Skip to main content

Full text of "USPTO Patents Application 09804625"

See other formats


per 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 5 
A61K 37/02, G01N 33/68 



Al 



(11) International Publication Number: WO 94/03200 

(43) International Publication Date: 17 February 1994 (17.02.94) 



(21) International Application Number: PCT/US93/07231 

(22) International Filing Date: 29 July 1993 (29.07.93) 



(30) Priority data: 
922,813 
029,335 
040,510 



31 July 1992(31.07.92) US 
4 March 1993(04.03.93) US 
31 March 1993(31.03.93) US 



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

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

(72) Inventors: RUEGER, David, C ; 19 Downey Street, Hop- 

kinton, MA 01748 (US). KUBERASAMPATH, Than- 
gavel ; Six Spring Street, Medway, MA 02053 (US). OP- 
PERMANN, Hermann ; 25 Summer Hill Road, Med- 
way, MA 02053 (US). OZKAYNAK, Engin ; 44 Purdue 
Drive, Milford, MA 01757 (US). PANG, Roy, H., L. ; 15 
Partridge Road, Etna, NH 03750 (US). COHEN, 
Charles, M. ; 98 Winthrop Street, Medway, MA 02053 
(US). 



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



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



Published 

With international search report. 

Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 
amendments. 



(54) Title: MORPHOGEN-INDUCED NERVE REGENERATION AND REPAIR 



(57) Abstract 



Disclosed are therapeutic treatment methods, compositions and devices for maintaining neural pathways in a mammal, in- 
cluding enhancing survival of neurons at risk of dying, inducing cellular repair of damaged neurons and neural pathways, and 
stimulating neurons to maintain their differentiated phenotype. In one embodiment, the invention provides means for stimulating 
CAM expression in neurons. The invention also provides means for evaluating the status of nerve tissue, including means for de- 
tecting and monitoring neuropathies in a mammal. The methods, devices and compositions include a morphogen-stimulating 
agent provided to the mammal in a therapeutically effective concentration. 



FOR THE PURPOSES OF INFORMATION ONLY 



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



AT 


Austria 


PR 


France 


MR 


Mauritania 


All 


Australia 


CA 


Gabon 


MW 


Malawi 


BB 


Barbados 


CB 


United Kingdom 


NE 


Niger 


BE 


Belgium 


CN 


Guinea 


NL 


Netherlands 


BF 


Burkina Faso 


GR 


Greece 


NO 


Norway 


BC 


Bulgaria 


HU 


Hungary 


NZ 


New Zealand 


BJ 


Benin 


IE 


Ireland 


PL 


Poland 


BR 


Brazil 


IT 


Italy 


PT 


Portugal 


BY 


Belarus 


JP 


Japan 


RO 


Romania 


CA 


Canada 


KP 


Democratic People's Republic 


RU 


Russian Federation 


CP 


Central African Republic 




of Korea 


5D 


Sudan 


CG 


Congo 


KR 


Republic of Korea 


SE 


Sweden 


CH 


Switzerland 


KZ 


Kazakhstan 


SI 


Slovenia 


a 


Cole d'l voire 


LI 


Liechtenstein 


SK 


Slovak Republic 


CM 


Cameroon 


LK 


Sri Lanka 


SN 


Senegal 


CN 


China 


LU 


Luxembourg 


TD 


Chad 


cs 


Czechoslovakia 


LV 


Latvia 


TC 


Togo 
Ukraine 


C2 


Gech Republic 


MC 


Monaco 


UA 


DE 


Germany 


MG 


Madagascar 


US 


United States of America 


DK 


Denmark 


ML 


Mali 


UZ 


Uzbekistan 


BS 


Spain 


MN 


Mongolia 


VN 


Viet Nam 


PI 


Finland 











WO 94/03200 



PCT/US93/07231 



Morphogen- Induced Nerve Regeneration and Repair 

5 

BACKGROUND OF THE INVENTION 

The present invention relates to methods for 
enhancing the survival of neuronal cells in vivo and to 

10 methods, compositions and devices for maintaining 
neural pathways in vivo * More particularly, the 
invention provides methods for enhancing survival of 
neuronal cells at risk of dying, including methods for 
redif ferentiating transformed cells of neural origin 

15 and methods for maintaining phenotypic expression of 
differentiated neuronal cells. The invention also 
provides means for repairing damaged neural pathways, 
including methods for stimulating axonal growth over 
extended distances, and methods for alleviating 

20 immunologically-related nerve tissue damage. In a 

particular embodiment of the invention, this invention 
provides a method for stimulating cell adhesion 
molecule expression in cells, and particularly nerve 
cell adhesion molecule expression in neurons. Finally, 

25 the invention provides means for evaluating nerve 

tissue stasis and identifying neural dysfunction in a 
mammal • 

The mammalian nervous system comprises a peripheral 
30 nervous system (PNS) and a central nervous system (CNS, 
comprising the brain and spinal cord), and is composed 
of two principal classes of ceils: neurons and glial 
cells. The glial cells fill the spaces between 
neurons, nourishing them and modulating their function. 
35 Certain glial cells, such as Schwann cells in the PNS 



WO 94/03200 



PCI7US93/07231 



and oligodendrocytes in the CNS, also provide a 
protective myelin sheath that surrounds and protects 
neuronal axons, which are the processes that extend 
from the neuron cell body and through which the 
5 electric impulses of the neuron are transported. In 
the peripheral nervous system, the long axons of 
multiple neurons are bundled together to form a nerve 
or nerve fiber. These, in turn, may be combined into 
fascicles, wherein the nerve fibers form bundles 

10 embedded, together with the intraneural vascular 
supply, in a loose collagenous matrix bounded by a 
protective multilamellar sheath. In the central 
nervous system, the neuron cell bodies are visually 
distinguishable from their myelin-ensheathed processes, 

15 and are referenced in the art as grey and white matter, 
respectively. 

During development, differentiating neurons from 
the central and peripheral nervous systems send out 

20 axons that must grow and make contact with specific 

target cells. In some cases, growing axons must cover 
enormous distances; some grow into the periphery, 
whereas others stay confined within the central nervous 
system. In mammals, this stage of neurogenesis is 

25 complete during the embryonic phase of life and 

neuronal cells do not multiply once they have fully 
differentiated. 

Accordingly, the neural pathways of a mammal are 
30 particularly at risk if neurons are subjected to 
mechanical or chemical trauma or to neuropathic 
degeneration sufficient to put the neurons that define 
the pathway at risk of dying. A host of neuropathies, 
some of which affect only a subpopulation or a system 
35 of neurons in the peripheral or central nervous systems 



WO 94/03200 



PCT/US93/07231 



have been identified to date. The neuropathies, which 
may affect the neurons themselves or the associated 
glial cells, may result from cellular metabolic 
dysfunction, infection, exposure to toxic agents, 
5 autoimmunity dysfunction, malnutrition or ischemia. In 
some cases the cellular dysfunction is thought to 
induce cell death directly. In other cases, the 
neuropathy may induce sufficient tissue necrosis to 
stimulate the body's immune/inflammatory system and the 
10 mechanisms of the body's immune response to the initial 
neural injury then destroys the neurons and the pathway 
defined by these neurons. 

- Currently no satisfactory method exists to repair 

15 the damage caused by these neuropathies, which include 
multiple sclerosis, amyotrophic lateral sclerosis 
(ALS), Huntington's chorea, Alzheimer's disease, 
Parkinson's disease (parkinsonism), and metabolically 
derived disorders, such as hepatic encephalopathy. 

20 Current attempts to counteract the effects of severe 
traumatic or neural degenerative lesions of the brain 
and/or spinal cord have to date primarily involved 
implantation of embryonic neurons in an effort to 
replace functionally, or otherwise compensate for, lost 

25 or deficient neurons. Currently, however, human fetal 
cell transplantation research is severely restricted. 
Administration of neurotrophic factors such as nerve 
growth factor and insulin-like growth factor also have 
been suggested to stimulate neuronal growth within the 

30 CNS. (See, for example, Lundborg, (1987) Acta Orthop. 
Scand. 58:145*169 and US Pat. No. 5,093,317.) 
Administration of neurotrophic factors to the CNS 
requires bypassing the blood-brain barrier. The 
barrier may be overcome by direct infusion, or by 

35 modifying the molecule to enhance its transport across 



WO 94/03200 



PCT/US93/07231 



the barrier, as by chemical modification or 
conjugation, or by molecule truncation. Schwann cells 
also have been grafted to a site of a CNS lesion in an 
attempt to stimulate and maintain growth of damaged 
5 neuronal processes (Paino et al. (1991) Exp, Neurology 
H4(2):254-257). 

Where the damaged neural pathway results from CNS 
axonal damage, autologous peripheral nerve grafts have 

10 been used to bridge lesions in the central nervous 
system and to allow axons to make it back to their 
normal target area. In contrast to CNS neurons, neurons 
of the peripheral nervous system can extend new 
peripheral processes in response to axonal damage. This 

15 regenerative property of peripheral nervous system 

axons is thought to be sufficient to allow grafting of 
these segments to CNS axons. Successful grafting 
appears to be limited, however, by a number of factors, 
including the length of the CNS axonal lesion to be 

20 bypassed, and the distance of the graft sites from the 
CNS neuronal cell bodies, with successful grafts 
occurring near the cell body. 

Within the peripheral nervous system, this cellular 
25 regenerative property of neurons has limited ability to 
repair function to a damaged neural pathway. 
Specifically, the new axons extend randomly, and are 
often misdirected, making contact with inappropriate 
targets that can cause abnormal function. Tor example, 
30 if a motor nerve is dame ad, regr owing axons may 

contact the wrong muscles, resulting in paralysis. In 
addition, where severed nerve processes result in a gap 
of longer than a few millimeters, e.g., greater than 10 



WO 94/03200 - PCT/US93/07231 



millimeters (nun), appropriate nerve regeneration does 
not occur, either because the processes fail to grow 
the necessary distance, or because of misdirected 
axonal growth. Efforts to repair peripheral nerve 
5 damage by surgical means has met with mixed results, 
particularly* where damage extends over a significant 
distance. In some cases, the suturing steps used to 
obtain proper alignment of severed nerve ends 
stimulates the formulation of scar tissue which is 

10 thought to inhibit axon regeneration. Even where scar 
tissue formation has been reduced, as with the use of 
nerve guidance channels or other tubular prostheses, 
successful regeneration generally still is limited to 
nerve damage of less than 10 millimeters in distance. 

15 In addition, the reparative ability of peripheral 

neurons is significantly inhibited where an injury or 
neuropathy affects the cell body itself or results in 
extensive degeneration of a distal axon. 

20 Mammalian neural pathways also are at risk due to 

damage caused by neoplastic lesions. Neoplasias of 
both the neurons and glial cells have been identified. 
Transformed cells of neural origin generally lose their 
ability to behave as normal differentiated cells and 

25 can destroy neural pathways by loss of function. In 
addition, the proliferating tumors may induce lesions 
by distorting normal nerve tissue structure, inhibiting 
pathways by compressing nerves, inhibiting cerbrospinal 
fluid or blood supply flow, and/or by stimulating the 

30 body's immune response. Metastatic tumors, which are a 
significant cause of neoplastic lesions in the brain 
and spinal cord, also similarly may damage neural 
pathways and induce neuronal cell death. 



WO 94/03200 



PCT/US93/07231 



One type of morphoregulatory molecule associated 
with neuronal cell growth/ differentiation and 
development is the cell adhesion molecule ("CAM"), most 
notably the nerve cell adhesion molecule (N-CAM) . CAMs 
5 belong to the immunoglobulin super-family and mediate 
cell-cell interactions in developing and adult tissues 
through homophilic binding, i.e., CAM-CAM binding on 
apposing cells. A number of different CAMs currently 
have been identified. Of these, the most thoroughly 

10 studied to date are N-CAM and L-CAM (liver cell 

adhesion molecules), both of which have been identified 
on all cells at early stages of development, as well as 
in different adult tissues. In neural tissue 
development, N-CAM expression is believed to be 

15 important in tissue organization, neuronal migration, 
nerve-muscle tissue adhesion, retinal formation, 
synaptogenesis, and neural degeneration. Reduced N-CAM 
expression also is thought to be associated with nerve 
dysfunction. For example, expression of at least one 

20 form of N-CAM, N-CAM- 180, is reduced in a mouse 

dysmyelinating mutant (Bhat (1988) Brain Res. 452:373- 
377). Reduced levels of N-CAM also have been 
associated with normal pressure hydrocephalus (Werdelin 
(1989) Acta Neurol. Scand . 2i :177 - 181 )/ and with t YP e 

25 XI schizophrenia (Lyons et al., (1988) Biol. Psychiatry 
23:769-775.) In addition, antibodies to N-CAM have 
been shown to disrupt functional recovery in injured 
nerves (Remsen (1990) Exp. Neurobiol. 110 :268-273). 

30 It is an object of this invention to provide 

methods for enhancing survival of neurons at risk of 
dying in a mammal. Another object is to provide 
methods for maintaining neural pathways in vivo at risk 
of injury, or following damage to nerve tissue due to 

35 mechanical or chemical trauma, a neuropathy, or a 



WO 94/03200 



PCT/US93/07231 



neoplastic lesion. Another object is to provide 
compositions and devices for repairing gaps in a neural 
pathway of the peripheral nervous system. Yet another 
object is to provide a means for redif ferentiating 
5 transformed cells defining neural pathways, 

particularly transformed cells of neural origin. 
Another object is to provide a means for stimulating 
CAM expression, particularly N-CAM expression in a 
cell. Yet another object is to provide methods for 

10 monitoring the status of nerve tissue by monitoring 
fluctuations in protein levels present in nerve 
tissue, serum and/or cerebrospinal fluid. These and 
other objects and features of the invention will be 
apparent from the description, drawings, and claims 

15 which follow. 



WO 94/03200 



PCT/US93/07231 



- 8 - 

Summary of the Invention 

The present invention provides methods and 
5 compositions for maintaining neural pathways in a 
mammal in vivo, including methods for enhancing the 
survival of neural cells. 

In one aspect, the invention features compositions 
10 and therapeutic treatment methods that comprise the 
step of administering to a mammal a therapeutically 
effective amount of a morphogenic protein 
( "morphogen" ) , as defined herein, upon injury to a 
neural pathway, or in anticipation of such injury, for 
15 a time and at a concentration sufficient to maintain 
the neural pathway, including repairing damaged 
pathways, or inhibiting additional damage thereto. 

