 



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reduction in the systemic concentration of calcium 
(CA^*) is seen^ which correlates with the presence of 
the provided morphogen and can be shown to correspond 
to increased alkaline phosphatase activity • 

5 

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

10 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 of the claims are therefore intended to be 

15 embraced therein. 



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SEQUENCE LISTING 



(1) GENERAL INFOBHATION: 

(i)APFLICANT: COHEN, CHARLES H. 

KUBERASAHPATH, THANGAVEL 
PANG, ROY H.L. 
OPPERHANN, HERMANN 
RDEGER, OATID C. 



(li) TITLE OF INVENTION: PROTEIN- INDUCED MORPHOGENESIS 
(iii) NUMBER OF SEQUENCES: 23 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: TESTA, HURVITZ & THIBEAULT 

(B) STREET: 53 STATE STREET 

(C) CIT7: BOSTON 

(D) STATE: MASSACHUSETTS 

(E) COUNTRY: U.S.A. 

(F) ZIP: 02109 

(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 

(vii) PRIOR APPLICATION DATA: 

(A) APPLICATION NUMBER: US 667,274 

(B) FILING DATE: ll.HAR-1991 

(Tii) PRIOR APPLICATION DATA: 

(A) APPLICATION NUMBER: US 752,764 

(B) FILING DATE: 30-AUG-1991 

(2) INFORMATION FOR SEQ ID N0:1: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH; 97 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 



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PCT/US92/019d8 



(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: Generic Sequence 1 
(D) OTHER INFORMATION: Each Xaa 

indicates one of the 20 naturally- 
occurring L-isoner/ a-amino acids 
or a derivative thereof, 
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: 

Xaa Xaa Xaa Xaa Xaa Xaa 
1 5 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

10 15 
Xaa Xaa Xaa Xaa xaa Xaa Xaa Cys Xaa Xaa Xaa 

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

30 35 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
40 45 50 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

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

65 70 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

75 80 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

85 90 
Xaa Cys Xaa 
95 

(2) INFORMATION FOR SEQ ID NO: 2: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 



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PCr/US92/01968 



(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: Generic Sequence 2 
(D) OTHER INFORMATION: Each Xaa 

indicates one of the 20 naturally- 
occurring L-isomer, a-amino acids 
or a derivative thereof, 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 

Xaa Xaa Xaa Xaa Xaa Xaa 
1 5 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

10 15 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 

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

30 35 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
40 45 50 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

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

65 70 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

75 80 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

85 90 
Xaa Cys Xaa 
95 

(2) INFORMATION FOR SEQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 



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(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: Generic Sequence 3 
(D) OTHER INFORMATION: wherein each 
Xaa is independently selected from 
a group of one or more specified 
amino acids as defined in the 
specification. 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 

Leu Tyr Val Xaa Phe 
1 5 
Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

10 

Xaa Ala Pro Gly Xaa Xaa Xaa Ala 

15 20 
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 
25 30 
Xaa Pro Xaa Xaa Xaa Xaa Xaa 

35 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 
40 45 
Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 

50 

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 

70 75 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
80 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

85 90 
Xaa Cys Gly Cys Xaa 
95 



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(2) INFORMATION FOR SEQ ID NO: 4: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: Generic Sequence 4 

(D) OTHER INFORMATION: wherein each 
Xaa is independently selected from 
a group of one or more specified 
amino acids as defined in the 
specification . 

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



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

15 

Xaa Ala Pro Xaa Gly Xaa Xaa Ala 

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

40 

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

55 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

60 65 
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
70 



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PCT/US92/01968 



Xaa. Xaa Xaa Leu Xaa Xaa Xaa 

75 BO 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
85 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

90 95 
Xaa Cys Gly Cys Xaa 
100 

(2) INFORMATION FOR SEQ ID NO: 5: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 
(iz) FEATURE: 

(A) NAME: bOP-1 (mature form) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 

Ser Thr Gly Ser Lys Gin Arg Ser Gin 

1 5 
Asn Arg Ser Lys Thr Pro Lys Asn Gin 

10 15 
Glu Ala Leu Arg Met Ala Asn Val Ala 

20 25 
Glu Asn Ser Ser Ser Asp Gin Arg Gin 

30 35 
Ala Cys Lys Lys His Glu Leu Tyr Val 
40 45 
Ser Phe Arg Asp Leu Gly Trp Gin Asp 

50 

Trp lie lie Ala Pro Glu Gly Tyr Ala 

55 60 
Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 
65 70 



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PCr/US92/01968 



Phe Pro Leu Asn Ser 
75 

Thr Asn His Ala lie 
85 

Val His Phe lie Asn 

95 

Pro Lys Pro Cys Cys 
100 

Leu Asn Ala lie Ser 
110 

Asp Asp Ser Ser Asn 
120 

Lys Tyr Arg Asn Met 
130 

Cys Gly Cys His 



Tyr Met Asn Ala 
80 

Val Gin Thr Leu 
90 

Pro Glu Thr Val 

Ala Pro Thr Gin 
105 

Val Leu Tyr Phe 
115 

Val lie Leu Lys 
125 

Val Val Arg Ala 
135 



INFORMATION FOR SEQ ID NO: 6: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: mOP-1 (mature form) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 

Ser Thr Gly Gly Lys Gin Arg Ser Cln 

1 5 

Asn Arg Ser Lys Thr Pro Lys Asn Gin 

10 15 

Glu Ala Leu Arg Met Ala Ser Val Ala 

20 25 



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Glu Asn Ser 
30 

Ala Cys Lys 

Ser Phe Arg 

Trp lie lie 
55 

Ala Tyr Tyr 
65 

Phe Pro Leu 
75 

Thr Asn His 

Val His Phe 

Pro Lys Pro 
100 

Leu Asn Ala 
110 

Asp Asp Ser 
120 

Lys Tyr Arg 

Cys Gly Cys 



-84- 



Ser Ser Asp 

Lys His Glu 
40 

Asp Leu Gly 
50 

Ala Pro Glu 
60 

Cys Glu Gly 

Asn Ser Tyr 

Ala He Val 
85 

He Asn Pro 
95 

Cys Cys Ala 
105 

He Ser Val 

Ser Asn Val 

Asn Met Val 

130 

His 



PCT/US92/01968 



Gin Arg Gin 
35 

Leu Tyr Val 
45 

Trp Gin Asp 

Gly Tyr Ala 

Glu Cys Ala 
70 

Met Asn Ala 
80 

Gin Thr Leu 
90 

Asp Thr Val 

Pro Thr Gin 

Leu Tyr Phe 
115 

He Leu Lys 
125 

Val Arg Ala 
135 



(2) INFORMATION FOR SEQ ID NO: 7: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: hOP-2 (mature form) 



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

Ala Val Arg Pro Leu Arg Arg Arg Gin 

1 5 
Pro Lys Lys Ser Asn Glu Leu Pro Gin 

10 15 
Ala Asn Arg Leu Pro Gly lie Phe Asp 

20 25 
Asp Val His Gly Ser His Gly Arg Gin 

30 35 
Val Cys Arg Arg His Glu Leu Tyr Val 
40 45 
Ser Phe Gin Asp Leu Gly Trp Leu Asp 

50 

Trp Val lie Ala Pro Gin Gly Tyr Ser 

55 60 
Ala Tyr Tyr Cys Glu Gly Glu Cys Ser 

65 70 
Phe Pro Leu Asp Ser Cys Met Asn Ala 

75 80 
Thr Asn His Ala lie Leu Gin Ser Leu 
85 90 
Val His Leu Met Lys Pro Asn Ala Val 