In another aspect, the invention features 
20 compositions and therapeutic treatment methods for 

maintaining neural pathways in a mammal in vivo which 
include administering to the mammal, upon injury to a 
neural pathway or in anticipation of such injury, a 
compound that stimulates in vivo a therapeutically 
25 effective concentration of an endogenous morphogen 
within the body of the mammal sufficient to maintain 
the neural pathway, including repairing damaged 
pathways or inhibiting additional damage thereto. 
These compounds are referred to herein as morphogen- 
30 stimulating agents, and are understood to include 

substances which, when administered to a mammal, act on 
tissue(s) or organ(s) that normally are responsible 



WO 94/03200 



PCT/US93/07231 



for, or capable of , producing a morphogen and/or 
secreting a morphogen/ and which cause the endogenous 
level of the morphogen to be altered. The agent may 
act # for example , by stimulating expression and/or 
5 secretion of an endogenous morphogen. 

In particular , the invention provides methods for 
enhancing the survival of neurons at risk of dying, 
including protecting neurons from the tissue 

10 destructive effects associated with the body's immune/ 
inflammatory response to a nerve injury. The invention 
also provides methods for stimulating neurons to 
maintain their differentiated phenotype, including 
inducing the redif ferentiation of transformed cells of 

15 neuronal origin to a morphology characteristic of 
untrans formed neurons. In one embodiment, the 
invention provides means for stimulating production of 
cell adhesion molecules in cells, particularly nerve 
cell adhesion molecules (N-CAM) in neurons. The 

20 invention also provides methods, compositions and 
devices for stimulating cellular repair of damaged 
neurons and neural pathways, including regenerating 
damaged axons of the peripheral and central nervous 
systems. In addition, the invention also provides 

25 means for evaluating the status of nerve tissue, and 
for detecting and monitoring neuropathies in a mammal 
by monitoring fluctuations in the morphogen levels or 
endogenous morphogen antibody levels present in a 
mammal's serum or cerebrospinal fluid. 

30 

As used herein, a "neural pathway" describes a 
nerve circuit for the passage of electric signals from 
a source to a target cell site. The pathway includes 
the neurons through which the electric impulse is . 



WO 94/03200 



PCT/US93/07231 



transported, including groups of interconnecting 
neurons, the nerve fibers formed by bundled neuronal 
axons, and the glial cells surrounding and associated 
with the neurons. 

5 

In one aspect of the invention, the morphogens 
described herein are useful in repairing damaged neural 
pathways of the peripheral nervous system. In 
particular, the morphogens are useful for repairing 

10 damaged pathways, including transected or otherwise 

damaged nerve fibers (nerves) requiring regeneration of 
neuronal processes, particularly axons, over extended 
distances to bridge a gap in the nerve itself, or 
between the nerve and a post-synaptic cell. 

15 Specifically, the morphogens described herein are 
capable of stimulating complete axonal nerve 
regeneration, including vascularization and reformation 
of the protective myelin sheath. The morphogen 
preferably is provided to the site of injury dispersed 

20 in a biocompatible, bioresorbable carrier material 
capable of maintaining the morphogen at the site and, 
where necessary, means for directing axonal growth from 
the proximal to the distal ends of a severed neuron or 
nerve. For example, means for directing axonal growth 

25 may be required where nerve regeneration is to be 

induced over an extended distance, such as greater than 
10 mm. Many carriers capable of providing these 
functions are envisioned. For example, useful carriers 
include substantially insoluble materials or viscous 

30 solutions prepared as disclosed herein comprising 

laminin, hyaluronic acid or collagen, or other suitable 
synthetic, biocompatible polymeric materials such as 
polylactic, polyglycolic or polybutyric acids and/or 
copolymers thereof. The currently preferred carrier 

35 comprises an extracellular matrix composition, such as 



WO 94/03200 



PCT/US93/07231 



- 11 - 

one described herein derived/ for example , from mouse 
sarcoma cells. Also envisioned as especially useful 
are brain tissue-derived extracellular matrices. 

5 In a particularly preferred embodiment , the 

morphogen is provided to the site as part of a device 
wherein the morphogen is disposed in a nerve guidance 
channel which spans the distance of the damaged 
pathway. The channel acts both as a protective 

10 covering and a physical means for guiding growth of a 
neuronal process such as an axon. Useful channels 
comprise a biocompatible membrane or casing, which may 
be tubular in structure, having a dimension sufficient 
to span the gap or break in the nerve to be repaired, 

15 and having openings adapted to receive severed nerve 
ends. The casing or membrane may be made of any 
biocompatible, nonirritating material, such as silicone 
or a biocompatible polymer such as polyethylene or 
polyethylene vinyl acetate. The casing also may be 

20 composed of biocompatible, bioresorbable polymers, 
including, for example, collagen, hyaluronic acid, 
poly lactic, polybutyric and polyglycolic acids. In a 
currently preferred embodiment, the outer surface of 
the channel is substantially impermeable. 

25 

The morphogen may be disposed in the channel in 
association with a biocompatible carrier material, or 
it may be adsorbed to or otherwise associated with the 
inner surface of the casing, such as is described in 

30 U.S. Pat. No. 5,011,486, provided that the morphogen is 
accessible to the severed nerve ends. Additionally, 
although the nerve guidance channels described herein 
generally are tubular in shape, it should be evident to 
those skilled in the art that various alternative 

35 shapes may be employed. The lumen of the guidance 



WO 94/03200 



PCI7US93/07231 



- 12 - 

channels may, for example, be oval or even square in 
cross section. Moreover the guidance channels may be 
constructed of two or more parts which may be clamped 
together to secure the nerve stumps. Nerve endings may 
5 be secured to the nerve guidance channels by means of 
sutures, biocompatible adhesives such as fibrin glue, 
or other means known in the medical art. 

The morphogens described herein also are envisioned 
10 to be useful in autologous peripheral nerve segment 
implants to bypass damaged neural pathways in the 
central nervous system, such as in the repair of 
damaged or detached retinas, or other damage to the 
optic nerve. Here the morphogen is provided to the 
15 site of attachment to stimulate axonal growth at the 
graft site, particularly where the damaged axonal 
segment to be bypassed occurs far from the neuronal 
cell body. 

20 The morphogens described herein also are useful for 

enhancing survival of neuronal cells at risk of dying, 
thereby preventing, limiting or otherwise inhibiting 
damage to neural pathways. Non-mitotic neurons are at 
risk of dying as a result of a neuropathy or other 

25 cellular dysfunction of a neuron or glial cell inducing 
cell death, or following a chemical or mechanical 
lesion to the cell or its surrounding tissue. The 
chemical lesions may result from known toxic agents, 
including lead, ethanol, ammonia, formaldehyde and many 

30 other organic solvents, as well as the toxins in 

cigarette smoke and opiates. Excitatory amino acids, « 
such as glutamate also may play a role in the 
pathogenesis of neuronal cell death (see Freese et al. 
(1990) Brain Res. 521 :254-264). Neuronal cell death 

35 also is thought to be a significant contributing factor 



WO 94/03200 



PCT/US93/07231 



- 13 - 

in a number of neurodegenerative diseases, including 
Alzheimer's disease, Huntington's chorea, and 
Parkinson's disease, amyotrophic lateral sclerosis and 
multiple sclerosis. The etiology of these neuropathies 
5 may be metabolic, as results in hepatic encephalopathy, 
infectious, toxic, autoimmune, nutritional or ischemic. 
In addition, ethanol and a number of other toxins also 
have been identified as significant contributing 
factors in neurodegenerative diseases. The morphogens 

10 described herein may be provided to cells at risk of 
dying to enhance their survival and thereby protect the 
integrity of the neural pathway. The morphogens may be 
provided directly to the site, or they may be provided 
systemically • Alternatively, as described above, an 

15 agent capable of stimulating endogenous morphogen 
expression and/or secretion, preferably in cells 
associated with the nerve tissue of interest, may be 
administered to the mammal. 

20 In another aspect of the invention, the method 

disclosed is useful for redif f erentiating transformed 
cells, particularly transformed cells of neuronal or 
glial origin, such that the morphogen- treated cells are 
induced to display a morphology characteristic of 

25 untrans formed cells. Where the transformed cells are 
cells of neuronal origin, morphogen treatment 
preferably induces cell rounding and cell aggregation 
(clumping), cell-cell adhesion, neurite outgrowth 
formation and elongation, and N-CAM production. The 

30 methods described herein are anticipated to 

substantially inhibit or reduce neural cell tumor 
formation and/or proliferation in nerve tissue. It is 
anticipated that the methods of this invention will be 
useful in substantially reducing the effects of various 

35 carcinomas of nerve tissue origin such as 



WO 94/03200 



PCT/US93/07231 



- 14 - 

retinoblastomas, neuroblastomas, and gliomas or 
glioblastomas. In addition, the method also is 
anticipated to aid in inhibiting neoplastic lesions 
caused by metastatic tissue. Metastatic tumors are one 
5 of the most common neoplasms of the CNS, as they can 
reach the intracranial compartment through the 
bloodstream. Metastatic tumors may damage neural 
pathways for example, by distorting normal nerve tissue 
structure, compressing nerves, blocking flow of 
10 cerebrospinal fluid or the blood supply nourishing 
brain tissue, and/or by stimulating the body's immune 
response. 

In another aspect of the invention, the morphogens 

15 described herein are useful for providing 

neuroprotective effects to alleviate neural pathway 
damage associated with the body's immune/inflammatory 
response to an initial injury to nerve tissue. Such a 
response may follow trauma to nerve tissue, caused, for 

20 example, by an autoimmune dysfunction, neoplastic 
lesion, infection, chemical or mechanical trauma, 
disease, by interruption of blood flow to the neurons 
or glial cells, for example following ischemia or 
hypoxia, or by other trauma to the nerve or surrounding 

25 material. For example, the primary damage resulting 
from hypoxia or ischemia-reperfusion following 
occlusion of a neural blood supply, as in an embolic 
stroke, is believed to be immunologically associated. 
In addition, at least part of the damage associated 

30 with a number of primary brain tumors also appears to 
be immunologically related. Application of the 
morphogen directly to the cells to be treated, or 
providing the morphogen to the mammal systemically, for 
example, intravenously or indirectly by oral 

35 administration, may be used to alleviate and/or inhibit 



WO 94/03200 



PCT/US93/07231 



- 15 - 

the immunologically related response to a neural 
injury. Alternatively, administration of an agent 
capable of stimulating morphogen expression and/or 
secretion in vivo , preferably at the site of injury, 
5 also may be used. Where the injury is to be induced, 
as during surgery or other aggressive clinical 
treatment, the morphogen or agent may be provided prior 
to induction of the injury to provide a neuroprotective 
effect to the nerve tissue at risk. 

10 

In still another aspect, the invention described 
herein provides methods for supporting the growth and 
maintenance of differentiated neurons, including 
inducing neurons to continue expressing their 

15 phenotype. It is anticipated that this activity will 
be particularly useful in the treatment of nerve tissue 
disorders where loss of function is caused by reduced 
or lost cellular metabolic function and cells become 
senesent or quiescent, such as is thought to occur in 

20 aging cells and to be manifested in Alzheimer's 

disease. Application of the morphogen directly to 
cells to be treated, or providing it systemically by 
parenteral or oral administration stimulates these 
cells to continue expressing their phenotype, 

25 significantly inhibiting and/or reversing the effects 
of the cellular metabolic dysfunction, thereby 
maintaining the neural pathway at risk. Alternatively, 
administration of an agent capable of stimulating 
endogenous morphogen expression and/or secretion in 

30 vivo may be used. 



WO 94/03200 PCT/US93/07231 



- 16 - 

In still another aspect, the invention provides 
methods for stimulating CAM expression levels in a 
cell, particularly N-CAM expression in neurons. CAMs 
are molecules defined as carrying out cell-cell 
5 interactions necessary for tissue formation. CAMs are 
believed to play a fundamental regulatory role in 
tissue development, including tissue boundary 
formation, embryonic induction and migration, and 
tissue stabilization and regeneration. Altered CAM 
10 levels have been implicated in a number of tissue 
disorders, including congenital defects, neoplasias, 
and degenerative diseases. 

In particular, N-CAM expression is associated with 

15 normal neuronal cell development and differentiation, 
including retinal formation, synaptogenesis, and nerve- 
muscle tissue adhesion. Inhibition of one or more of 
the N-CAM isoforms is known to prevent proper tissue 
development. Altered N-CAM expression levels also are 

20 associated with neoplasias, including neuroblastomas 
(see infra), as well as with a number of neuropathies, 
including normal pressure hydrocephalous and type II 
schizophrenia. Application of the morphogen directly 
to the cells to be treated, or providing the morphogen 

25 to the mammal systemically, for example, parenterally, 
or indirectly by oral administration, may be used to 
induce cellular expression of one or more CAMs, 
particularly N-CAMs. Alternatively, administration of 
an agent capable of stimulating morphogen expression 

30 and/or secretion in vivo , preferably at the site of 
injury, also may be used to induce CAM production. 



35 



CAMs also have been postulated as part of a 
morphoregulatory pathway whose activity is induced by a 
to date unidentified molecule (See, for example, 



WO 94/03200 



PCT/US93/07231 



Edelman, G.M. (1986) Ann. Rev. Cell Biol* 2;81-116). 
Without being limited to any given theory/ the 
morphogens described herein may act as the inducer of 
this pathway. 

5 

Finally/ modulations of endogenous morphogen levels 
may be monitored as part of a method of detecting nerve 
tissue dysfunction. Specifically/ modulations in 
endogenous morphogen levels are anticipated to reflect 

10 changes in nerve tissue status. Morphogen expression 
may be monitored directly in biopsied cell samples, in 
cerebrospinal fluid, or serum. Alternatively, 
morphogen levels may be assessed by detecting changes 
in the levels of endogenous antibodies to the 

15 morphogen. For example, one may obtain serum samples 
from a mammal/ and then detect the concentration of 
morphogen or antibody present in the fluid by standard 
protein detection means known to those skilled in the 
art. As an example/ binding protein capable of 

20 interacting specifically with the morphogen of interest 
such as an anti-morphogen antibody may be used to 
detect a morphogen in a standard immunoassay. The 
morphogen levels detected then may be compared to a 
previously determined standard or reference level, with 

25 changes in the detected levels being indicative of the 
status of the tissue. 

In one preferred embodiment of the invention, the 
morphogen or morphogen-stimulating agent is 
30 administered systemically to the individual, e.g./ 

orally or parenterally. In another embodiment of the 
invention, the morphogen may be provided directly to 
the nerve tissue, e.g., by injection to the cerebral 
spinal fluid or to a nerve tissue locus. 



35 



WO 94/03200 



PCT/US93/07231 



- 18 - 

In any treatment method of the invention , 
"administration of morphogen" refers to the 
administration of the morphogen, either alone or in 
combination with other molecules. For example, the 
5 mature form of the morphogen may be provided in 

association with its precursor "pro" domain, which is 
known to enhance the solubility of the protein. Other 
useful molecules known to enhance protein solubility 
include casein and other milk components, as well as 

10 various serum proteins. Additional useful molecules 
which may be associated with the morphogen or 
morphogen- stimulating gent include tissue targeting 
molecules capable of directing the morphogen or 
morphogen-stimulating agent to nerve tissue. Tissue 

15 targeting molecules envisioned to be useful in the 
treatment protocols of this invention include 
antibodies, antibody fragments or other binding 
proteins which interact specifically with surface 
molecules on nerve tissue cells. 