95 

Pro Lys Ala Cys Cys Ala Pro Thr Lys 

100 105 

Leu Ser Ala Thr Ser Val Leu Tyr Tyr 

110 115 
Asp Ser Ser Asn Asn Val lie Leu Arg 

120 125 
Lys His Arg Asn Met Val Val Lys Ala 
130 135 
Cys Gly Cys His 



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(2) INFORMATION FOR SEQ ID NO: 8: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: mOP-2 (mature form) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 

Ala Ala Arg Pro Leu Lys Arg Arg Gin 

1 5 
Pro Lys Lys Thr Asn Glu Leu Pro His 

10 15 
Pro Asn Lys Leu Pro Gly lie Phe Asp 

20 25 
Asp Gly His Gly Ser Arg Gly Arg Glu 

30 35 
Val Cys Arg Arg His Glu Leu Tyr Val 
40 45 
Arg Phe Arg Asp Leu Gly Trp Leu Asp 

50 

Trp Val lie Ala Pro Gin Gly Tyr Ser 

55 60 
Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 

65 70* 
Phe Pro Leu Asp Ser Cys Met Asn Ala 

75 80 
Thr Asn His Ala He Leu Gin Ser Leu 
85 ' 90 

Val His Leu Met Lys Pro Asp Val Val 

95 

Pro Lys Ala Cys Cys Ala Pro Thr Lys 
100 105 



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PCr/US92/01968 



Leu Ser Ala Thr Ser Val Leu Tyr Tyr 

110 115 

Asp Ser Ser Asn Asn Val lie Leu Arg 

120 125 

Lys His Arg Asn Met Val Val Lys Ala 
130 135 

Cys Gly Cys His 

(2) INFORMATION FOR SEQ ID NO: 9: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 96 amino acids 

(B) TYPE: eunino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: CBMP-2A(fx) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 

Cys Lys Arg His Pro Leu Tyr Val Asp Phe Ser 

15 10 
Asp Val Gly Tip Asn Asp Trp lie Val Ala Pro 

15 20 
Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu 

25 30 
Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser 

35 40 
Thr Asn His Ala lie Val Gin Thr Leu Val Asn 
45 50 55 

Ser Val Asn Ser Lys lie Pro Lys Ala Cys Cys 

60 65 
Val Pro Thr Glu Leu Ser Ala lie Ser Met Leu 

70 75 
Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys 
80 85 



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PCT/US92/01968 



Asn Tyr Gin Asp Met Val Val Glu Gly Cys Gly 

90 95 
Cys Arg 
100 



(2) INFORMATION FOR SEQ ID NO: 10: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 101 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: CBMP-2B(fx) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 

Cys Arg Arg His Ser 
1 5 
Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn 

10 15 
Asp Trp lie Val Ala Pro Pro Gly Tyr Gin Ala 

20 25 
Phe Tyr Cys His Gly Asp Cys Pro Phe Pro Leu 

30 35 
Ala Asp His Leu Asn Ser Thr Asn His Ala lie 

40 45 
Val Gin Thr Leu Val Asn Ser Val Asn Ser Ser 
50 55 60 

lie Pro Lys Ala Cys Cys Val Pro Thr Glu Leu 

65 70 
Ser Ala lie Ser Met Leu Tyr Leu Asp H31u Tyr 

75 80 
Asp Lys Val Val Leu Lys Asn Tyr Gin Glu Met 
85 90 



89 



PCT/US92/01968 



Val Val Glu Gly Cys Gly Cys Arg 
95 100 



INFORMATION FOR SEQ ID NO; 11: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: DPP(fx) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 

Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser 

15 10 
Asp Val Gly Trp Asp Asp Trp He Val Ala Pro 

15 20 
Leu Gly Tyr Asp Ala Tyr Tyr Cys His Gly Lys 

25 30 
Cys Pro Phe Pro Leu Ala Asp His Phe Asn Ser 

35 40 
Thr Asn His Ala Val Val Gin Thr Leu Val Asn 
45 50 55 

Asn Asn Asn Pro Gly Lys Val Pro Lys Ala Cys 

60 65 
Cys Val Pro Thr Gin Leu Asp Ser Val Ala Met 

70 75 
Leu Tyr Leu Asn Asp <31n Ser Thr Val Val Leu 

80 85 
Lys Asn Tyr Gin Glu Met Thr Val Val Gly Cys 

90 95 
Gly Cys Arg 
100 



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( 2 ) INFORMATION FOR SEQ ID NO : 12 : 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: Vgl(fx) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 

Cys Lys Lys Arg His Leu Tyr Val Glu Phe Lys 

1 5 10 

Asp Val Gly Trp Gin Asn Trp Val lie Ala Pro 

15 20 
Gin Gly Tyr Met Ala Asn Tyr Cys Tyr Gly Glu 

25 30 
Cys Pro Tyr Pro Leu Thr Glu lie Leu Asn Gly 

35 40 
Ser Asn His Ala He Leu Gin Thr Leu Val His 
45 50 55 

Ser He Glu Pro Glu Asp He Pro Leu Pro Cys 

60 65 
Cys Val Pro Thr Lys Met Ser Pro He Ser Met 

70 75 
Leu Phe Tyr Asp Asn Asn Asp Asn Val Val Leu 

80 85 
Arg His Tyr Glu Asn Met Ala Val Asp Glu Cys 

90 95 
Gly Cys Arg 
100 



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(2) INFORMATION FOR SEQ ID NO: 13: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: Vgr-l(fx) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 



Cys Lys Lys His Glu Leu Tyr Val Ser Phe Gin 

1 5 10 

Asp Val Gly Trp Gin Asp Trp He He Ala Pro 

15 20 
Xaa Gly Tyr Ala Ala Asn Tyr Cys Asp Gly Glu 

25 30 
Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala 

35 40 
Thr Asn His Ala He Val Gin Thr Leu Val His 
45 50 55 

Val Met Asn Pro Glu Tyr Val Pro Lys Pro Cys 

60 65 
Cys Ala Pro Thr Lys Val Asn Ala He Ser Val 

70 75 
Leu Tyr Phe Asp Asp Asn Ser Asn Val He Leu 

80 85 
Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys 

90 95 
Gly Cys His 
100 

(2) INFORMATION FOR SEQ ID NO: 14: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 106 amino acids 

(B) TYPE: protein 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



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(vi) ORIGINAL SOURCE: 
(A) ORGANISM: human 
(F) TISSUE TYPE: BRAIN 

(ix) FEATURE: 
(D) OTHER INFORMATION: 

/product" "GDF-1 (fx)'' 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1A: 

Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly 
15 10 

Trp His Arg Trp Val He Ala Pro Arg Gly Phe Leu Ala Asn Tyr 
15 20 25 

Cys Gin Gly Gin Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly 
30 35 40 

Gly Pro Pro Ala Leu Asn His Ala Val Leu Arg Ala Leu Met His 
45 50 55 

Ala Ala Ala Pro Gly Ala Ala Asp Leu Pro Cys Cys Val Pro Ala 
60 65 70 

Arg Leu Ser Pro He Ser Val Leu Phe Phe Asp Asn Ser Asp Asn 
75 80 85 

Val Val Leu Arg Gin Tyr Glu Asp Met Val Val Asp Glu Cys Gly 
90 95 100 

Cys Arg 
105 



(2) INFORMATION FOR SEQ ID NO: 15: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 5 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

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



Cys Xaa Xaa laa Xaa 
1 5 



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(2) INFORMATION FOR SEQ ID NO: 16: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1822 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) HOLECULE TYPE: cDNA 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HOMO SAPIENS 
. (F) TISSUE TYPE: HIPPOCAMPUS 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 49.. 1341 

(D) OTHER INFORMATION: /standard_name= "hOPl" 

(Xi) SEQUENCE DESCRIPTION: SEQ ID N0:16: 

GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA €CGCGTAGAG CCGGCGCG ATG CAC GTG 57 

Met His Val 
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 Pro His Ser Phe Val Ala Leu Trp Ala 
5 10 15 

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 

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

CGG GAG ATG CAG CGC GAG ATC CTC TCC ATT TTG GGC TTG.CCC CAC CGC 249 
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 
70 75 80. 