20 

Still another useful tissue targeting molecule is 
part or all of the morphogen precursor "pro" domain, 
particularly that of OP-1 or GDF-1. These proteins are 
found naturally associated with nerve tissue but also 

25 may be synthesized in other tissues and targeted to 
nerve tissue after secretion from the synthesizing 
tissue. For example, while the protein has been shown 
to be active in bone tissue, the primary source of OP-1 
synthesis appears to be the tissue of the urogenic 

30 system (e.g., renal and bladder tissue), with secondary 
expression levels occurring in the brain, heart and 
lungs (see below.) Moreover, the protein has been 
identified in serum, saliva and various milk forms. In 
addition, the secreted form of the protein comprises 

35 the mature dimer in association with the pro domain of 



WO 94/03200 PCT/US93/07231 



the intact morphogen sequence. Accordingly, the 
associated morphogen pro domains may act to target 
specific morphogens to different tissues in vivo . 

5 Associated tissue targeting or solubility-enhancing 

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

Finally, the morphogens or morphogen- stimulating 
agents provided herein also may be administered in 
combination with other molecules known to be beneficial 
15 in maintaining neural pathways, including, for example, 
nerve growth factors and ant i- inflammatory agents. 

Where the morphogen is intended for use as a 
therapeutic for disorders of the CNS, an additional 

20 problem must be addressed: overcoming the so-called 
"blood-brain barrier", the brain capillary wall 
structure that effectively screens out all but selected 
categories of molecules present in the blood, 
preventing their passage into the brain. The 

25 blood-brain barrier may be bypassed effectively by 
direct infusion of the morphogen or morphogen- 
stimulating agent into the brain. Alternatively, the 
morphogen or morphogen- stimulating agent may be 
modified to enhance its transport across the 

30 blood-brain barrier. For example, truncated forms of 
the morphogen or a morphogen-stimulating agent may be 
most successful. Alternatively, the morphogen or 



WO 94/03200 



PCT/US93/07231 



- 20 - 



morphogen-stimulating agent may be modified to render 
it more lipophilic, or it may be conjugated to another 
molecule which is naturally transported across the 
barrier, using standard means known to those skilled in 
5 the art, as, for example, described in Pardridge, 
Endocrine Reviews 7 :314-330 (1986) and U.S. Pat* 
No. 4,801,575. 

Accordingly, as used herein, a functional "analog" 
10 of a morphogen refers to a protein having morphogenic 
biological activity but possessing additional 
structural differences compared to a morphogen as 
defined herein, e.g., having additional chemical 
moieties not normally a part of a morphogen. Such 
15 moieties (introduced, for example, by acylation, 

alkylation, cationization, or glycosylation reactions, 
or other means for conjugating the moiety to the 
morphogen) may improve the molecule's solubility, 
absorption, biological half-life, or transport, e.g., 
20 across the blood-brain barrier. 

Among the morphogens useful in this invention 
are proteins originally identified as osteogenic 
proteins, such as the OP-1, OP-2 and CBMP2 proteins, as 

25 well as amino acid sequence-related proteins such as 
DPP (from Drosophila), Vgl (from Xenopus), Vgr-1 (from 
mouse, see U.S. 5,011,691 to Oppermann et al.), GDF-1 
(from mouse, see Lee (1991) PNAS 88:4250-4254), all of 
which are presented in Table II and Seq. ID Nos.5-14), 

30 and the recently identified 60A protein (from 

Drosophila, Seq. ID No. 24, see Wharton et al. (1991) 
PNAS 88:9214-9218. ) The members of this family, which 
include members of the TGF-0 super-family of proteins, 
share substantial amino acid sequence homology in their 

35 C-terminal regions. The proteins are translated as a 



WO 94/03200 



PCT/US93/07231 



- 21 - 

precursor, having an N- terminal signal peptide 
sequence, typically less tahn about 30 residues, 
followed by a "pro" domain that is cleaved to yield the 
mature sequence. The signal peptide is cleaved rapidly 
5 upon translation, at a cleavage site that can be 

predicted in a given sequence using the method of Von 
Heijne ( ( 1986) Nucleic Acids Research 14:4683-469'l« ) 
Table I, below, describes the various morphogens 
identified to date, including their nomenclature as 

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

15 TABLE I 

"OP-1" Refers generically to the group of 

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

20 encoding OP-1 protein, including allelic 

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

25 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 

30 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 

35 proteins, cleaved to yield the mature, 



PCT/US93/07231 



- 22 - 

morphogenically active proteins are 
defined essentially by residues 30-292 
(hOPl) and residues 30-291 (mOPl). 

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

refers generically to the morphogenically 
active proteins expressed from a DNA 
sequence encoding the CBMP2 proteins, 
including allelic and species variants 
thereof, e.g., human CBMP2A ( "CBMP2A(fx) ", 
Seq ID No. 9) or human CBMP2B DNA 



WO 94/03200 



PCT/US93/07231 



- 23 - 

( ,, CBMP2B(fx) M , Seq. ID No. 10). The amino 
acid sequence for the full length 
proteins , referred to in the literature as 
BMP2A and BMP2B, or BMP 2 and BMP4, appear 
5 in Wozney, et al. (1988) Science 242 :1528- 

1534. The pro domain for BMP2 (BMP2A) 
likely includes residues 25-248 or 25-282; 
the mature protein, residues 249-396 or 
283-396. The pro domain for BMP4 (BMP2B) 
10 likely includes residues 25-256 or 25-292; 

the mature protein, residues 257-408 or 
293-408. 



"DPPffx)'' refers to protein sequences encoded by the 
15 Drosophila DPP gene and defining the 

conserved seven cysteine skeleton (Seq. ID 
No. 11). The amino acid sequence for the 
full length protein appears in Padgett, et 
al (1987) Nature 325: 81-84. The pro 
20 domain likely extends from the signal 

peptide cleavage site to residue 456; the 
mature protein likely is defined by 
residues 457-588. 

25 n Vgl(fx) M refers to protein sequences encoded by the 

Xenopus Vgl gene and defining the 
conserved seven cysteine skeleton (Seq. ID 
No. 12). The amino acid sequence for the 
full length protein appears in 

30 Weeks (1987) Cell 51: 861-867. The 

prodomain likely extends from the signal 
peptide cleavage site to residue 246; the 
mature protein likely is defined by 
residues 247-360. 



35 



WO 94/03200 



PCT/US93/07231 



- 24 - 



"Vgr-l(fx) " refers to protein sequences encoded by the 
murine Vgr-1 gene and defining the 
conserved seven cysteine skeleton (Seg. ID 
No. 13). The amino acid sequence for the 
5 full length protein appears in Lyons, et 

al, (1989) PNAS 86: 4554-4558. The 
prodomain likely extends from the signal 
peptide cleavage site to residue 299; the 
mature protein likely is defined by 
10 residues 300-438. 



"GDF-l(fx) " refers to protein sequences encoded by the 
human GDF-1 gene and defining the 
conserved seven cysteine skeleton (Seq. ID 
No. 14). The cDNA and encoded amino 
sequence for the full length protein is 
provided in Seq. ID. No. 32. The 
prodomain likely extends from the signal 
peptide clavage site to residue 214; the 
mature protein likely is defined by 
residues 215-372. 

"60A" refers generically to the morphogenically 

active proteins expressed from part or all 
25 of a DNA sequence (from the Drosophila 60A 

gene) encoding the 60A proteins (see Seq. 
ID No. 24 wherein the cDNA and encoded 
amino acid sequence for the full length 
protein is provided). n 60A(fx)" refers to 
30 the protein sequences defining the 

conserved seven cysteine skeleton 
(residues 354 to 455 of Seq. ID No. 24.) 



15 



20 



WO 94/03200 



PCT/US93/07231 



- 25 - 

The prodoraain likely extends from the 
signal peptide cleavage site to residue 
324; the mature protein likely is defined 
by residues 325-455. 

5 

M BMP3(fx)" refers to protein sequences encoded by the 
human BMP 3 gene and defining the conserved 
seven cysteine skeleton (Seq. ID No. 26). 
The amino acid sequence for the full 

10 length protein appears in Wozney et al. 

(1988) Science 242 ; 1528-1534. The pro 
domain likely extends from the signal 
peptide cleavage site to residue 290; the 
mature protein likely is defined by 

15 residues 291-472. 

M BMP5(fx)" refers to protein sequences encoded by the 
human BMP5 gene and defining the conserved 
seven cysteine skeleton (Seq. ID No. 27). 

20 The amino acid sequence for the full 

length protein appears in Celeste, et al. 
(1991) PNAS 87: 9843-9847. The pro domain 
likely extends from the signal peptide 
cleavage site to residue 316; the mature 

25 protein likely is defined by residues 

317-454. 



M BMP6(fx) ,f refers to protein sequences encoded by the 
human BMP6 gene and defining the conserved 
30 seven cysteine skeleton (Seq. ID No. 28). 

The amino acid sequence for the full 
length protein appear sin Celeste, et al. 



WO 94/03200 * PCT/US93/07231 



- 26 - 

(1990) PNAS 87: 9843-5847 . The pro domain 
likely includes extends from the signal 
peptide cleavage site to residue 374; the 
mature sequence likely includes 
5 residues 375-513. 



The OP-2 proteins have an additional cysteine 
residue in this region (e.g., see residue 41 of Seq. ID 

10 Nos. 7 and 8), in addition to the conserved cysteine 
skeleton in common with the other proteins in this 
family. The GDF-1 protein has a four amino acid insert 
within the conserved skeleton (residues 44-47 of Seq. 
ID No. 14) but this insert likely does not interfere 

15 with the relationship of the cysteines in the folded 

structure. In addition, the CBMP2 proteins are missing 
one amino acid residue within the cysteine skeleton. 

The morphogens are inactive when reduced, but are 

20 active as oxidized homodimers and when oxidized in 
combination with other morphogens of this invention. 
Thus, as defined herein, a morphogen is a dimeric 
protein comprising a pair of polypeptide chains, 
wherein each polypeptide chain comprises at least the 

25 C-terminal six cysteine skeleton defined by residues 
43-139 of Seq. ID No. 5, including functionally 
equivalent arrangements of these cysteines (e.g., amino 
acid insertions or deletions which alter the linear 
arrangement of the cysteines in the sequence but not 

30 their relationship in the folded structure), such that, 
when the polypeptide chains are folded, the dimeric 
protein species comprising the pair of polypeptide 
chains has the appropriate three-dimensional structure, 
including the appropriate intra- or inter-chain 

35 disulfide bonds such that the protein is capable of 



WO 94/03200 



PCT/US93/07231 



- 27 - 

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

In one preferred aspect, the morphogens of 
this invention comprise one of two species of generic 

15 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 

20 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- 

25 terminus: 

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

30 Preferred amino acid sequences within the 

foregoing generic sequences include: Generic Sequence 
3 (Seq. ID No. 3), Generic Sequence 4 (Seq. ID No. 4), 
Generic Sequence 5 (Seq. ID No. 30) and Generic 
Sequence 6 (Seq. ID No* 31), listed below. These 



WO 94/03200 PCT/US93/07231 



- 28 - 

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

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

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

20 4, respectively, providing an appropriate cysteine 

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

25 Generic Sequence 3 

Leu Tyr Val Xaa Phe 

1 5 

Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

10 



30 



Xaa Ala Pro Xaa Gly Xaa Xaa Ala 
15 20 



WO 94/03200 PCI7US93/07231 



- 29 - 

Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 

25 30 
Xaa Pro Xaa Xaa Xaa Xaa Xaa 

. 35 

5 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 
10 55 60 

Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
65 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 
70 75 
15 Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 

80 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

85 90 
Xaa Cys Gly Cys Xaa 
20 95 

wherein each Xaa is independently selected from a group 
of one or more specified amino acids defined as 
follows: "Res." means "residue" and Xaa at res. 4 = 
(Ser, Asp or Glu); Xaa at res. 6 = (Arg, Gin, Ser or 
25 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); 
Xaa at res. 12 = (Asp, Arg or Asn); Xaa at res. 14 = (lie 
or Val); Xaa at res. 15 = (He or Val); Xaa at res. 18 = 



WO 94/03200 * PCT/US93/07231 



(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 
res. 26 = (Glu, His, Tyr, Asp or Gin); Xaa at res. 28 « 
5 (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); 
Xaa at res. 36 = (Tyr, Cys, His, Ser or lie); Xaa at 

10 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 - (lie or 
Val); Xaa at res. 4 5 = (Val or Leu); Xaa at res .46 - 
(Gin or Arg); Xaa at res. 47 = (Thr, Ala or Ser); Xaa at 

15 res. 49 = (Val or Met); Xaa at res. 50 = (His or Asn); 
Xaa at res. 51 = (Phe, Leu, Asn, Ser, Ala or Val); Xaa 
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 

20 = (Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at 
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 

25 or Glu); Xaa at res. 67 = (Leu, Met or Val); Xaa at 
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 

30 or Leu); Xaa at res. 76 » (Asp or Asn); Xaa at res. 77 = 
(Asp, Glu, Asn or Ser); Xaa at res. 78 = (Ser, Gin, Asn 
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 = 

35 (Lys, Asn, Gin or His); Xaa at res. 86 = (Tyr or His); 



WO 94/03200 



PCT/US93/07231 



- 31 - 

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

Generic Sequence 4 

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

Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

15 

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

30 35 
Xaa Pro Xaa Xaa Xaa Xaa Xaa 

40 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 
20 45 50 

Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 

55 

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

70 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 

75 80 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
30 B5 

Xaa Xaa Xaa Xaa Net Xaa Val Xaa 

90 95 
Xaa Cys Gly Cys Xaa 
100 



WO 94/03200 



PCT/US93/07231 



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

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

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

20 lie); Xaa at res. 42 = (Met, Phe, Gly or Leu); Xaa at 
res. 44 = (Ala, Ser or Gly); Xaa at res .45 = (Thr, Leu 
or Ser); Xaa at res. 49 = (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 

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

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

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

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



WO 94/03200 



PCT/US93/07231 



- 33 - 

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

15 

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

20 Generic Sequences 5 and 6 are composite amino acid 

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

25 (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, 
Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), 
and GDF-1 (from mouse, Seq. ID No. 14), human BMP3 
(Seq. ID No. 26), human BMP 5 (Seq. ID No. 27), human 
BMP6 (Seq. ID No. 28) and 60(A) (from Drosophila, Seq. 

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

35 variable positions within the sequence. As for Generic 



WO 94/03200 



PCT/US93/07231 



10 



- 34 - 

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

Generic Sequence 5 



Leu Xaa Xaa Xaa Phe 
1 5 
Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

10 

15 Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala 

15 20 
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 

25 30 
Xaa Pro Xaa Xaa Xaa Xaa Xaa 
20 35 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 

40 45 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

50 

25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

55 60 
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
65 



WO 94/03200 PCT/US93/07231 



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 Xaa 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. 2 « 
(Tyr or Lys); Xaa at res .3 = Val or lie); Xaa at res. 4 
= (Ser, Asp or Glu); Xaa at res. 6 = (Arg, Gin, Ser, Lys 
or Ala); Xaa at res. 7 « (Asp, Glu or Lys); Xaa at res. 8 

15 « (Leu, Val or He); Xaa at res. 11 « (Gin, Leu, Asp, 
His, Asn or Ser); Xaa at res. 12 = (Asp, Arg, Asn or 
Glu); Xaa at res. 14 = (He or Val); Xaa at res. 15 = 
(He or Val); Xaa at res .16 (Ala or Ser); Xaa at res. 18 
= (Glu, Gin, Leu, Lys, Pro or Arg); Xaa at res. 19 « 

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

25 Gin or Asn); Xaa at res. 31 ■« (Phe, Leu or Tyr); Xaa at 
res. 33 « (Leu, Val or Met); Xaa at res. 34 ■« (Asn, Asp, 
Ala, Thr or Pro); Xaa at res. 35 - (Ser, Asp, Glu, Leu, 
Ala or Lys); Xaa at res. 36 = (Tyr, Cys, His, Ser or 
He); Xaa at res. 37 - (Met, Phe, Gly or Leu); Xaa at 

30 res. 38 - (Asn, Ser or Lys); Xaa at res. 39 » (Ala, Ser, 
Gly or Pro); Xaa at res. 40 « (Thr, Leu or Ser); Xaa at 
res. 44 = (He, Val or Thr); Xaa at res. 45 = (Val, Leu 



WO 94/03200 



PCT/US93/07231 



- 36 - 

or lie); Xaa at res. 46 = (Gin or Arg); Xaa at res. 47 « 
(Thr, Ala or Ser); Xaa at res. 48 = (Leu or lie); Xaa at 
res. 49 « (Val or Met); Xaa at res. 50 - (His, Asn or 
Arg); Xaa at res. 51 - (Phe, Leu, Asn, Ser, Ala or Val); 
5 Xaa at res. 52 • (He, Met, Asn, Ala, Val or Leu); Xaa 
at res. 53 = (Asn, Lys, Ala, Glu, Gly or Phe); Xaa at 
res. 54 = (Pro, Ser or Val); Xaa at res. 55 = (Glu/ Asp, 
Asn, Gly, Val or Lys); Xaa at res. 56 = (Thr, Ala, Val, 
Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at res. 57 = 

10 (Val, Ala or He); Xaa at res. 58 = (Pro or Asp); Xaa at 
res. 59 = (Lys, Leu or Glu); Xaa at res. 60 = (Pro or 
Ala); Xaa at res. 63 = (Ala or Val); Xaa at res. 65 = 
(Thr, Ala or Glu); Xaa at res. 66 » (Gin, Lys, Arg or 
Glu); Xaa at res. 67 = (Leu, Met or Val); Xaa at res. 68 

15 = (Asn, Ser, Asp or Gly); Xaa at res. 69 *= (Ala, Pro or 
Ser); Xaa at res. 70 ■ (He, Thr, Val or Leu); Xaa at 
res. 71 = (Ser, Ala or Pro); Xaa at res. 72 = (Val, Met 
or He); Xaa at res. 74 = (Tyr or Phe); Xaa at res. 75 = 
(Phe, Tyr, Leu or His); Xaa at res. 76 «= (Asp, Asn or 

20 Leu); Xaa at res. 77 «= (Asp, Glu, Asn or Ser); Xaa at 
res. 78 = (Ser, Gin, Asn, Tyr or Asp); Xaa at res. 79 - 
(Ser, Asn, Asp, Glu or Lys); Xaa at res. 80 = (Asn, Thr 
or Lys); Xaa at res. 82 * (He, Val or Asn); Xaa at 
res. 84 = (Lys or Arg); Xaa at res .85 « (Lys, Asn, Gin, 

25 His or Val); Xaa at res. 86 « (Tyr or His); Xaa at 

res. 87 - (Arg, Gin, Glu or Pro); Xaa at res. 88 = (Asn, 
Glu or Asp); Xaa at res. 90 = (Val, Thr, Ala or He); 
Xaa at res. 92 ■ (Arg, Lys, Val, Asp or Glu); Xaa at 
res. 93 = (Ala, Gly, Glu or Ser); Xaa at res. 95 = (Gly 

30 or Ala) and Xaa at res. 97 = (His or Arg). 



WO 94/03200 



PCT/US93/07231 



- 37 - 
Generic Sequence 6 

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

5 Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

15 

Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala 

20 25 
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 
10 30 35 

Xaa Pro Xaa Xaa Xaa Xaa Xaa 

40 

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

55 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

60 65 
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
20 70 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 

75 80 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
85 

25 Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

90 95 
Xaa Cys Xaa Cys Xaa 
100 



30 wherein each Xaa is independently selected from a group 
of one or more specified amino acids as defined by the 
following: "Res." means "residue" and Xaa at res. 2 
(Lys, Arg, Ala or Gin); Xaa at res. 3 - (Lys, Arg or 
Met); Xaa at res. 4 «= (His, Arg or Gin); Xaa at res. 5 = 

35 (Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr); Xaa at 



WO 94/03200 



PCT/US93/07231 



res .7 » (Tyr or Lys); Xaa at res. 8 = (Val or He); Xaa 
at res. 9 = (Ser, Asp or Glu); Xaa at res. 11 = (Arg, 
Gln> Ser, Lys or Ala); Xaa at res. 12 « (Asp, Glu, or 
Lys); Xaa at res. 13 = (Leu, Val or He); Xaa at res. 16 
5 = (Gin, Leu, Asp, His, Asn or Ser); Xaa at res. 17 - 
(Asp, Arg, Asn or Glu); Xaa at res. 19 = (He or Val); 
Xaa at res. 20 = (He or Val); Xaa at res. 21 * (Ala or 
Ser); Xaa at res. 23 « (Glu, Gin, Leu, Lys, Pro or Arg); 
Xaa at res. 24 = (Gly or Ser); Xaa at res. 2 5 = (Tyr or 

10 Phe); Xaa at res. 26 = (Ala, Ser, Asp, Met, His, Gin, 

Leu, or Gly); Xaa at res. 28 « (Tyr, Asn or Phe); Xaa at 
res. 31 = (Glu, His, Tyr, Asp, Gin or Ser); Xaa at 
res .33 = Glu, Lys, Asp, Gin or Ala); Xaa at res. 35 = 
(Ala, Ser, Pro, Gin or Asn); Xaa at res. 36 = (Phe, Leu 

15 or Tyr); Xaa at res. 38 = (Leu, Val or Met); Xaa at 
res. 39 « (Asn, Asp, Ala, Thr or Pro); Xaa at res. 40 
(Ser, Asp, Glu, Leu, Ala or Lys); Xaa at res. 41 = (Tyr, 
Cys, His, Ser or He); Xaa at res. 42 = (Met, Phe, Gly 
or Leu); Xaa at res. 43 = (Asn, Ser or Lys); Xaa at 

20 res. 44 = (Ala, Ser, Gly or Pro); Xaa at res. 45 = (Thr, 
Leu or Ser); Xaa at res. 4 9 = (He, Val or Thr); Xaa at 
res. 50 = (Val, Leu or He); Xaa at res. 51 « (Gin or 
Arg); Xaa at res. 52 = (Thr, Ala or Ser); Xaa at res. 53 
= (Leu or He); Xaa at res. 54 = (Val or Met); Xaa at 

25 res. 55 « (His, Asn or Arg); Xaa at res. 56 = (Phe, Leu, 
Asn, Ser, Ala or Val); Xaa at res. 57 = (He, Met, Asn, 
Ala, Val or Leu); Xaa at res. 58 = (Asn, Lys, Ala, Glu, 
Gly or Phe); Xaa at res. 59 « (Pro, Ser or Val); Xaa at 
res. 60 = (Glu, Asp, Gly, Val or Lys); Xaa at res. 61 = 

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

35 (Gin, Lys, Arg or Glu); Xaa at res. 72 « (Leu, Met or 



WO 94/03200 



PCI7US93/07231 



- 39 - 

Val); Xaa at res. 73 « (Asn, Ser, Asp or Gly); Xaa at 
res. 74 ~ (Ala, Pro or Ser); Xaa at res. 75 ■ (lie, Thr, 
Val or Leu); Xaa at res. 76 « (Ser, Ala or Pro); Xaa at 
res. 77 = (Val, Met or He); Xaa at res. 79 = (Tyr or 
5 Phe); Xaa at res. 80 = (Phe, Tyr, Leu or His); Xaa at 
res. 81 « (Asp, Asn or Leu); Xaa at res. 82 * (Asp, Glu, 
Asn or Ser); Xaa at res. 83 « (Ser, Gin, Asn, Tyr 'or 
Asp); xaa at res. 84 = (Ser, Asn, Asp, Glu or Lys); Xaa 
at res. 85 - (Asn, Thr or Lys); Xaa at res. 87 - (He, 

10 Val or Asn); Xaa at res. 89 ■ (Lys or Arg); Xaa at 

res. 90 «= (Lys, Asn, Gin, His or Val); Xaa at res. 91 = 
(Tyr or His); Xaa at res. 92 - (Arg, Gin, Glu or Pro); 
Xaa at res. 93 •<= (Asn, Glu or Asp); Xaa at res. 95 = 
(Val, Thr, Ala or He); Xaa at res. 97 = (Arg, Lys, Val, 

15 Asp or Glu); Xaa at res. 98 - (Ala, Gly, Glu or Ser); 
Xaa at res. 100 - (Gly or Ala); and Xaa at res. 102 ■ 
(His or Arg). 

Particularly useful sequences for use as 

20 morphogens in this invention include the C- terminal 
domains, e.g., the C-terminal 96-102 amino acid 
residues of Vgl, Vgr-1, DPP, OP-1, OP-2, CBMP-2A, 
CBMP-2B, GDF-1 (see Table II, below, and Seq. ID 
Nos. 5-14), as well as proteins comprising the 

25 C- terminal domains of 60A, BMP3, BMP5 and BMP 6 (see 
Seq. ID Nos. 24-28), all of which include at least the 
conserved six or seven cysteine skeleton. In addition, 
biosynthetic constructs designed from the generic 
sequences, such as COP-1, 3-5, 7, 16, disclosed in U.S. 

30 Pat. No. 5,011,691, also are useful. Other sequences 
include the inhibins/activin proteins (see, for 
example, U.S. Pat. Nos. 4,968,590 and 5,011,691). 
Accordingly, other useful proteins are those exhibiting 
morphogenic activity and having amino acid sequences 

35 sharing at least 70% amino acid sequence homology or 



WO 94/03200 



PCT/US93/07231 



"similarity" , and preferably 80% homology or similarity 
with any of the sequences above. These are anticipated 
to include allelic variants , species variants and other 
sequence variants (e.g., "muteins" or "mutant 
5 proteins" ), whether naturally occurring or 

biosynthetically produced, as well as novel members of 
this morphogenic family of proteins. 

As used herein, "amino acid sequence homology" is 

10 understood to mean amino acid sequence similarity, and 
homologous sequences share identical or similar amino 
acids, where similar amino acids are conserved amino 
acids as defined by Dayoff et al., Atlas of Protein 
Sequence and Structure ; vol.5, Suppl.3, pp. 34 5-362 

15 (M.O. Dayoff, ed., Nat'l BioMed. Research Fdn., 

Washington D.C. 1978.) Thus, a candidate sequence 
sharing 70% amino acid homology with a reference 
sequence requires that, following alignment of the 
candidate sequence with the reference sequence, 70% of 

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

25 sequences. Thus, a candidate sequence sharing 60% 

amino acid identity with a reference sequence requires 
that, following alignment of the candidate sequence 
with the reference sequence, 60% of the amino acids in 
the candidate sequence are identical to the 

30 corresponding amino acid in the reference sequence. 



WO 94/03200 



PCT/US93/07231 



- 41 - 

As used herein, all homologies and identities 
calculated use OP-1 as the reference sequence. Also as 
used herein, sequences are aligned for homology and 
identity calculations using the method of Needleman et 
5 al. (1970) J.Mol. Biol. 48 :443-453 and identities 

calculated by the Align program (DNAstar, Inc.) In all 
cases, internal gaps and amino acid insertions in the 
candidate sequence as aligned are ignored when making 
the homology/identity calculation. 

10 

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 

15 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 OP-1 and OP-2 proteins, including the 
Drosophila 60A protein. Accordingly, in another 

20 preferred aspect of the invention, useful morphogens 
include active proteins comprising species of 
polypeptide chains having the generic amino acid 
sequence herein referred to as "OPX", which 
accommodates the homologies between the various 

25 identified species of 0P1 and OP2 (Seq. ID No. 29). 

In still another preferred aspect of the invention, 
useful morphogens include active proteins comprising 
polypeptide chains encoded by nucleic acids which 

30 hybridize to DNA or RNA sequences encoding the C- 
terroinal sequence defining the consumed cysteine 
domain, e.g., nucleotides 1036-1341 and nucleotides 
1390-1695 of Seq. Id. Nos. 16 and 20, respectively, t>f 
0P1 or OP2 under stringent hybridization conditions. 

35 As used herein, stringent hybridization conditions are 



WO 94/03200 



PCT/US93/07231 



- 42 - 

defined as hybridization in 40% formamide, 5 X SSPE, 
5 X Denhardt's Solution, and 0.1% SDS at 37°C 
overnight, and wshing in 0.1 X SSPE, 0.1% SDS at 50°C. 

5 The morphogens useful in the methods, composition 

and devices of this invention include proteins 
comprising any of the polypeptide chains described 
above, whether isolated from naturally-occurring 
sources, or produced by recombinant DNA or other 

10 synthetic techniques, and 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, including 

15 those 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 

20 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 

25 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 

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

35 description of the morphogens useful in the methods, 



WO 94/03200 



PCT/US93/07231 



- 43 - 

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

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

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

15 mammal, including enhancing the survival of neurons at 
risk of dying and stimulating nerve regeneration and 
repair in a variety of mammals, including humans. 



The foregoing and other objects, features and 
20 advantages of the present invention will be made more 
apparent from the following detailed description of the 
invention. 