CTG GAC CTG TAC AAC GCC ATG GCG GTG GAG GA€ GGC GGC GGG CCC GGC 345 
Leu Asp Leu Tyx Asn Ala Met Ala Val Glu Glu Gly Gly Gly Pro Gly 
85 90 95 



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



393 



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CCC CCT CTG GCC AGC CTG CAA GAT AGC CAT TTC CTC ACC GAC GCC GAC AAl 
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 A89 
Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe 
135 lAO 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 
150 155 160 

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 

TAC ATC CGG GAA CGC TTC GAC AAT GAG ACG TTC CGG ATC AGC GTT TAT 633 
IVr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg He Ser Val Tyr 
180 185 190 195 

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 729 
Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp 
215 220 225 

ATC ACA GCC ACC AGC AAC CAC TGG GTG GTC AAT CCG CGG CAC AAC CTG 777 
He Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu 
230 235 240 

GGC CTG CAG CTC TCG GTG GAG ACG CTG GAT GGG CAG AGC ATC AAC CCC 825 
Gly Leu Gin Leu Ser Val Glu Thr Leu Asp <ily Gin Ser He Asn Pro 
245 250 255 

AAG TTG GCG GGC CTG ATT GGG CGG CAC GGG CCC CAG AAC AAG CAG CCC 873 
Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys Gin Pro 
260 265 270 275 

TTC ATG GTG GCT TTC TTC AAG GCC ACG GAG XSTC CAC TTC CGC AGC ATC 921 
Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser He 
280 285 290 

CGG TCC ACG GGG AGC AAA CAG CGC AGC CAG AAC CGC TCC AAG ACG CCC 969 
Arg Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro 
295 300 305 

AAG AAC CAG GAA GCC CTG CGG ATG GCC AAC GTG GCA GAG AAC AGC AGC 1017 
Lys Asn Gin Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser 
310 315 320 



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AGC GAC CAG AGG CAG GCC TGT AAG AAG CAC GAG CTG TAT GTC AGC TTC 1065 
Set 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 Tyr 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 lyr Het 
360 365 370 

AAC GCC ACC AAC CAC GCC ATC GTG CAG ACG CTG GTC CAC TTC ATC AAC 1209 
Asn Ala Thr Asn His Ala He Val Gin Thr Leu Val His Phe He Asn 
375 380 385 

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

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

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 

GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA ACTTTAAAGG 1471 

TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CAGTGGCAGC 1531 

ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AAAAAACAAC 1591 

GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TGCACGGACT 1651 

CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GGAGGAA(;GG 1711 

GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTGAC CCGGAAGTTC 1771 

CTGTAATAAA TGTCACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A 1822 



(2) INFORMATION FOR SEQ ID NO: 17: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 431 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



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(ix) FEATURE: 

(D) OTHER INFORMATION: /Product-"OPl-PP" 

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

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

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 

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

Pro His Arg Fro Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Fro 
65 70 75 80 

Met Phe Met Leu Asp Leu T3^ Asn Ala Met Ala Val Glu Glu Gly Gly 
85 90 95 

Gly Fro Gly Gly Gin Gly Phe Ser Tyr Pro lyr Lys Ala Val Phe Ser 
100 105 110 

Thr Gin Gly Fro Fro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Ihr 
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 Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu 
145 150 155 160 

Ser Lys He Pro Glu Gly Glu Ala Val Ihr Ala Ala Glu Phe Arg He 
165 170 • 175 

Tyr Lys Asp Tyr He Arg Glu Arg Phe Asp Asn Glif Thr Phe Arg He 
180 185 190 

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 
225 230 235 240 

His Asn Leu Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser 
245 250 255 



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He Asn Pro Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn 
260 265 270 

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 Glh 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 
305 310 315 320 

Asn Ser Ser Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr 
325 330 335 

Val Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu 
340 345 350 

Gly Tyr Ala Ala Tyr lyx Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn 
355 360 365 

Ser Tyr Met 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 Thx Gin 
385 390 395 400 

Leu Asn Ala He Ser Val Leu lyr Phe Asp Asp Ser Ser Asn Val He 
405 410 415 

Leu Lys 'Lys Tyr Arg Asn Het Val Val Arg Ala Cys Gly Cys His 
420 425 430 



(2) INFORMATION FOR SEQ ID NO: 18: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1873 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) T0P0L0G7: linear 

(ii) MOLECULE TYPE: cDNA 

(vi) ORIGINAL SOURCE: 

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

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 104.. 1393 

(D) OTHER INFORMATION: /note= "MOPl (CDNA)" 



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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: 

CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CCACCTGGGG 60 

CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCC^SGC GCG ATG CAC GIG CGC 115 

Met His Val Arg 
1 

TCG CTG CGC GCT GCG GCG CCA CAC AGC TIC 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 

GTG CAC TCC AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG CGG 259 
Val His Ser Ser Phe He His Arg Arg Leu Arg Ser Gin Glu Arg Arg 
40 45 50 

GAG ATG CAG CGG GAG ATC CTG TCC ATC TTA GGG TTG CCC CAT CGC CCG 307 
Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu Pro His Arg Pro 
55 60 65 

CGC CCG CAC CTC CAG GGA AAG CAT AAT TCG GC€ CCC ATG TTC ATG TTG 355 
Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro Met Phe Met Leu 
70 75 80 

GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG AGC GGG CCG GAC GGA CAG 403 
Asp Leu lyr Asn Ala Met Ala Val Glu Glu Ser Gly Pro Asp Gly Gin 
85 90 95 100 

GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AGT ACC CAG GGC CCC CCT 451 
Gly Phe Ser Tjrr Pro TJrr Lys Ala Val Phe Ser Thr Gin Gly Pro Pro 
105 110 115 

TTA GCC AGC CTG CAG GAC AGC CAT TTC CTC ACT GAC GCC GAC ATG GTC 499 
Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp Ala Asp Met Val 
120 125 130 

ATG AGC TTC GTC AAC CTA GTG GAA CAT GAC AAA GAA TTC TTC CAC CCT 547 
Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe His Pro 
135 140 145 

CGA TAC CAC CAT CGG GAG TTC CGG TTT GAT CTT TCC AAG ATC CCC GAG 595 
Arg lyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys He Pro Glu 
150 155 160 

GGC GAA GCG GTG ACC GCA GCC GAA TTC AGG ATC TAT AAG GAC TAC ATC 643 
Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He Tyr Lys Asp Tyr He 
165 170 175 180 



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CGG GAG CGA TTT GAC AAC GAG ACC TTC CAG ATC ACA GTC TAT CAG TGG 
Arg Glu Arg Phe Asp Asn Glu Thr Phe Gin lie Thr Val Tyr Gin Trp 
185 190 195 