WO 94/03200 PCT/US93/07231 



- 44 - 

Brief Description of the Drawings: 

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

Fig. 1(A and B) are photomicrographs illustrating 
10 the ability of morphogen (OP-1) to induce transformed 
neuroblastoma x glioma cells (1A) to redif ferentiate to 
a morphology characteristic of untrans formed neurons 
(IB); 

15 Fig. 2A is a dose response curve for the induction 

of the 180 kDa and 140 kDa N-CAM isoforms in morphogen- 
treated NG108-15 cells; 

Fig. 2B is a photomicrograph of a Western blot of 
20 whole cell extracts from morphogen-treated NG108-15 
cells with an N-CAM- specific antibody; and 

Fig. 3 is a plot of the mean number of cell 
aggregates counted in 20 randomly selected fields as a 
25 function of morphogen concentration. 

Fig. 4 is a photomicrograph of an immunoblot 
demonstrating the presence of OP-1 in human serum. 

30 



WO 94/03200 



PCT/US93/07231 



- 45 - 

Detailed Description of the Invention 

It now has been discovered that the proteins 
described herein are effective agents for enhancing the 
5 survival of neurons, particularly neurons at risk of 
dying, and for maintaining neural pathways in a mammal. 
As described herein, these proteins ("morphogens") are 
capable of enhancing survival of non-mitotic neurons, 
stimulating neuronal CAM expression, maintaining the 

10 phenotypic expression of differentiated neurons, 

inducing the redif ferentiation of transformed cells of 
neural origin, and stimulating axonal growth over 
breaks in neural processes, particularly large gaps in 
distal axons. The piroteins also are capable of 

15 providing a neuroprotective effect to alleviate the 
tissue destructive effects associated with 
immunologically-related nerve tissue damage. Finally, 
the proteins may be used as part of a method for 
monitoring the viability of nerve tissue in a mammal. 

20 

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

25 nonlimiting examples which 1) illustrate the 

suitability of the morphogens and morphogen-stimulating 
agents described herein as therapeutic agents for 
maintaining nerual pathways in a mammal and enhancing 
survival of neuronal cells at risk of dying; and 

30 2) provide assays with which to test candidate 

morphogens and morphogen- stimulating agents for their 
efficacy. 



WO 94/03200 



PCT/US93/0723I 



- 46 - 

I. Useful Morphogens 

As defined herein a protein is morphogenic if it is 
capable of inducing the developmental cascade of 
5 cellular and molecular events that culminate in the 
formation of new, organ-specific tissue and comprises 
at least the conserved C-terminal six cysteine skeleton 
or its functional equivalent (see supra). 
Specifically, the morphogens generally are capable of 

10 all of the following biological functions in a 

morphogenically permissive environment: stimulating 
proliferation of progenitor cells; stimulating the 
differentiation of progenitor cells; stimulating the 
proliferation of differentiated cells; and supporting 

15 the growth and maintenance of differentiated cells. 
Details of how the morphogens useful in the method of 
this invention first were identified, as well as a 
description on how to make, use and test them for 
morphogenic activity are disclosed in international 

20 application US92/01968 (W092/15323 ) , the disclosure of 
which is hereby incorporated by reference. As 
disclosed therein, the morphogens may be purified from 
naturally- sourced material or recombinant ly produced 
from procaryotic or eucaryotic host cells, using the 

25 genetic sequences disclosed therein. Alternatively, 

novel morphogenic sequences may be identified following 
the procedures disclosed therein. 



Particularly useful proteins include those which 
30 comprise the naturally derived sequences disclosed in 
Table II. Other useful sequences include biosynthetic 
constructs such as those disclosed in U.S. Pat. 



WO 94/03200 * PCT/US93/07231 



- 47 - 

5,011/691/ the disclosure of which is incorporated 
herein by reference (e.g., COP-1, COP-3, COP-4, COP-5, 
COP- 7/ and COP- 16). 



5 Accordingly, the morphogens useful in the methods 

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

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

Cys Xaa Xaa Xaa Xaa (Seq. ID No. 15) 
20 15 

Table II, set forth below, compares the amino acid 
sequences of the active regions of native proteins that 
have been identified as morphogens, including human 

25 OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP- 1 

(mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 
(Seq. ID Nos. 7, 8, and 20-23), CBMP2A (Seq. ID No. 9), 
CBMP2B (Seq. ID No. 10), BMP3 (Seq. ID No. 26), DPP 
(from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, 

30 Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), 
GDF-1 (from mouse, Seq. ID Nos. 14/ 32 and 33)/ 60A 
protein (from Drosophila, Seq. ID Nos. 24 and 25), BMP 5 
(Seq. ID No. 27) and BMP 6 (Seq. ID No. 28). The 
sequences are aligned essentially following the method 

35 of Needleman et al. (1970) J. Mol. Biol. , £8:443-453, 



WO 94/03200 



PCT/US93/07231 



- 48 - 

calculated using the Align Program (DNAstar, Inc.) In 
the table, three dots indicates that the amino acid in 
that position is the same as the amino acid in hOP-1. 
Three dashes indicates that no amino acid is present in 
5 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 these amino acid sequences in this region 
comprise Asn-Ser (residues 58, 59), with CBMP-2A then 
10 comprising Lys and lie, whereas CBMP-2B comprises Ser 
and lie. 



15 



TABLE II 





h0P-l 


Cys 


Lys 


Lys 


His 


Glu Leu 


Tyr 




mOP-1 






• * • 




• • • * • • 






hOP-2 




Arg 


Arg 


• • • 


• • • • • • 


• # * 




mOP-2 




. Arg 


Arg 


• • • 


• • * • • * 


• • « 


20 


DPP 




Arg 


Arg 




Ser 






Vgl 




• • * 


Lys 


Arg 


His 






Vgr-1 






• • « 


* * • 


Gly ... 






CBMP-2A 




* • • 


Arg 


• # • 


Pro ... 


• • • 




CBMP-2B 




Arg 


Arg 




Ser ... 




25 


BMP3 




Ala 


Arg 


Arg 


Tyr 


Lys 




GDF-1 




Arg 


Ala 


Arg 


Arg ... 


• * • 




60A 




Gin 


Met 


Glu 


Thr ... 


• * • 




BMP5 




« • • 


• • • 


• • • 


• • • • « • 


• • • 




BMP6 




Arg 


• • • 


• • • 


• * » • • • 


• • • 


30 




1 








5 





h0P-l 
m0P-l 
35 h0P-2 



Ser Pbe Arg Asp Leu Gly Trp Gin Asp 



» • • * • i 



* * • • • 



Gin 



i • • • i 



Leu 



WO 94/03200 



PCT/US93/07231 



- 49 - 



10 



mOP-2 

DPP 

Vgl 

Vgr-1 

CBMP-2A 

CBMP-2B 

BMP 3 

GDF-1 

60A 

BMP 5 

BMP 6 



Ser 
Asp 
Glu 

• » • 

Asp 
Asp 
Asp 

• • • 
Asp 



Ser 
Lys 
Gin 
Ser 
Ser 
Ala 

• • • 

Lys 
Gin 



Glu 



Val 
Val 
Val 
Val 
Val 
lie 
Val 



Leu 
Asp 



Asn 



• . « • • • 

Asn . . • 

Asn ... 

Ser ' Glu 

His Arg 
His 



10 



15 



15 



20 



25 



hOP-1 

mOP-1 

hOP-2 

mOP-2 

DPP 

Vgl 

Vgr-1 

CBMP-2A 

CBHP-2B 

BMP3 

GDF-1 

60A 

BMP5 

BHP6 



Trp He He Ala Pro Glu Gly Tyr Ala 



Val 
Val 

Val 



Val 



Val 



Val 
Val 



Ser 



Gin 
Gin 
Leu 
Gin 
Lys 
Pro 
Pro 
Lys 
Arg 



Lys 



Ser 



Pbe 
Phe 



Ser 
Ser 
Asp 
Het 

His 
Gin 
Asp 
Leu 
Gly 



20 



25 



30 



35 



hOP-1 
mOP-1 
h0P-2 
mOP-2 
DPP 



Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 



• • • • < 



► * • • • 



• • • • • • 



• * * • • • Hi s 



Ser 

• • • 

Lys ... Pro 



• • • • • * 



WO 94/03200 



PCT/US93/07231 



- 50 - 



Vgl 

Vgr-1 

CBHF-2A 

CBKP-2B 

BHF3 

GDF-1 

60A 

BHP5 

BHP6 



Asn 
Asn 
Phe 
Phe 

• • • 

Asn 
Phe 
Phe 
Asn 



Tyr 
Asp 
His 
His 
Ser 
Gin 
Ser 
Asp 
Asp 



Glu 
Asp 
Ala 
Gin 



10 



30 



Pro 
Ser 
Pro 
Pro 
Gin 

Asn 
Ser 
Ser 
35 



15 



20 



25 



hOP-1 

mOP-1 

hOP-2 

m0P-2 

DPP 

Vgl 

Vgr-1 

CBMP-2A 

CBMP-2B 

GDF-1 

BHP3 

60A 

BHP5 

BMP 6 



Phe Pro Leu Asn Ser Tyr Met Asn Ala 



Tyr 



Leu 



Val 
Met 



Asp 
Asp 
Ala 
Thr 

• • * 

Ala 
Ala 
Ala 
Pro 



Asp 
Glu 
Ala 
Asp 
Asp 
Leu 
Lys 
Ala 
Ala 
Ala 
40 



Cys 
Cys 
His 
He 
His 
His 
His 
Ser 
Ser 
His 
His 
His 



Phe 
Leu 

• • • 

Leu 
Leu 
Gly 
Leu 

• • • - 
Met 
Met 



Ser** 
Lys 



Ser 
Gly 

• • • 

Ser 
Ser 

• • • 

Pro 



30 



35 



hOP-1 

dOP-1 

h0P-2 

mOP-2 

DPP 

Vgl 

Vgr-1 

CBMP-2A 



Thr Asn His Ala He Val Gin Thr Leu 



Ser 



Val 



Leu 
Leu 

• » • 

Leu 



Ser 
Ser 



WO 94/03200 * PCT/US93/07231 



- 51 - 



CBHP-2B 

BMP3 

GDF-1 

60A 

BMP 5 

BHP6 



i • * ••• • • • 



Ser 
Leu 



Thr lie ... Ser lie 
Val Leu Arg Ala 



• • . • • » 



• • « . • # • < 



45 



50 



25 



nur- i 


Vol 


Hi c; 


Phe 


lie 


Asn 


Pro 


Glu 


Thr 


Val 


■nflD 1 

mur-i 


... 








... 




Asp 






nOF-2 


• . * 


nis 


Leu 


Mot- 

net. 


T vc 




Asn 


Ala 




mOP-2 




HIS 


Leu 


Mot- 


T vc 




Act) 


Val 




DFF 


• • • 


Asn 


Asn 


Asn 






Glv 


Lys 




Vgl 






Del 




Glu 






Asd 


He 


vgr-i 


* • • 


• • • 


Val 


Met 








Tyr 




CBnr-zA 


• . . 


Asn 


Cor 


Val 




0G A. 




Lys 


He 


CBMP-2B 




Asn 


Ser 


val 


• • • 


Car 

aer 




Del 


lie 


BMP 3 


... 


Arg 


Ala** 


Gly 


Val 


Val 


Pro 


Gly 


He 


GDF-1 


Met 




Ala 


Ala 


Ala 




Gly 


Ala 


Ala 


60A 






Leu 


Leu 


Glu 




Lys 


Lys 




BMP5 






Leu 


Met 


Phe 




Asp 


His 




BMP 6 






Leu 


Met 








Tyr 








55 










60 






bOP-1 


Pro 


Lys 


Pro 


Cys 


Cys 


Ala 


Pro 


Thr 


Gin 


mOP-1 


• • » 


• • . 


... 


• . • 




... 






• . . 


hOP-2 


• • . 


* • • 


Ala 


• • • 




... 






Lys 


mOP-2 


• • • 


• « • 


Ala 


* . • 




• . • 






Lys 


DPP 


• • • 


* . • 


Ala 






Val 








Vgl 


• . • 


Leu 


• • • 


• • . 




Val 






Lys 


Vgr-1 


• • • 


* • • 


• • • 






... 






Lys 


CBMP-2A 


• • • 


.• • • 


Ala 






Val 






Glu 


CBMP-2B 


... 


• • . 


Ala 






Val 






Glu 



WO 94/03200 



PCT/US93/07231 



- 52 - 



20 



BHF3 


... 


Glu 


• • • 


... 




Val 




Glu 


Lys 


GDF-1 


Asp 


Leu 


... 


... 


... 


Val 


... 


Ala 


Arg 


60A 


• • . 


• • • 






... 


• • • 


... 


• • • 


Arg 


BMP5 


• • • 


• • • 






... 


* • • 


... 


• • • 


Lys 


BMP 6 




• . • 








• . • 


... 


• . • 


Lys 


• 






65 










70 






Lpti 


Asn 


Ala 


He 


Ser 


Val 


Leu 


Tyr 


Phe 


mOP-1 
















• . . 


... 






Ser 




Thr 








... 


Tyr 


mOP-2 




Ser 


... 


Thr 




... 




... 


Tyr 


Vgl 


Met 


Ser 


Pro 


... 




Met 




Phe 


Tyr 


Ver-1 


Val 






... 




... 




• • * 


... 


DPP 


• * • 


Asp 


Ser 


Val 


Ala 


Met 


... 


... 


Leu 


CBMP-2A 


... 


Ser 


» • • 


• • . 


• . . 


Met 


• • • 


• • • 


Leu 


CBMP-2B 


• • • 


Ser 


• . • 


... 


• • • 


Met 




... 


Leu 


BHP3 


Met 


Ser 


Ser 


Leu 


... 


He 


. * • 


Phe 


Tyr 


GDF-1 


• • • 


Ser 


Pro 


... 


... 


• . * 


... 


Phe 


. * . 


60A 




Gly 


• « • 


Leu 


Pro 


... 




• . • 


His 


BMP5 








... 


... 


... 




• • . 


... 


BMP 6 


• • • 


• • • 


. « • 


... 


• • . 


• • • 


... 


• . . 


... 










75 










80 


hOP-1 


Asp 


Asp 


Ser 


Ser 


Asn 


Val 


He 


Leu 


Lys 


mOP-1 

WIS* — J. 














... 






hOP-2 


• • » 


Ser 


... 


Asn 


• . • 




... 




Arg 


mOP-2 


♦ • • 


Ser 


. • • 


Asn 


• • • 




• * • 




Arg 


DPP 


Asn 


... 


Gin 


* • * 


Thr 




Val 




... 


Vgl 


• • • 


Asn 


Asn 


Asp 


... 




Val 




Arg 


Vgr-1 


• • • 


• • • 


Asn 


... 


• . • 




... 




• . * 


CBMP-2A 




Glu 


Asn 


Glu 


Lys 




Val 




... 