691 



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 



739 



CGC ACC ATC TGG OCT TCT GAG GAG GGC TGG TTG GTG TTT GAT ATC ACA 787 
Arg Thr He Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp He Thr 
215 220 225 

GCC ACC AGC AAC CAC TGG GTG GTC AAC CCT CGG CAC AAC CTG GGC TTA 835 
Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu Gly Leu 
230 235 240 

CAG CTC TCT GTG GAG ACC CTG GAT GGG CAG AGC ATC AAC CCC AAG TTG 883 
Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser He Asn Pro Lys Leu 
245 250 255 260 

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 

GTG GCC TTC TTC AAG GCC ACG GAA GTC CAT CTC CGT ACT ATC CGG TCC 979 
Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg Ser He Arg Ser 
280 285 290 

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 
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 Are Asp 
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 

TAC TGT GAG GGA GAG TGC GCC TTC CCT CTG AAC TCC TAC ATG AAC GCC 1219 
Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala 
360 365 37<l 

ACC AAC CAC GCC ATC GTC CAG ACA CTG -GTr 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 



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ACA GTA CCC AAG CCC TGC TGT GCG CCC ACC CAtJ CTC AAC GCC ATC TCT 1315 
Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala He Ser 
390 395 ^00 

GTC CTC TAC TTC GAC GAC AGC TCT AAT GTC ATC CTG AAG AAG TAC AGA 1363 
Val Leu Tyr Phe Asp Asp Ser Ser Asn Val He Leu Lys Lys Tyr Arg 
405 410 A15 A20 

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 

ACCTTTGCGG GGCCACACCT TTCCAAATCT TCGATGTCTC ACCATCTAAG TCTCTCACTG 1473 

CCCACCTTGG CGAGGAGAAC AGACCAACCT CTCCTGAGCC TTCCCTCACC TCCCAACCGG 1533 

AAGCATGIAA GGGTTCCA€A AACCTGAGCG TGCAGCAGCT GATGAGCGCC CTTTCCTTCT 1593 

GGCACGTGAC GGACAAGATC CTACCAGCTA CCACAGCAAA CGCCTAAGAG CAGGAAAAAT 1653 

GTCTGCCAGG AAAGTGTCCA GTGTCCACAT GGCCCCTGGC GCTCTGACTC TTTGAGGAGT 1713 

AATCGCAAGC CTCGTTCAGC TGCAGCAGAA GGAAGGGCTT AGCCAGGGTG CGCGCTGGCG 1773 

TCTGTGTTGA AGGGAAACCA AGCAGAAGCC ACTGTAATGA TAT^TCACAA TAAAACCCAT 1833 

GAATGAAAAA AAAAAAAAAA AAAAAAAAAA AAAAGAATTC 1873 

(2) INFORMATION FOR SEQ ID NO: 19: 

• (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 430 amino acids 

(B) TYPE: amino acid 
(D) TOPOUKJY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(D) OTHER INFORMATION: /product^ "mOPl-PP" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: 

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

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 He His Arg Arg Leu Arg Ser 
35 40 45 



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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 
65 70 75 80 

Het Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Ser Gly 
85 90 95 

Pro Asp Gly Gin Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr 
100 105 110 

Gin Gly Pro Pro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp 
115 120 125 

Ala Asp Het Val Het Ser Phe Val Asn Leu Val Glu His Asp Lys Glu 
130 135 140 

Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser 
145 150 155 160 

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

Lys Asp Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Gin He Thr 
180 185 190 

Val lyr Gin Trp Leu Gin Glu His Ser Gly Arg Glu Ser Asp Leu Phe 
195 200 205 

Leu Leu Asp Ser Arg Thr He Trp Ala Ser Glu Glu Gly Trp Leu Val 
210 215 220 

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 Thr Leu Asp Gly Gin Ser He 
245 250 255 

Asn Pro Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys 
260 265 270 

Gin Pro Phe Het Val Ala Phe Phe Lys Ala Thr <>lu Val His Leu Arg 
275 280 285 

Ser He Arg Ser Thr Gly Gly Lys Gin Arg Ser Gin Asn Arg Ser Lys 
290 295 300 

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 Cln Ala Cys Lys Lys His Glu Leu Tyr Val 
325 330 335 



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Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Fro Glu Gly 
340 3^5 330 

Tyr Ala Ala lyx Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser 
355 360 365 

Tyr Het Asn Ala Thr Asn His Ala He Val Gin Thr Leu Val His Phe 
^ 370 375 380 

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 He Leu 
405 410 415 

Lys Lys Tyr Arg Asn Het Val Val Arg Ala Cys Gly Cys His 
^ 420 A25 430 

(2) INFOEHATION FOR SEQ ID NQ:20: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1723 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) HOLECULE TYPE: cDNA 

(vi) ORIGINAL SOURCE: 

(A) ORGANISH: Homo sapiens 
(F) TISSUE TYPE: HIPPOCAHPUS 

(ix)FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 490.. 1696 

(D) OTHER INFORHATION: /note* ''hOP2 (cDNA) 



If 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: 

GGCGCGGGCA GAGCAGGAGT GGCTGGAGGA GCTGTGGTTG GAGCAGGAGG TGGCACGGCA 60 

GGGCTGGAGG GCTCCCTATG AGTGGCGGAG ACGGCCCAGG AGQCGCTGGA GCAACAGCTC 120 

CCACACCGCA CCAAGCGGTG GCTGCAGGAG CTCGCCCATC GCCCCTGCGC TGCTCGGACC 180 

GCGGCCACAG CCGGACTGGC GGGTACGGCG GCGACAGAGG CATTGGCCGA GAGTCCCAGT 240 

CCGCAGAGTA GCCCCGGCCT CGAGGCGGTG GCGTCCCGGT CCTCTCCGTC CAGGAGCCAG 300 

GACAGGTGTC GCGCGGCGGG GCTCCAGGGA CCOCGCCTGA GGCCGGCTGC CCGCCCGTCC 360 

CGCCCCGCCC CGCCGCCCGC CGCCCGCCGA GCCCAGCCTC CTTGCCGTCG CGGCGTCCCC 420 



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AGGCCCTGGG TCGGCCGCGG AGCCGATGCG CGCCCGCTGA GCGCCCCAGC TGAGCGCCCC 480 

CGGCCTGCC ATG ACC GCG CTC CCC GGC CCG CTC TGG CTC CTG GGC CTG 528 
het Thr Ala Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu 
1 5 10 

GCG CTA TGC GCG CTG GGC GGG GGC 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 

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 

CGC GAG ATC CTG GCG GTG CTC GGG CTG CCT GGG CGG CCC CGG CCC CGC 672 
Arg Glu lie 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 GCG CCG CTC TTC ATG 720 
Ala Pro Pro Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro Leu Phe Het 
65 70 75 

CTG GAC CTG TAC CAC GCC ATG GCC GGC GAC GAC GAC GAG GAC GGC GCG 768 
Leu Asp Leu Tyr His Ala Met Ala Gly Asp Asp Asp Glu Asp Gly Ala 
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 

AAC ATG GTG GAG CGA GAC C€T GCC CTG GGC CAC CAG GAG CCC CAT TGG 864 
Asn Het Val Glu Arg Asp Arg Ala Leu Gly His Gin Glu Pro His Trp 
110 115 120 125 

AAG GAG TTC CGC TTT GAC CTG ACC CAG ATC CCG GCT GGG KSAG GCG GTC 912 
Lys Glu Phe Arg Phe Asp Leu Thr Gin He Pro Ala Gly Glu Ala Val 
130 135 ^ 140 

ACA GCT GCG GAG TTC CGG ATT TAC AAG GTG CCC AGC ATC CAC CTG CTC 960 
Thr Ala Ala Glu Phe Arg He lyx Lys Val Pro Ser He His Leu Leu 
145 150 155 

AAC AGG ACC CTC CAC GTC AGC ATG TTC CAG GTG GTC CAG. CAG CAG JCC 1008 
Asn Arg Thr Leu His Val Ser Het Phe Gin Val Val Gin Glu Gin Ser 
160 165 170 

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 CAT GTC ACA GCA GCC AGT GAC TGC 
Gly Asp Glu Gly Trp Leu Val Leu Asp Val Thr Ala Ala Ser Asp Cys 
190 195 200 • 205 



1104 



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TGG TTG CTG AAG CGT CAC AAG GAC CTG GGA CTC CGC CTC TAT GTG GAG 1152 
Trp Leu leu Lys Arg His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu 
210 215 220 

ACT GAG GAC GGG CAC AGC GTG GAT CCT GGC CTG GCC GGC CTG CTG GGT 1200 
Thr Glu Asp Gly His Ser Val Asp Pro Gly Leu Ala Gly Leu Leu Gly 
225 230 235 

CAA CGG GCC CCA CGC TCC CAA CAG CCT TTC GTG GTC ACT TTC TTC AGG 1248 
Gin Arg Ala Pro Arg Ser Gin Gin Pro Phe Val Val Ihr Phe Phe Arg 
240 245 250 

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 
Are Arg Gin Pro Lys Lys Ser Asn Glu Leu Pro Gin Ala Asn Arg Leu 
270 275 280 285 

CCA GGG ATC TTT GAT GAC GTC CAC GGC TCC CAC GGC CGG CAG GTC TGC 1392 
Pro Gly He Phe Asp Asp Val Bis Gly Ser His Gly Arg Gin Val Cys 
290 295 300 

CGT CGG CAC GAG CTC TAC GTC AGC TTC CAG GAC CTC GGC TGG CTG GAC 1440 
Arg Arg His Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Leu Asp 
305 310 315 

TGG GTC ATC GCT CCC CAA GGC TAC TCG GCC TAT TAC TGT GAG GGG GAG 1488 
Trp Val He Ala Pro Gin Gly Tyt Ser Ala lyr lyr Cys Glu Gly Glu 
320 325 330 

TGC 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 

CTG CAG TCC CTG GTG CAC CTG ATG AAG CCA AAC GCA GTC CCC AAG GCG 1584 
Leu Gin Ser Leu Val His Leu Het Lys Pro Asn Ala Val Pro Lys Ala 
350 355 360 365 

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 Tyr Tyr Asp 
370 375 380 

AGC AGC AAC AAC GTC ATC CTG CGC AAA CAC CGC AAC ATG GTG GTC AAG 1680 
Ser Ser Asn Asn Val He Leu Arg Lys His Arg Asn Met Val Val Lys 
385 390 395 

GCC TGC GGC TGC CAC T GAGTCAGCCC GCCCAGCCCT ACTGCAG 1723 
Ala Cys Gly Cys His 
400 



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(2) INFOSUATION FOR SEQ ID N0:21: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 402 amino acids 

(B) TYPE: amino acid 
(0) TOPOLOGY: linear 

(ii) IiOLECULE TYPE: protein 

(ix)FEATURE: 

(A)OTHER INFORMATION: /product- '•hOP2-PP" 

(xi)SEQUENCE DESCRIPTION: SEQ ID N0:21: 

Het Thr Ala Leu Pro Gly Fro Leu Trp Leu Leu Gly Leu Ala Leu Cys 
15 10 15 

Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pro Pro Pro Gly Cys Pro 
20 25 30 

Gin Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Val Gin Arg Glu lie 
35 40 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 Pbe Met Leu Asp Leu 
65 70 75 80 

Tyr His Ala Met Ala Gly Asp Asp Asp Glu Asp Gly Ala Pro Ala Glu 
85 90 95 

Arg Arg Leu Gly Arg Ala Asp Leu Val Met Ser Phe Val Asn Met Val 
100 105 110 

Glu Arg Asp Arg Ala Leu Gly His Gin Glu Pro His Trp Lys Glu Phe 
115 120 125 

Arg Phe Asp Leu Thr Gin He Pro Ala Gly Glu Ala Val Thr Ala Ala 
130 135 140 

Glu Phe Arg He Tyr Lys Val Pro Ser He His Leu Leu Asn Are Thr 
145 150 155 160 

Leu His Val Ser Met 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 



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Lys Are His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Glu Asp 
' 210 215 220 

Gly His Ser Val Asp Pro Gly Leu Ala Gly Leu Leu Gly Gin Arg Ala 
225 230 235 2A0 

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 Gly Arg Gin Val Cys Arg Arg His 
290 295 300 

Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Leu Asp Trp Val He 
305 310 315 320 

Ala Pro Gin Gly IVr 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 Met Lys Pro Asn Ala Val Pro Lys Ala Cys Cys Ala 
355 360 365 

Pro Thr Lys Leu Ser Ala Thr Ser Val Leu lyr Tyr Asp Ser Ser Asn 
370 375 380 

Asn Val He Leu Arg Lys His Arg Asn Het Val Val Lys Ala Cys Gly 
385 390 395 400 

Cys His 

(2) ZNFOBHATION FOR SEQ ID NO: 22: 

(i) SEQUENCE CHARACTERISTICS; 

(A) LENGTH: 1926 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 
(D TOPOLOGT: linear 

(ii) MOLECULE TYPE: cONA 

(vi) ORIGINAL SOURCE: 

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

(ix) FEATURE: 



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(A) NAHE/KEY: COS 

(B) LOCATION: 93.. 1289 

(D) OTHER INFORMATION: /note» "nOP2 cDNA" 
(xi) SEQUENCE OESCRIFTION: SEQ ID NO: 22: 

GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC 50 

CCGACCAGCT ACCAGTGGAT GCGCGCCGGC TGAAAGTCCG AG ATG GCT AT6 CGT 104 

Met Ala Met Arg 
1 

CCC GGG CCA CTC TGG CTA TTG GGC CTT GCT CTG TGC GCG CT6 GGA GGC 152 
Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys Ala Leu Gly Gly 
5 10 15 20 

GGC CAC GGT CCG CGT CCC CCG CAC ACC TGT CCC CAG CGT CGC CTG GGA 200 
Gly His Gly Pro Arg Pro Pro His Thr Cys Pro Gin Arg Arg Leu Gly 
25 30 35 

GCG CGC GAG CGC CGC GAC ATG CAG CGT GAA ATC CTG GCG 6TG CTC GGG 248 
Ala Arg Glu Arg Arg Asp Met Gin Arg Glu lie Leu Pro Val Leu Gly 
40 45 50 

CTA CCG GGA CGG CCC CGA CCC CGT GCA CAA CCC GCG GCT GCC CGG CAG 296 
Leu Pro Gly Arg Fro Arg Pro Arg Ala Gin Pro Ala Ala Ala Arg Gin 
55 60 65 

CCA GCG TCC GCG CCC CTC TTC ATG TTG GAC CTA TAC CAC GCC ATG ACC 344 
Pro Ala Ser Ala Pro Leu Phe Met Leu Asp Leu Tyr His Ala Met Thr 
70 75 80 