CBMP-2B 




Glu 


Tyr 


Asp 


Lys 




Val 




■ . • 


BHP3 


• • • 


Glu 


Asn 


Lys 


• • • 




Val 




• • • 



WO 94/03200 



PCT/US93/07231 



- 53 - 



GDF-1 
60A 
BMP 5 
BHP6 



Asn . . . Asp 

Leu Asn Asp Glu 

• • • • • • • 

• • • • • • Asn • • • 



Val 
Asn 



85 



Arg 



10 



15 



20 



hOP-1 

mOP-1 

hOP-2 

mOP-2 

DPP 

Vgl 

Vgr-1 

CBMP-2A 

CBMP-2B 

BMP3 

GDF-1 

60A 

BMP 5 

BMP 6 



Lys Tyr Arg Asn Met Val Val Arg 



Asn 
His 

* • • 

Asn 
Asn 
Val 
Gin 



His 
His 



Gin 
Glu 



Glu 



Gin Asp 

Gin Glu 
Pro 

Glu Asp 



Trp 



Thr 
Ala 



90 



Thr 



He 



95 



Lys 
Lys 
Val 
Asp 

Glu 
Glu 
Glu 
Asp 
Lys 



25 



30 



35 



hOP-1 

mOP-1 

h0P-2 

m0P-2 

DPP 

Vgl 

Vgr-1 

CBMP-2A 

CBHP-2B 

BHP3 

GDF-1 



Ala Cys Gly Cys His 



Gly 
Glu 

• • • 

Gly 
Gly 
Ser 
Glu 



Ala 



Arg 
Arg 

♦ * • 

Arg 
Arg 
Arg 
Arg 



WO 94/03200 PCT/US93/07231 



10 



- 54 - 

6 OA Sex 

BHP5 Sex ••• ••• ••• 

6HP6 ••• • • • ••■ 

100 

★★Between residues 56 and 57 of BHF3 is a Val residue; 

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



As is apparent from the foregoing amino acid 
sequence comparisons, significant amino acid changes 
can be made within the generic sequences while 
retaining the morphogenic activity. For example, while 

15 the GDF-1 protein sequence depicted in Table II shares 
only about 50% amino acid identity with the hOPl 
sequence described therein, the GDF-1 sequence shares 
greater than 70% amino acid sequence homology (or 
"similarity") with the hOPl sequence, where "homology" 

20 or "similarity" includes allowed conservative amino 
acid changes within the sequence as defined by Dayoff , 
et al., Atlas of Protein Sequence and Structure vol.5, 
supp.3, pp. 345-362, (M.O. Dayoff, ed., Nat'l BioMed. 
Res. Fd'n, Washington D.C. 1979.) 

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 OP-1 and OP-2 proteins, including the 
Drosophila 60A protein. Accordingly, in still another 

35 preferred aspect, the invention includes morphogens 



WO 94/03200 



PCT/US93/07231 



- 55 - 

comprising species of polypeptide chains having the 
generic amino acid sequence referred to herein as 
"OPX", which defines the seven cysteine skeleton and 
accommodates the identities between the various 
5 identified mouse and human OPl and OP2 proteins. OPX 
is presented in Seq. ID No. 29. As described therein, 
each Xaa at a given position independently is selected 
from the residues occurring at the corresponding 
position in the C-terminal sequence of mouse or human 
10 OPl or OP2 (see Seq. ID Nos. 5-8 and/or Seq. ID Nos. 
16-23). 

II. Formulations and Methods for Administering 
Therapeutic Agents 

15 

The morphogens may be provided to an individual by 
any suitable means, preferably directly (e.g., locally, 
as by injection to a nerve tissue locus) or 
systemically (e.g., parenterally or orally). Where the 

20 morphogen is to be provided parenterally, such as by 
intravenous , subcutaneous , intramuscular , intraorbital , 
ophthalmic , intraventricular , intracranial , 
intracapsular, intraspinal , intracisternal , 
intraperitoneal, buccal, rectal, vaginal, intranasal or 

25 by aerosol administration, the morphogen preferably 

comprises part of an aqueous solution. The solution is 
physiologically acceptable so that in addition to 
delivery of the desired morphogen to the patient, the 
solution does not otherwise adversely affect the 

30 patient's electrolyte and volume balance. The aqueous 
medium for the morphogen thus may comprise normal 
physiologic saline (9.85% NaCl, 0.15M), pH 7-7.4. The 
aqueous solution containing the morphogen can be made, 
for example, by dissolving the protein in 50% ethanol 

35 containing acetonitrile in 0.1% trif luoroacetic acid 



WO 94/03200 



PCT/US93/07231 



- 56 - 

(TFA) or 0.1% HC1, or equivalent solvents. One volume 
of the resultant solution then is added, for example, 
to ten volumes of phosphate buffered saline (PBS), 
which further may include 0.1-0.2% human serum albumin 
5 (HSA). The resultant solution preferably is vortexed 
extensively. If desired, a given morphogen may be made 
more soluble by association with a suitable molecule. 
For example, association of the mature dimer with the 
pro domain of the morphogen increases solubility of the 

10 protein significantly (see Section II. 1, below). In 
fact, the endogenous protein is thought to be 
transported in this form. Another molecule capable of 
enhancing solubility and particularly useful for oral 
administrations, is casein. For example, addition of 

15 0.2% casein increases solubility of the mature active 
form of OP-1 by 80%. Other components found in milk 
and/or various serum proteins also may be useful. 

Useful solutions for parenteral administration may 

20 be prepared by any of the methods well known in the 
pharmaceutical art, described, for example, in 
Remington's Pharmaceutical Sciences (Gennaro, A., ed.), 
Mack Pub., 1990. Formulations may include, for 
example, polyalkylene glycols such as polyethylene 

25 glycol, oils of vegetable origin, hydrogenated 

naphthalenes, and the like. Formulations for direct 
administration, in particular, may include glycerol and 
other compositions of high viscosity. Biocompatible, 
preferably bioresorbable, polymers, including, for 

30 example, hyaluronic acid, collagen, polybutyrate , 
tricalcium phosphate, lactide and lactide/glycolide 
copolymers, may be useful excipients to control the 
release of the morphogen in vivo * Other potentially 
useful parenteral delivery systems for these morphogens 

35 include ethylene-vinyl acetate copolymer particles, 



WO 94/03200 • PCT/US93/07231 



- 57 - 



osmotic pumps, implantable infusion systems, and 
liposomes. Formulations for inhalation administration 
contain as excipients, for example, lactose, or may be 
aqueous solutions containing, for example, 
5 polyoxyethylene-9-lauryl ether, glycocholate and 

deoxycholate, or oily solutions for administration in 
the form of nasal drops, or as a gel to be applied 
intranasally. Formulations for parenteral 
administration may also include glycocholate for buccal 
10 administration, methoxysalicylate for rectal 
administration, or cutric acid for vaginal 
administration* 

Alternatively, the morphogens described herein may 

15 be administered orally. Oral administration of 

proteins as therapeutics generally is not practiced as 
most proteins are readily degraded by digestive enzymes 
and acids in the mammalian digestive system before they 
can be absorbed into the bloodstream. However, the 

20 morphogens described herein typically are acid stable 
and protease-resistant (see, for example, U.S. Pat. No. 
4,968,590.) In addition, at least one morphogen, OP-1, 
has been identified in mammary gland extract, colostrum 
and 57-day milk. Moreover, the OP-1 purified from 

25 mammary gland extract is morphogenically active. 

Specifically, this protein induces endochondral bone 
formation in mammals when implanted subcutaneous ly in 
association with a suitable matrix material, using a 
standard in vivo bone assay, such as is disclosed in 

30 U.S. Pat. No. 4,968,590. Moreover, the morphogen also 
is detected in the bloodstream (see Example 9, below). 
Finally, soluble form morphogen, e.g., mature morphogen 
associated with the pro domain, is capable of 
maintaining neural pathways in a mammal (See Examples 4 

35 and 6 below). These findings indicate that oral and 



WO 94/03200 



« 



PCT/US93/07231 



- 58 - 

parenteral administration are viable means for 
administering morphogens to an individual. In 
addition, while the mature forms of certain morphogens 
described herein typically are sparingly soluble, the 
5 morphogen form found in milk (and mammary gland extract 
and colostrum) is readily soluble, probably by 
association of the mature, morphogenically active form 
with part or all of the pro domain of the intact 
sequence and/or by association with one or more milk 
10 components. Accordingly, the compounds provided herein 
also may be associated with molecules capable of 
enhancing their solubility in vitro or in vivo . 

The compounds provided herein also may be 
15 associated with molecules capable of targeting the 
morphogen or morphogen-stimulating agent to nerve 
tissue. For example, an antibody, antibody fragment, 
or other binding protein that interacts specifically 
with a surface molecule on nerve tissue cells, 
20 including neuronal or glial cells, may be used. Useful 
targeting molecules may be designed, for example, using 
the single chain binding site technology disclosed, for 
example, in U.S. Pat. No. 5,091,513. 

25 As described above, the morphogens provided herein 

share significant sequence homology in the C- terminal 
active domains. By contrast, the sequences typically 
diverge significantly in the sequences which define the 
pro domain. Accordingly, the pro domain is thought to 

30 be morphogen-specif ic. As described above, it is also 
known that the various morphogens identified to date 
are differentially expressed in the different tissues. 
Accordingly, without being limited to any given theory, 
it is likely that, under natural conditions in the 

35 body, selected morphogens typically act on a given 



WO 94/03200 



PCT/US93/07231 



- 59 - 

tissue. Accordingly , part or all of the pro domains 
which have been identified associated with the active 
form of the morphogen in solution, may serve as 
targeting molecules for the morphogens described 
5 herein. For example, the pro domains may interact 
specifically with one or more molecules at the target 
tissue to direct the morphogen associated with the pro 
domain to that tissue. Accordingly, another useful 
targeting molecule for targeting morphogen to nerve 
10 tissue is part or all of a morphogen pro domain, 

particularly part or all of the pro domains of OP-1 or 
GDF-1, both of which proteins are found naturally 
associated with nerve tissue. 

15 Finally, the morphogens or morphogen-stimulating 

agents provided herein may be administered alone or in 
combination with other molecules known to be beneficial 
in maintaining neural pathways, including nerve growth 
factors and anti-inflammatory agents. 

20 

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

30 

The compositions can be formulated for parenteral 
or oral administration to humans or other mammals in 
therapeutically effective amounts, e.g., amounts which 
provide appropriate concentrations for a time 
35 sufficient to eliminate or reduce the patient's 



WO 94/03200 PCT/US93/07231 



- 60 - 

pathological condition , to provide therapy for the 
neurological diseases and disorders described above , 
and amounts effective to enhance neural cell survival 
an/or to protect neurons and neural pathways in 
5 anticipation of injury to nerve tissue. 

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

10 number of factors/ including the dosage of the drug to 
be administered, the chemical characteristics (e.g., 
hydrophobicity ) of the compounds employed, and the 
route of administration. The preferred dosage of drug 
to be administered also is likely to depend on such 

15 variables as the type and extent of progression of the 
neurological disease, the overall health status of the 
particular patient, the relative biological efficacy of 
the compound selected, the formulation of the compound 
excipients, and its route of administration. In general 

20 terms, the compounds of this invention may be provided 
in an aqueous physiological buffer solution containing 
about 0.1 to 10% w/v compound for parenteral 
administration. Typical dose ranges are from about 10 
ng/kg to about 1 g/kg of body weight per day; a 

25 preferred dose range is from about 0.1 /jg/kg to 
100 mg/kg of body weight per day. Optimally, the 
morphogen dosage given in all cases is between 2-20 pg 
of protein per kilogram weight of the patient per day. 
No obvious OP-1 induced pathological lesions are 

30 induced when mature morphogen (e.g., OP-1, 20 yq) is 
administered daily to normal growing rats for 



WO 94/03200 * PCT/US93/07231 



- 61 - 

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

5 

Since the ability of proteins and protein fragments 
to penetrate the blood-brain barrier may be related to 
their size, lipophilicity or their net ionic charge, 
suitable modifications of the morphogens may be 

10 formulated (e.g., by substituting 

pentaf luorophenylalanine for phenylalanine, or by 
conjugation to a cationized protein such as albumin) to 
increase their transportability across the barrier, 
using standard methodologies known in the art. See, 

15 for example, Kastin et al., Pharmac. Biochem. Behav. 
(1979) 11:713-716; Rapoport et al.. Science (1980) 
207:84-86; Pardridge et al., (1987) Biochem. Biophys. 
Res. Commun. 146 :307-313; Riekkinen et al.,(1987) 
Peptides 8: 261-:265. The efficacy of these functional 

20 analogs may be assessed for example, by evaluating the 
ability of these compounds to induce redif f erentiation 
of transformed cells, or enhance survival of neurons at 
risk of dying, as described in the Examples provided 
herein. 

25 

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



WO 94/03200 



PCT/US93/07231 



- 62 - 

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

administered prophylactically in a surgical setting. 
Optimally, the morphogen dosage given in all cases is 
between 2-20 /jg of protein per kilogram weight of the 
patient • 

10 

Alternatively, an effective amount of an agent 
capable of stimulating endogenous morphogen levels may 
be administered by any of the routes described above. 
For example, an agent capable of stimulating morphogen 

15 production and/or secretion from nerve tissue cells may 
be provided to a mammal, e.g., by direct administration 
of the morphogen to glial cells associated with the 
nerve tissue to be treated. A method for identifying 
and testing agents capable of modulating the levels of 

20 endogenous morphogens in a given tissue is described 
generally herein in Example 13, and in detail in 
internatinal application US92/07359 (WO93/015172 ) , the 
disclosure of which is incorporated herein by 
reference. Briefly, candidate compounds can be 

25 identified and tested by incubating the compound in 
vitro with a test tissue or cells thereof, for a time 
sufficient to allow the compound to affect the 
production, i.e., the expression and/or secretion, of a 
morphogen produced by the cells of that tissue. Here, 

30 suitable tissue or cultured cells of a tissue 

preferably would comprise neurons and/or glial cells. 
For example, suitable tissue for testing may include 
cultured cells isolated from the substantia nigra, 
adendema glial cells, and the like. 



35 



WO 94/03200 



PCI7US93/07231 



A currently preferred detection means for 
evaluating the level of the morphogen in culture upon 
exposure to the candidate compound comprises an 
immunoassay utilizing an antibody or other suitable 
5 binding protein capable of reacting specifically with a 
morphogen and being detected as part of a complex with 
the morphogen. Immunoassays may be performed using 
standard techniques known in the art and antibodies 
raised against a morphogen and specific for that 

10 morphogen. As described herein, morphogens may be 
isolated from natural-sourced material or they may be 
recombinantly produced. Agents capable of stimulating 
endogenous morphogens then may formulated into 
pharmaceutical preparations and administered as 

15 described herein. 

Where the morphogen is to be provided to a site to 
stimulate axon regeneration, the morphogen preferably 
is provided to the site in association with a 

20 biocompatible, preferably bioresorbable carrier 

suitable for maintaining a protein at a site in vivo , 
and through which a neural process may regenerate. A 
currently preferred carrier also comprises sufficient 
structure to assist direction of axonal growth. 