GAT GAC GAC GAC GGC GGG CCA CCA CAG GCT CAC TTA CGC CGT HCC GAC 392 
Asp Asp Asp Asp Gly Gly Pro Pro Gin Ala His Leu Cly Arg Ala Asp 
85 90 95 100 

CTG GTC ATG AGC TTC GTC AAC ATG GTG GAA CGC GAC CGT ACC CTG GGC 440 
Leu Val Met Ser Phe Val Asn Met Val Glu Arg Asp Arg Thr Leu Gly 
105 110 115 

TAC CAG GAG CCA CAC TGG AAG GAA TTC CAC TTT GAC CTA ACC CAG ATC 488 
Tyr Gin Glu Pro His Trp Lys Glu Phe His Phe Asp Leu Thr Gin He 
120 125 130 

CCT GCT GGG GAG GCT GTC ACA GCT GCT GAG TTC CGG ATC TAC AAA GAA 536 
Pro Ala Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He Tyr Lys Glu 
135 140 145 

CCC AGC ACC CAC CCG CTC AAC ACA ACC CTC CAC ATC AGC ATG TTC GAA 584 
Pro Ser Thr His Pro Leu Asn Thr Thr Leu His He Ser Met Phe Glu 
150 155 160 



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GTG GTC CAA GAG CAC TCC AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT 632 
Val Val Gin Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp 
165 170 175 180 

CTT CAG ACG CTC CGA TCT GGG GAC GAG GGC TGG CTG GTG CTG GAC ATC 680 
Leu Gin Thr Leu Arg Ser Gly Asp Glu Gly Trp Leu Val Leu Asp lie 
185 190 195 

ACA GCA GCC AGT GAC CGA TGG CTG CTG AAC CAT CAC AAG GAC CTG GGA 728 
Thr Ala Ala Ser Asp Arg Trp Leu Uu Asn His His Lys Asp Leu Gly 
200 205 210 

CTC CGC CTC TAT GTG GAA ACC GCG GAT GGG CAC AGC ATG GAT CCT GGC 776 
Leu Arg Leu Tyr Val Glu Thr Ala Asp Gly His Ser Met Asp Pro Gly 
215 220 225 

CTG GCT GGT CTG CTT GGA CGA CAA GCA CCA CGC TCC AGA CAG CCT TTC 824 
Leu Ala Gly Leu Leu Gly Arg Gin Ala Pro Arg Ser Arg Gin Pro Phe 
230 235 240 

ATG GTA ACC TTC TTC AGG GCC AGC CAG AGT CCT GTG CGG *GCC CCT CGG 872 
Met Val Thr Phe Phe Arg Ala Ser Gin Ser Pro Val Arg Ala Pro Arg 
245 250 255 260 

GCA GCG AGA CCA CTG AAG AGG AX^G CAG CCA AAG AAA ACG AAC GAG CTT 920 
Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn Glu Leu 
265 270 275 

• 

CCG CAC CGC AAC AAA CTC CCA GGG ATC TTT GAT GAT GGC CAC GGT TCC 968 
Pro His Pro Asn Lys Leu Pro Gly lie Phe Asp Asp Gly His Gly Ser 
280 285 290 

CGC GGC AGA GAG GTT TGC CGC AGG CAT GAG CTC TAC GTC AGC TTC CCT 1016 
Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Arg 
295 300 305 

GAC CTT GGC TGG CTG GAC TGG GTC ATC GCC CCC CAG GGC TAC TCT GCC 1064 
Asp Leu Gly Trp Leu Asp Trp Val He Ala Pro Gin Gly Tyr Ser Ala 
310 315 320 

TAT TAC TGT GAG GGG GAG TGT GCT TTC CCA CTG GAC TCC TGT ATG AAC 1112 
Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys Met Asn 
325 330 335 340 

GCC ACC AAC CAT GCC ATC TTG CAG TCT CTG GTG CAC CTG ATG AAG CCA 1160 
Ala Thr Asn His Ala He Leu Gin Ser Leu Val His Leu Met Lys Pro 
345 350 355 

GAT CTT GTC CCC AAG GCA TGC TGT GCA CCC ACC AAA CTG AGT GCC ACC 1208 
Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr 
360 365 370 



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TCT GTG CTG lAC TAT GAC AGC AGC AAC AAT GTC ATC CTG CGT AAA CAC 1256 
Ser Val Leu lyr Tyr Asp Ser Ser Asn Asn Val lie Leu Arg Lys His 
375 380 385 

CGT AAC ATG GTG GTC AAG GCC TGT GGC TGC CAC TGAGGCCCCG CCCAGCATCC 1309 
Arg Asn Met Val Val Lys Ala Cys Gly Cys His 
390 395 

TGCTTCTACT ACCTTACCAT CTGGCCGGGC CCCTCTCCAG AGGCAGAAAC CCTTCTATGT 1369 

TATCATAGCT CAGACAGGGG CAATGGGAGG CCCTTCACTT CCCCTGGCCA CTTCCTGCTA 1429 

AAATTCTGGT CTTTCCCAGT TCCTCTGTCC TTCATGGGGT TTCGGGGCIA TCACCCCGCC 1489 

CTCTCCATCC TCCTACCCCA AGCATAGACT GAATGCACAC AGCATCCCAG AGCTATGCTA 1549 

ACTGAGAGGT CTGGGGTCAG CACTGAAGGC CCACATGAGG AAGACTGATC CTTGGCCATC 1609 

CTCAGCCCAC AATGGCAAAT TCTGGATGGT CTAAGAAGGC CGTGGAATTC TAAACTAGAT 1669 

GATCTGGGCT CTCTGCACCA TTCATTGTGG CAGTTGGGAC ATTTTTAGGT ATAACAGACA 1729 

CATACACTTA GATCAATGCA TCGCTGTACT CCTTGAAATC AGAGCTAGCT T6TTAGAAAA 1789 

AGAATCAGAG CCAGGTATAG CGGTGCATGT CATTAATCCC AGCGCTAAAG AGACAGAGAC 1849 

AGGAGAATCT CTGTGAGTTC AAGGCCACAT AGAAAGAGCC TGTCTCGGGA GCAGGAAAAA 1909 

AAAAAAAAAC GGAATTC 1926 

(2) INFORHATION FOR SEQ ID NO: 23: 

(i) SEQUENCE CHAKACTERISTICS: 

(A) LENGTH: 399 amino acids 

(B) TYPE: amino acid 
(D) TOFOLOGT: linear 

(ii) HOLECULE TYPE: protein 

(ix) FEATORE: 

(0) OTHER INFORMATION: /product- "mOF2-PP" 

(zi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: 

Net Ala Met Arg Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys 
15 10 15 

Ala Leu Gly Gly Gly His Gly Pro Arg Pro Pro His Thr Cys Pro Gin 
20 25 30 

Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Met Gin Arg Glu He Leu Ala 
35 40 45 



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Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg Ala Gin Pro Ala Ala 
50 55 60 65 

Ala Are Gin Pro Ala Ser Ala Pro Leu Phe het Leu Asp Leu Tyr His Ala 
70 75 80 

Het Thr Asp Asp Asp Asp Gly Gly Pro Pro Gin Ala His Leu Gly Arg 
85 90 95 

Ala Asp Leu Val Met Ser Phe Val Asn Met Val Glu Arg Asp Arg Thr 
100 105 110 

Leu Gly Tyr Gin Glu Pro His Trp Lys Glu Phe His Phe Asp Leu Thr 
115 120 125 130 

Gin lie Pro Ala Gly Glu Ala Val "Oa Ala Ala Glu Phe Arg He Tyr 
135 lAO 145 

Lys Glu Pro Ser Thr His Pro Leu Asn Thr Thr Leu His He Ser Met 
150 155 160 

Phe Glu Val Val Gin Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe 
165 170 175 

Leu Asp Leu Gin Thr Leu Arg Ser Gly Asp Glu Gly Trp Leu Val Leu 
180 185 190 

Asp lie Thr Ala Ala Ser Asp Arg Trp Leu Leu Asn His His Lys Asp 
195 200 205 210 

Leu Gly Leu Arg Leu lyr Val Glu Thr Ala Asp Gly His Ser Met Asp 
215 220 225 

Pro Gly Leu Ala Gly Leu Leu Gly Arg Gin Ala Pro Arg Ser Arg Gin 
230 235 240 

Pro Phe Met Val Thr Phe Phe Arg Ala Ser Gin Ser Pro Val Arg Ala 
245 250 255 

Pro Arg Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn 
260 265 270 

Glu Leu Pro His Pro Asn Lys Leu Pro Gly He Phe Asp Asp Gly His 
275 280 285 290 

Gly Ser Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr Val Ser 
295 300 305 

Phe Arg Asp Leu Gly Trp Leu Asp Trp Val He Ala Pro Gin Gly Tyr 
310 315 320 



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Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys 
325 330 335 

Het Asn Ala Thr Asn His Ala lie Leu Gin Ser Leu Val His Leu Met 
340 345 350 

Lys Fro Asp Val Val Pro Lys Ala Cys Cys Ala Fro Thr Lys Leu Ser 
355 360 365 370 

Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val He Leu Arg 
375 380 385 

Lys His Arg Asn Het Val Val Lys Ala Cys Gly Cys His 
390 395 



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What is claimed is: 

1. A composition for increasing the 

progenitor cell population in a mammal comprising: 

progenitor cells, stimulated ex vivo by 
exposure to a morphogen at a concentration and for a 
time sufficient such that said progenitor cells are 
stimulated to proliferate. 

2^ A composition for inducing non- 

chondrogenic tissue growth in a mammal comprising: 

progenitor cells, stimulated by 
exposure to a morphogen at a concentration and for a 
time sufficient such that said progenitor cells, when 
disposed in vivo within a tissue locus, are capable 
of non-chondrogenic tissue-specific differentiation 
and proliferation within said locus. 

3^ The composition of claim 1 or 2 wherein 

20 said progenitor cells are hemopoietic pluripotential 
stem cells. 

4, The composition of claim 1 or 2 wherein 
said progenitor cells are of mesenchymal origin. 

25 

5. A composition for inducing the 
formation of non-chondrogenic replacement tissue at a 
tissue locus in a mammal comprising: 

a biocompatible, acellular matrix 
30 having components specific for said tissue and 

capable of providing a morphogenically permissive, 
tissue-specific environment; and 

a morphogen such that said morphogen, 
when absorbed on said matrix and provided to a 



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tissue-specific locus requiring replacement tissue, 
is capable of inducing the developmental cascade of 
tissue morphogenesis at said locus. 

5 6. A composition for inducing the 

formation of non-chondrogenic replacement tissue at a 
tissue locus in a mammal comprising: 

a biocompatible, acellular matrix 
capable of providing a morphogenically permissive 
10 environment; and 

a morphogen such that said morphogen, 
when absorbed on said matrix and provided to a 
tissue-specific locus requiring replacement tissue, 
is capable of inducing the developmental cascade of 
15 tissue morphogenesis at said locus. 

7. The composition of claim 5 or 6 wherein 

said matrix is biodegradable. 

20 8. The composition of claim 5 or 6 wherein 

said matrix is derived from organ-specific tissue. 

9. The composition of claim 5 or 6 wherein 
said matrix comprises collagen and cell attachment 

25 factors selected from the group consisting of 
glycosaminoglycans and proteoglycans. 

10. The composition of claim 5 or 6 wherein 
said matrix defines pores of a dimension sufficient 

30 to permit the influx, differentiation and 

proliferation of migratory progenitor cells from the 
body of said mammal. 



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11. The composition of claim 1, 2, 5 or 6 
wherein said morphogen comprises an amino acid 
sequence sharing at least 70% homology with one of 
the sequences selected from the group consisting of: 

5 hOPl (Seq. ID No. 5); mOPl (Seq. ID No. 6); hOP2 
(Seq. ID No. 7); mOP2 (Seq. ID No. 8); CBMP2A(fx) 
(Seq. ID No. 9); CBMP2B(fx) (Seq. ID No. 10); DPP(fx) 
(Seq. ID No. 11); Vgl(fx) (Seq. ID No. 12); Vgr-l(fx) 
(Seq. ID No. 13); and GDF-l(fx) (Seq. ID No. 14). 

10 

12. The composition of claim 11 wherein 
said morphogen comprises an amino acid sequence 
sharing at least 80% homology with one of the 
sequences selected from said group. 

15 

13. The composition of claim 12 wherein said 
morphogen conprises an amino acid sequence having 
greater than 60% amino acid identity with the 
sequence defined by residues 43-139 of Seq. ID No. 5 

20 (hOPl). 

14. The composition of claim 13 wherein said 
morphogen comprises an amino acid sequence having 
greater than 65% identity with the sequence defined 

25 by residues 43-139 of Seq. ID No. 5 (hOPl). 

15. A method of increasing a population of 
progenitor cells comprising the step of: 

contacting progenitor cells with a 
30 morphogen at a concentration and for a time 

sufficient such that said progenitor cells are 
stimulated to proliferate. 



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16. The method of claim 15 for increasing 
progenitor cells in a mammal comprising the 
additional step of supplying said stimulated 
progenitor cells to a mammal to increase the 

5 progenitor cell population in said mammal. 

17. A method of inducing non-chondrogenic 
tissue growth in a mammal comprising the step of: 

contacting progenitor cells with a 
10 morphogen at a concentration and for a time 

sufficient such that said progenitor cells / when 
provided to a tissue-specific locus in a mammal, are 
capable of nonchondrogenic tissue-specific 
differentiation and proliferation at said locus. 

15 

18. The method of claim 14 or 16 wherein 
said progenitor cells are of mesenchymal origin. 

19. A method of maintaining the phenotypic 
20 expression of differentiated cells in a mammal 

comprising the steps of: 

contacting said differentiated cells 
with a morphogen at a concentration and for a time 
sufficient such that said cells are stimulated to 
25 express their phenotype. 

20. The method of claim 19 wherein said 
differentiated cells are senescent or quiescent 
cells. 

30 

21. A method of inducing non-chondrogenic 
tissue growth at a tissue locus in a mammal 
comprising: 



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providing said locus with a morphogen 
at a concentration and for a time sufficient such 
that said protein, when provided to a inorphogenically 
permissive tissue-specific locus, is capable of 
5 inducing the developmental cascade of tissue 
morphogenesis at said locus. 

22. The method of claim 21 wherein said 
nonchondrogenic tissue is hepatic tissue, and said 

10 tissue locus is the liver. 

23. The method of claim 22 wherein said 
protein is provided to said locus in association with 
a biocompatible, acellular matrix. 

15 

24. The method of claim 23 wherein said 
matrix has components specific for said tissue. 

25. The method of claim 23 wherein said 
20 matrix is biodegradable. 

26. The method of claim 23 wherein said 
matrix is derived from organ-specific tissue. 

25 27. The method of claim 23 wherein said 

matrix comprises collagen and cell attachment factors 
specific for said tissue. 