25 Currently preferred carriers include structural 

molecules such as collagen, hyaluronic acid or laminin, 
and/or synthetic polymers or copolymers of, for 
example, polylactic acid, polyglycolic acid or 
polybutyric acid. Currently most preferred are 

30 carriers comprising tissue extracellular matrix. These 
may be obtained commercially. In addition, a brain 



WO 94/03200 



PCT/US93/07231 



- 64 - 

tissue-derived extracellular matrix may be prepared as 
described in international application US92/01968 
(W092/15323) , incorporated hereinabove by reference, 
and/or by other means known in the art. 

5 

The currently preferred means for repairing breaks 
in neural pathways , particularly pathways of the * 
peripheral nervous system, include providing the 
morphogen to the site as part of a device that includes 

10 a biocompatible membrane or casing of a dimension 
sufficient to span the break and having openings 
adapted to receive severed nerve ends. The morphogen is 
disposed * within the casing, preferably dispersed 
throughout a suitable carrier, and is accessible to the 

15 severed nerve ends. Alternatively, the morphogen may 
be adsorbed onto the interior surface of the casing, or 
otherwise associated therewith. In addition, currently 
preferred casings have an impermeable exterior surface. 
The casing acts as a nerve guidance channel, aiding in 

20 directing axonal growth. In addition, the casing also 
protects the damaged nerve from immunologically-related 
agents which may assist in scar tissue formation. 
Suitable channel or casing materials include silicone 
or bioresorbable materials such as collagen, hyaluronic 

25 acid, laminin, polylactic acid, polyglycolic acid, 

polybutyric acid and the like. Additionally, although 
the nerve guidance channels described herein generally 
are tubular in shape, it should be evident to those 
skilled in the art that various alternative shapes may 

30 be employed. The lumen of the guidance channels may, 
for example, be oval or even square in cross section. 



WO 94/03200 



PCT/US93/07231 



- 65 - 

Moreover the guidance channels may be constructed of 
two or more parts which may be clamped together to 
secure the nerve stumps. Nerve endings may be secured 
to the nerve guidance channels by means of sutures, 
5 biocompatible adhesives such as fibrin glue, or other 
means known in the medical art. 

II. 1 Soluble Morphoqen Complexes 

10 A currently preferred form of the morphogen useful 

in therapeutic formulations, having improved solubility 
in aqueous solutions and consisting essentially of 
amino acids, is a dimeric morphogenic protein 
comprising at least the 100 amino acid peptide sequence 

15 having the pattern of seven or more cysteine residues 
characteristic of the morphogen family complexed with a 
peptide comprising part or all of a pro region of a 
member of the morphogen family, or an allelic, species 
or other sequence variant thereof. Preferably, the 

20 dimeric morphogenic protein is complexed with two 
peptides. Also, the dimeric morphogenic protein 
preferably is noncovalently complexed with the pro 
region peptide or peptides. The pro region peptides 
also preferably comprise at least the N-terminal 

25 eighteen amino acids that define a given morphogen 
pro region. In a most preferred embodiment, peptides 
defining substantially the full length pro region are 
used. 

30 Other soluble forms of morphogens include dimers of 

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



WO 94/03200 



PCT/US93/07231 



- 66 - 

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

5 

As described above, useful pro domains include the 
full length pro regions, as well as various truncated 
forms hereof, particularly truncated forms cleaved at 
proteolytic Arg-Xaa-Xaa-Arg cleavage sites. For 

10 example, in OP-1, possible pro sequences include 
sequences defined by residues 30-292 (full length 
form); 48-292; and 158-292. Soluble OP-1 complex 
stability is enhanced when the pro region comprises the 
full length form rather than a truncated form, such as 

15 the 48-292 truncated form, in that residues 30-47 show 
sequence homology to the N-terminal portions of other 
morphogens, and are believed to have particular utility 
in enhancing complex stability for all morphogens. 
Accordingly, currently preferred pro sequences are 

20 those encoding the full length form of the pro region 
for a given morphogen. Other pro sequences 
contemplated to have utility include biosynthetic pro 
sequences, particularly those that incorporate a 
sequence derived from the N-terminal portion of one or 

25 more morphogen pro sequences. 

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

35 



WO 94/03200 



PCT/US93/07231 



- 67 - 

In another preferred aspect , useful pro region 
peptides include polypeptide chains comprising an amino 
acid sequence encoded by a nucleic acid that hybridizes 
under stringent conditions with a DNA or RNA sequence 
5 encoding at least the N-terminal eighteen amino acids 
of the pro region sequence for OP1 or OP2, e.g., 
nucleotides 136-192 and 152-211 of Seq. ID No. 16* and 
20, respectively. 

10 A. Isolation of Soluble morphogen complex from 

conditioned media or body fluid 

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

15 disassociated during purification, generally by 

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

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

25 morphogen complexes in substantially pure form. 

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

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

35 purification of a variety of morphogens, all of which 



WO 94/03200 



PCT/US93/07231 



- 68 - 

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

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

In this experiment OP-1 was expressed in mammalian 

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

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

25 from the bulk of the contaminating serum proteins that 
elute in the flow through and 35 mM imidazole wash 
fractions. The Zn-IMAC purified soluble OP-1 is next 
applied to an S-Sepharose cation-exchange column 
equilibrated in 20 mM NaP0 4 (pH 7.0) with 50 mM NaCl. 

30 This S-Sepharose step serves to further purify and 

concentrate the soluble OP-1 complex in preparation for 
the following gel filtration step. The protein was 



WO 94/03200 



PCT/US93/07231 



applied to a Sephacryl S-200HR column equilibrated in 
TBS. Using substantially the same protocol, soluble 
morphogens also may be isolated from one or more body 
fluids, including serum, cerebro-spinal fluid or 
5 peritoneal fluid. 

IMAC was performed using Chelating- Sepharose 
(Pharmacia) that had been charged with three column 
volumes of 0.2 M ZnSO^. The conditioned media was 

10 titrated to pH 7.0 and applied directly to the ZN-IMAC 
resin equilibrated in 20 mM HEPES (pH 7.0) with 500 mM 
NaCl. The Zn-IMAC resin was loaded with 80 mL of 
starting conditioned media per mL of resin. After 
loading, the column was washed with equilibration 

15 buffer and most of the contaminating proteins were 

eluted with 35 mM imidazole (pH 7.0) in equilibration 
buffer. The soluble OP-1 complex then is eluted with 
50 mM imidazole (pH 8.0) in 20 mM HEPES and 500 mM 
NaCl. 

20 

The 50 mM imidazole eluate containing the soluble 
OP-1 complex was diluted with nine volumes of 20 mM 
NaP0 4 (pH 7.0) and applied to an S- Sepharose 
(Pharmacia) column equilibrated in 20 mM NaP0 4 (pH 7.0) 

25 with 50 mM NaCl. The S-Sepharose resin was loaded with 
an equivalent of 800 mL of starting conditioned media 
per mL of resin. After loading the S-Sepharose column 
was washed with equilibration buffer and eluted with 
100 mM NaCl followed by 300 mM and 500 mM NaCl in 20 mM 

30 NaP0 4 (pH 7.0). The 300 mM NaCl pool was further 
purified using gel filtration chromatography. Fifty 
mis of the 300 mm NaCl eluate was applied to a 5.0 X 90 
cm Sephacryl S-200HR (Pharmacia) equilibrated in Tris 
buffered saline (TBS), 50 mM Tris, 150 mM NaCl 

35 (pH 7.4). The column was eluted at a flow rate of 5 



WO 94/03200 PCT/US93/07231 



- 70 - 

mL/minute collecting 10 mL fractions. The apparent 
molecular of the soluble OP-1 was determined by 
comparison to protein molecular weight standards 
(alcohol dehydrogenase (ADH, 150 kDa), bovine serum 
5 albumin (BSA, 68 kDa), carbonic anhydrase (CA, 30 kDa) 
and cytochrome C (cyt C, 12.5 kDa). The purity of the 
S-200 column fractions was determined by separation on 
standard 15% polyacrylamide SDS gels stained with 
coomassie blue. The identity of the mature OP-1 and 
10 the pro-domain was determined by N- terminal sequence 
analysis after separation of the mature OP-1 from the 
pro-domain using standard reverse phase C18 HPLC. 

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

The complex components can be verified by running 
the complex-containing fraction from the S-200 or S- 
200HR columns over a reverse phase C18 HPLC column and 

25 eluting in an acetonitrile gradient (in 0.1% TFA), 

using standard procedures. The complex is dissociated 
by this step, and the pro domain and mature species 
elute as separate species. These separate species then 
can be subjected to N-terminal sequencing using 

30 standard procedures (see, for example, Guide to 
Protein Purification , M. Deutscher, ed., Academic 
Press, San Diego, 1990, particularly pp. 602-613), and 
the identity of the isolated 36kD, 39kDa proteins 
confirmed as mature morphogen and isolated, cleaved pro 

35 domain, respectively. N-terminal sequencing of the 



WO 94/03200 



PCT/US93/07231 



- 71 - 

isolated pro domain from mammalian cell produced OP-1 
revealed 2 forms of the pro region, the intact form 
(beginning at residue 30 of Seq. ID No. 16) and a 
truncated form, (beginning at residue 48 of Seq* ID No. 
5 16.) N-terminal sequencing of the polypeptide subunit 
of the isolated mature species reveals a range of 
N- termini for the mature sequence, beginning at 
residues 293, 300, 313, 315, 316, and 318, of Seq. ID 
No. 16, all of which are active as demonstrated by the 
10 standard bone induction assay. 

B. In Vitro Soluble Morphogen Complex Formation 

As an alternative to purifying soluble complexes 

15 from culture media or a body fluid, soluble complexes 
may be formulated from purified pro domains and mature 
dimeric species. Successful complex formation 
apparently requires association of the components under 
denaturing conditions sufficient to relax the folded 

20 structure of these molecules, without affecting 

disulfide bonds. Preferably, the denaturing conditions 
mimic the environment of an intracellular vesicle 
sufficiently such that the cleaved pro domain has an 
opportunity to associate with the mature dimeric 

25 species under relaxed folding conditions. The 

concentration of denaturant in the solution then is 
decreased in a controlled, preferably step-wise manner, 
so as to allow proper refolding of the dimer and pro 
regions while maintaining the association of the pro 

30 domain with the dimer. Useful denaturants include 4-6M 
urea or guanidine hydrochloride (GuHCl), in buffered 
solutions of pH'4-10, preferably pH 6-8. The soluble 
complex then is formed by controlled dialysis or 
dilution into a solution having a final denaturant 

35 concentration of less than 0.1-2M urea or GuHCl, 



WO 94/03200 



PCT/US93/07231 



- 72 - 

preferably 1-2 M urea of GuHCl, which then preferably 
can be diluted into a physiological buffer. Protein 
purif ication/renaturing procedures and considerations 
are well described in the art, and details for 
5 developing a suitable renaturing protocol readily can 
be determined by one having ordinary skill in the art. 
One useful text one the subject is Guide to Protein 
Purification , M. Deutscher, ed., Academic Press, San 
Diego, 1990, particularly section V. Complex formation 
10 also may be aided by addition of one or more chaperone 
proteins . 

C. Stability of Soluble Morphoqen Complexes 

15 The stability of the highly purified soluble 

morphogen complex in a physiological buffer, e.g., 
tris-buf fered saline (TBS) and phosphate-buffered 
saline (PBS), can be enhanced by any of a number of 
means. Currently preferred is by means of a pro region 

20 that comprises at least the first 18 amino acids of the 
pro sequence (e.g., residues 30-47 of Seq. ID NO. 16 
for OP-1), and preferably is the full length pro 
region. Residues 30-47 show sequence homology to the 
N- terminal portion of other morphogens and are believed 

25 to have particular utility in enhancing complex 

stability for all morphogens. Other useful means for 
enhancing the stability of soluble morphogen complexes 
include three classes of additives. These additives 
include basic amino acids (e.g., L-arginine, lysine and 

30 betaine); nonionic detergents (e.g., Tween 80 or 
Nonldet P-120); and carrier proteins (e.g., serum 



WO 94/03200 



PCT/US93/07231 



- 73 - 

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

III. Examples 

10 Example 1. Identification of Morphoqen-Expressing 
Tissue 

Determining the tissue distribution of morphogens 
may be used to identify different morphogens expressed 

15 in a given tissue, as well as to identify new, related 
morphogens. Tissue distribution also may be used to 
identify useful morphogen-producing tissue for use in 
screening and identifying candidate morphogen- 
stimulating agents. The morphogens (or their roRNA 

20 transcripts) readily are identified in different 
tissues using standard methodologies and minor 
modifications thereof in tissues where expression may 
be low. For example, protein distribution may be 
determined using standard Western blot analysis or 

25 immunof luorescent techniques, and antibodies specific 
to the morphogen or morphogens of interest. Similarly, 
the distribution of morphogen transcripts may be 
determined using standard Northern hybridization 
protocols and transcript-specific probes. 

30 

Any probe capable of hybridizing specifically to a 
transcript, and distinguishing the transcript of 
interest from other, related transcripts may be used. 
Because the morphogens described herein share such high 
35 sequence homology in their active, C-terminal domains, 



WO 94/03200 



PCT/US93/07231 



- 74 - 

the tissue distribution of a specific morphogen 
transcript may best be determined using a probe 
specific for the pro region of the immature protein 
and/or the N- terminal region of the mature protein. 
5 Another useful sequence is the 3' non-coding region 
flanking and immediately following the stop codon. 
These portions of the sequence vary substantially among 
the morphogens of this invention, and accordingly, are 
specific for each protein. For example, a particularly 

10 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 and the N-tenninus of the 
mature sequence (see Lyons et al. (1989) PNA S 86:4554- 
4558 for a description of the cDNA sequence). 

15 Similarly, particularly useful mOP-l-specif ic probe 
sequences are the BstXl-Bgll fragment, a 0.68 Kb 
sequence that covers approximately two-thirds of the 
mOP-1 pro region; a StuI-StuI fragment, a 0.2 Kb 
sequence immediately upstream of the 7-cysteine domain; 

20 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 
essentially by residues 30-291.) Similar approaches 
may be used, for example, with hOP-1 (Seq. ID No. 16) 

25 or human or mouse OP- 2 (Seq. ID Nos. 20 and 22.) 