28. The method of claim 23 wherein said 

30 matrix defines pores of a dimension sufficient to 
permit the influx, differentiation and proliferation 
of migratory progenitor cells from the body of said 
mammal . 



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29. The method of claim 14, 16, 17 or 20 
where said mo'rphogen comprises an amino acid sequence 
sharing at least 70% homology with one of the 
sequences selected from the group consisting of hOPl 

5 (Seq. ID No, 5); mOPl (Seq. ID No. 6); hOP2 (Seq. ID 
No. 7); mOP2 (Seq. ID No. 8); CBMP2A(fx) (Seq. ID 
No. 9); CBMP2B{fx) (Seq. ID No. 10); DPP(fx) (Seq. ID 
No. 11); Vgl(fx) (Seq. ID No. 12); Vgr-l(fx) (Seq. ID 
No. 13); and GDF-l(fx) (Seq. ID No. 14). 

10 

30. A method for inducing hepatic tissue 
formation at a damaged tissue locus in a mammalian 
liver comprising providing to said locus a 
therapeutic amount of a morphogen comprising at least 

15 residues 43-139 of hOP-1 (Seq. ID No. 5). 

31. A method for diagnosing tissue 
dysfunction in a human, the method comprising the 
steps of : 

20 (a) repeating, at intervals, the step of 

detecting the concentration of endogenous anti- 
morphogen antibody present in a human; and 

(b) comparing said detected concentrations, 
wherein changes in the detected concentrations are 

25 indicative of status of said tissue. 

32. A method for evaluating the status of a 
tissue, the method comprising the step of detecting 
the concentration of a morphogen present in said 

30 tissue. 

33. The method of claim 32 <:omprising the 
additional steps of: 

(a) repeating, at intervals, the step of 
35 detecting the concentration of 

morphogen present in said tissue; and 



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(b) comparing said detected concentrations/ 
wherein changes in said detected 
concentrations are indicative of the 
status of said tissue. 

5 

34. The method of claim 33 wherein said 
morphogen is selected from the group consisting of: 
hOPl (Seq. ID No. 5); mOPl (Seq. ID No. 6); hOP2 
(Seq. ID No. 7); mOP2 (Seq. ID No. 8); CBMP2A(fx) 

10 (Seq. ID No. 9); CBMP2B(fx) (Seq. ID No. 10); DPP(fx) 
(Seq. ID No. 11); Vgl(fx) (Seq. ID No. 12); Vgr-l(fx) 
(Seq. ID No. 13); and GDF-l(fx) (Seq. ID No. 14). 

35. A morphogen useful in the manufacture 
15 of a pharmaceutical for use in the induction of non- 

chondrogenic mammalian tissue growth. 

36. A morphogen useful in the manufacture 
of a pharmaceutical for use as an inducer of 

20 progenitor cell proliferation. 

37. A morphogen useful in the manufacture 
of a pharmaceutical for use in maintaining the 
phenotypic expression of differentiated cells in a 

25 mammal. 

38. A morphogen useful in the manufacture 
of a pharmaceutical for use in the induction of 
hepatic tissue growth. 

30 

39. The morphogen of claims 35, 36, 21 , or 
38 wherein said morphogen comprises an amino acid 
sequence sharing at least 70% homology with a 
sequence selected from the group consisting of: hOPl 

35 (Seq. ID No. 5); mOPl (Seq. ID No. 6); hOP2 (Seq. ID 



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PCr/US92/01968 



No. 7); mOP2 (Seq. ID No. 8); CBMP2A(fx) (Seq. ID 
NO. 9); CBMP2B(fx) (Seq. ID No. 10); DPP(fx) (Seq. ID 
No. 11); Vgl(fx) (Seq. ID No. 12); Vgr-l(fx) (Seq. ID 
No. 13); and GDF-l(fx) (Seq. ID No. 14). 

5 

40. The morphogen of Claim 39 wherein said 
morphogen comprises an amino acid sequence sharing at 
least 80% homology with one of the sequences selected 
from said group. 

10 

41. A morphogen useful in the manufacture of a 
pharmaceutical to inhibit neoplastic cell growth. 

42. A cancer therapeutic agent comprising a 
15 morphogen. 

43. A therapeutic agent for tissue growth 
induction, the therapeutic agent comprising a 
morphogen. 

20 

44. A therapeutic agent for inducing phenotypic 
expression of differentiated cells, the therapeutic 
agent comprising a morphogen. 

25 45. A therapeutic agent for inducing progenitor 

cell proliferation, the therapeutic agent comprising 
a morphogen. 



30 



wo 92/15323 



1/11 



PC1/US92/01968 



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SUBSTITUTE SHEET 



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2.37- 
1.35- 



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SUBSTITUTE SHEET 



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Fig. 4A 




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SUBSTITUTE SHEET 



wo 92/15323 



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Fig. 5 A 




Fig. 5B 



SUBSTITUTE SHEET 




Fig. 6 A 




Fig. 6B 

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Fig. 6D 

SUBSTITUTE SHEET 




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SUBSTITUTE SHEET 



INTERNATIONAL SEARCH REPORT 

International Application No. PCT/0592/01966 



I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbo ls apply. Indieate all)^ 
Accofdino to International Patent Classification (IPCI or to both National Classification and IPC 
IPC (S): A61K 37/12; A61P 2/02; C07K 13/00 

US CL : 350/356, 402; 424/423, 426; 435/240.243 



II. FIELDS SEARCHED 



Minimum Documentation Searched^ 



Classification System 



Classification Symbols 



350/356, 402; 424/423, 426; 435/240.243 



Documentation Searched other than Minimum Documentation ^ 
to the extent that such Documents are included in the Fields Searched 



CHEMICAL ABSTRACTS, APS 



III. DOCUMENTS CONSIDERED TO BE RELEVANT'^ 



Category* 



Citation of Document'^ with indication, where appropriate, of the relevant passages^ 



Rdavant to Claim No. 



x/y 



x/y 



wo. A, 89/09788 (OPPERMANN ET AL.) 19 OCTOBER 1989, see 
entire document. 



WO, A, 89/09787 ( KUBERASAMPATH ET AL.) 19 OCTOBER 1989, 
see entire document. 



1/5-45 



1/5-45 



* Special cateoohes of cited documents: i& 

•A" document defiling tha general state of the art which is 

not considered to be of particular relevance 
T eariiar document but published on or after the 

international filing data 
"L" document which may throw doubts on priority daim(s) 

or wNch is cited to establish the publication date of 

anotfwr citation or other special reason (as specified) 
•O" document referring to an oral disclosure, use, exhibition 

or otfier means 

"P* document published prior to the imemational filing date 
but later than the priority date claimed 



later document published after the international filing 
date or priority date and not in conflict with the 
application but cited to understend the pnnctple or 
theory underiying the invention 
document of particular relevance; the claimed 
invention cannot be considered novel or cannot be 
considered to involve an inventive step 
document of particular relevance: the claimed 
invention cannot be considered to involve en 
inventive step when the document is combined with 
one or more other such documents, such combination 
being ob>riou8 to a parson skilled in tha art 
document member of the same patent family 



IV. CERTIFICATION 



Date of the Actual Completion of the international Search^ 

12 June 1992 



Oate of Mailing of this International Search Report^ 



international Searching Authority ^ 

ISA/US 



Signature of Authorized' Olffepr ^^// // 



JAMES KETTER 



Form PCT/iSA/210 {second sheetXMey 1986) & 



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