Using these morphogen- specif ic probes, which 
may be synthetically engineered or obtained from cloned 
sequences, morphogen transcripts can be identified in 

30 mammalian tissue, using standard methodologies well 
known to those having ordinary skill in the art. 
Briefly, total RNA is prepared from various adult 
murine tissues (e.g., liver, kidney, testis, heart, 
brain, thymus and stomach) by a standard methodology 

35 such as by the method of Chomczyaski et al. ((1987) 



WO 94/03200 



PCT/US93/07231 



- 75 - 

Anal* Biochem 162:156-159) and described below. Poly 
(A)+ RNA is prepared by using oligo (dT) -cellulose 
chromatography (e.g., Type 7, from Pharmacia LKB 
Biotechnology, Inc.). Poly (A)+ RNA (generally 15 pg) 
5 from each tissue is fractionated on a 1% 

agarose/f ormaldehyde gel and transferred onto a Nytran 
membrane (Schleicher & Schuell). Following the 
transfer, the membrane is baked at 80°C and the RNA is 
cross-linked under UV light (generally 30 seconds at 1 

10 mW/cm 2 ) . Prior to hybridization, the appropriate probe 
is denatured by heating. The hybridization is carried 
out in a lucite cylinder rotating in a roller bottle 
apparatus at approximately 1 rev/min for approximately 
15 hours at 37°C using a hybridization mix of 40% 

15 formamide, 5 x Denhardts, 5 x SSPE, and 0.1% SDS. 
Following hybridization, the non-specific counts are 
washed off the filters in 0.1 x SSPE, 0.1% SDS at 50°C. 

Examples demonstrating the tissue distribution of 

20 various morphogens, including Vgr-1, OP-1, BMP2, BMP3, 
BMP4, BMP 5, GDF-1, and OP-2 in developing and adult 
tissue are disclosed in international application 
US92/0196B (W092/15323), and in Ozkaynak, et al., 
(1991) Biochem. Biophys. Res. Commn * 179 : 116-123, and 

25 Ozkaynak, et al. (1992) J. Biol.Chem. 267: 25220-25227. 
Using the general probing methodology described herein, 
northern blot hybridizations using probes specific for 
these morphogens to probe brain, spleen, lung, heart, 
liver and kidney tissue indicate that kidney- related 

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

35 and heart tissue, while the liver appears to be a 



WO 94/03200 



PCT/US93/07231 



- 76 - 

seen in kidney and heart tissue, while the liver 
appears to be a secondary expression source for BMP5 f 
and the spleen appears to be a secondary expression 
source for BMP4. GDF-1 appears to be expressed 
5 primarily in brain tissue. To date, OP-2 appears to be 
expressed primarily in early embryonic tissue. 
Specifically, northern blots of murine embryos and 6- 
day post-natal animals shows abundant 0P2 expression in 
8-day embryos. Expression is reduced significantly in 
10 17 -day embryos and is not detected in post-natal 
animals. 

Example 2. Morphoqen Localization in the Nervous 
System 

15 

Morphogens have been identified in developing and 
adult rat brain and spinal cord tissue, as determined 
both by northern blot hybridization of morphogen- 
specif ic probes to mRNA extracts from developing and 

20 adult nerve tissue (see Example 1, above) and by 

immunolocalization studies. For example, northern blot 
analysis of developing rat tissue has identified 
significant OP-1 mRNA transcript expression in the CNS 
international application US92/01968 (W092/15323 ) , and 

25 Ozkaynak et al. (1991) Biochem. Biophys. Res. Comm. , 
129:11623 and Ozkaynak, et al. (1992) J. Biol. Chem. 
267:25220-25227. GDF-1 mRNA appears to be expressed 
primarily in developing and adult nerve tissue, 
specifically in the brain, including the cerebellum and 

30 brain stem, spinal cord and peripheral nerves (Lee, S. 
(1991) PNAS 88: 4250-4254). BMP2B (also referred in 
the art as BMP 4 ) and Vgr-1 transcripts also have been 
reported to be expressed in nerve tissue (Jones et al. 
(1991) Development 111 :531-542), although the nerve 

35 tissue does not appear to be the primary expression 



WO 94/03200 



PCT/US93/07231 



- 77 - 

tissue for these genes (Ozkaynak, et al., (1992) 
Biol. Chem. 267:25220-25227. Specifically, CBMP2 
transcripts are reported in the region of the 
diencephalon associated with pituitary development, and 
5 Vgr-1 transcripts are reported in the anteroposterior 
axis of the CNS, including in the roof plate of the 
developing neural tube, as well as in the cells 
immediately adjacent the floor plate of the developing 
neural tube. In older rats, Vgr-1 transcripts are 
10 reported in developing hippocampus tissue. In 

addition, the genes encoding OP-1 and BMP 2 originally 
were identified by probing human hippocampus cDNA 
libraries. 

15 Immunolocalization studies, performed using 

standard methodologies known in the art and disclosed 
in international application US92/01968 (W092/15323) , 
the disclosure of which is incorporated herein, 
localized OP-1 expression to particular areas of 

20 developing and adult rat brain and spinal cord tissue. 
Specifically, OP-1 protein expression was assessed in 
adult (2-3 months old) and five or six-day old mouse 
embryonic nerve tissue, using standard morphogen- 
specific antisera, specifically, rabbit anti-OPl 

25 antisera, made using standard antibody protocols known 
in the art and preferably purified on an OP-1 affinity 
column. The antibody itself was labelled using 
standard fluorescent labelling techniques, or a 
labelled anti-rabbit IgG molecule was used to visualize 

30 bound OP-1 antibody. 

As can be seen in FIG 1A and IB, immunofluorescence 
staining demonstrates the presence of OP-1 in adult rat 
central nervous system (CNS.) Similar and extensive 
35 staining is seen in both the brain (1A) and spinal cord 



WO 94/03200 



PCT/US93/07231 



- 78 - 

(IB). OP-1 appears to be localized predominantly to 
the extracellular matrix of the grey matter (neuronal 
cell bodies ) , distinctly present in all areas except 
the cell bodies themselves. In white matter, formed 
5 mainly of myelinated nerve fibers, staining appears to 
be confined to astrocytes (glial cells). A similar 
staining pattern also was seen in newborn rat (10 day 
old) brain sections. 

10 In addition, OP-1 has been specifically localized 

in the substantia nigra, which is composed primarily of 
striatal basal ganglia, a system of accessory motor 
neurons that function is association with the cerebral 
cortex and corticospinal system* Dysfunctions in this 

15 subpopulation or system of neurons are associated with 
a number of neuropathies, including Huntington's chorea 
and Parkinson's disease. 

OP1 also has been localized at adendema glial 
20 cells, known to secrete factors into the cerebrospinal 
fluid, and which occur around the intraventricular 
valve, coroid fissure, and central canal of the brain 
in both developing and adult rat. 

25 Finally, morphogen inhibition in developing embryos 

inhibits nerve tissue development. Specifically, 9-day 
mouse embryo cells, cultured in vitro under standard 
culturing conditions, were incubated in the presence 
and absence of an OP-l-specif ic monoclonal antibody 

30 prepared using recombinantly produced, purified mature 
OP-1 and the immunogen. The antibody was prepared 
using standard antibody production means well known in 
the art and as described generally in Example 13. 
After two days, the effect of the antibody on the 

35 developing embryo was evaluated by histology. As 



WO 94/03200 



PCT/US93/07231 



- 79 - 

determined by histological examination the OP-1- 
specific antibody specifically inhibits eye lobe 
formation in the developing embryo. In particular, the 
diencephalon outgrowth does not develop. In addition, 
5 the heart is malformed and enlarged. Moreover, in 
separate immunolocalization studies on embryo sections 
with labelled OP-1 specific antibody, the OP-l-specific 
antibody localizes to neural epithelia. 

10 The endogenous morphogens which act on neuronal 

cells may be expressed and secreted by nerve tissue 
cells, e.g., by neurons and/or glial cells associated 
with the neurons, and/or they may be transported to the 
neurons by the cerebrospinal fluid and/or bloodstream. 

15 Recently, OP-1 has been identified in the human blood 
(See Example 9, below). In addition, transplanted 
Schwann cells recently have been shown to stimulate 
nerve fiber formation in rat spinal cord, including 
inducing vascularization and myelin sheath formation 

20 around at least some of the new neuronal processes 
(Bunge (1991) Exp. Neurology 114 :254-257.) The 
regenerative property of these cells may be mediated by 
the secretion of a morphogen by the Schwann cells. 

25 Example 3. Morphogen Enhancement of Neuronal Cell 

Survival 

The morphogens described herein enhance cell 
survival, particularly of neuronal cells at risk of 

30 dying. For example, fully differentiated neurons are 
non-mitotic and die in vitro when cultured under 
standard mammalian cell culture conditions, using a 
chemically defined or low serum medium known in the 
art, (see, for example, Charness (1986) J. Biol. Chem. 

35 26:3164-3169 and Freese et al. (1990) Brain Res. 



WO 94/03200 



PCT/US93/07231 



- 80 - 

521 :254-264. ) However, if a primary culture of non- 
mitotic neuronal cells is treated with a morphogen, the 
survival of these cells is enhanced significantly. For 
example, a primary culture of striatal basal ganglia 
5 isolated from the substantia nigra of adult rat brain 
was prepared using standard procedures, e.g., by 
dissociation by trituration with pasteur pipette of 
substania nigra tissue, using standard tissue culturing 
protocols, and grown in a low serum medium, e.g., 

10 containing 50% DMEM (Dulbecco's modified Eagle's 

medium), 50% F-12 medium, heat inactivated horse serum 
supplemented with penicillin/streptomycin and 4 g/1 
glucose. Under standard culture conditions, these 
cells are undergoing significant cell death by three 

15 weeks when cultured in a serum-free medium. Cell death 
is evidenced morphologically by the inability of cells 
to remain adherent and by changes in their 
ultrastructural characteristics, e.g., by chromatin 
clumping and organelle disintegration. 

20 

In this example, the cultured basal ganglia were 
were treated with chemically defined medium conditioned 
with 0.1-100 ng/ml OP-1. Fresh, morphogen-conditioned 
medium was provided to the cells every 3-4 days. Cell 

25 survival was enhanced significantly and was dose 
dependent upon the level of OP-1 added: cell death 
decreased significantly as the concentration of OP-1 
was increased in cell cultures. Specifically, cells 
remained adherent and continued to maintain the 

30 morphology of viable differentiated neurons. In the 
absence of morphogen treatment, the majority of the 
cultured cells dissociated and underwent cell necrosis. 



WO 94/03200 



PCT/US93/07231 



- 81 - 

Dysfunctions in the basal ganglia of the sustantia 
nigra are associated with Huntington's chorea and 
parkinsonism in vivo . The ability of the morphogens 
defined herein to enhance neuron survival indicates 
5 that these morphogens will be useful as part of a 
therapy to enhance survival of neuronal cells at risk 
of dying in vivo due, for example, to a neuropathy or 
chemical or mechanical trauma. It is particularly 
anticipated that these morphogens will provide a useful 

10 therapeutic agent to treat neuropathies which affect 
the striatal basal ganglia, including Huntington's 
chorea and Parkinson's disease. For clinical 
applications, the morphogen may be administered or, 
alternatively, a morphogen- stimulating agent may be 

15 administered . 



20 



Example 4. Morphogen- Induced Redif f erentiation of 
Transformed Cells 



The morphogens described herein also induce 
redifferentiation of transformed cells to a morphology 
characteristic of untrans formed cells. In particular, 
the morphogens are capable of inducing 

25 redifferentiation of transformed cells of neuronal 

origin to a morphology characteristic of untrans formed 
neurons. The example provided below details morphogen 
induced redifferentiation of a transformed human cell 
line of neuronal origin, NG105-115. Morphogen- induced 

30 redifferentiation of transformed cells also has been 
shown in mouse neuroblastoma cells (N1E-115) and in 
human embryo carcimona cells (see international 
application US92/01968 (W092/15323) . 



WO 94/03200 



PCT/US93/07231 



NG108-15 is a transformed hybrid cell line produced 
by fusing neuroblastoma x glioma cells (obtained from 
America Type Tissue Culture, Rockville, MD), and 
exhibiting a morphology characteristic of transformed 
5 embryonic neurons, e.g., having a fibroblastic 

morphology. Specifically, the cells have polygonal 
cell bodies, short, spike-like processes and make few 
contacts with neighboring cells (see FIG. 1A). 
Incubation of NG108-15 cells, cultured in a chemically 
10 defined, serum-free medium, with 0.1 to 300 ng/ml of 
OP-1 for four hours induces an orderly, dose-dependent 
change in cell morphology. 

In the experiment NG108-15 cells were subcultured 

15 on poly-L-ly^ine coated 6-well plates. 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% trif luoroacetic was added to 
each well. OP-1 concentrations of 0-300 ng/ml were 

20 tested. Typically, the media was changed daily with new 
aliguots of OP-1, although morphogenesis can be induced 
by a single four hour incubation with OP-1. In 
addition, OP-1 concentrations of 10 ng/ml were 
sufficient to induce redif f erentiation. After one day, 

25 hOP-l-treated cells undergo a significant change in 
their cellular ultrastructure, including rounding of 
the soma, increase in phase brightness and extension of 
the short neurite processes. After two days, cells 
treated with OP-1 begin to form epithelioid sheets, 

30 which provide the basis for the growth of mutilayered 
aggregates at three day's post-treatment. By four 
days, the great majority of OP-l-treated cells are 
associated in tightiy-packed, mutilayered aggregates 



WO 94/03200 



PCT/US93/07231 



- 83 - 

(Fig, IB). Fig. 2 plots the mean number of multi- 
layered aggregates or cell clumps identified in twenty 
randomly selected fields from six independent 
experiments, as a function of morphogen concentration. 
5 Forty ng/ml of OP-1 is sufficient for half maximum 
induction of cell aggregation. 

The morphogen- induced redif f erentiation occurred 
without any associated changes in DNA synthesis, cell 

10 division, or cell viability, making it unlikely that 
the morphologic changes were secondary to cell 
differentiation or a toxic effect of hOP-1. Moreover, 
the OP-l-induced morphogenesis does not inhibit cell 
division, as determined by 3 H-thymidine uptake, unlike 

15 other molecules which have been shown to stimulate 

differentiation of transformed cells, such as butyrate, 
DM SO, retanoic acid or Forskolin. The data indicate 
that OP-1 can maintain cell stability and viability 
after inducing redif f erentiation. In addition, the 

20 effects are morphogen specific, and redif ferentiation 
is not induced when NG108-15 cells are incubated with 
0.1-40 ng/ml TGF-0. 

The experiments also have been performed with 
25 highly purified soluble morphogen (e.g., mature OPl 

associated with its pro domain) which also specifically 
induced redif ferentiation of NG108-15 cells. 

The morphogens described herein accordingly provide 
30 useful therapeutic agents for the treatment of 
neoplasias and neoplastic lesions of the nervous 
system, particularly in the treatment of 



WO 94/03200 



PCI7US93/07231 



- 84 - 

neuroblastomas, including retinoblastomas, and gliomas. 
The morphogens themselves may be administered or, 
alternatively, a morphogen- stimulating agent may be 
administered. 

5 

Example 5. Nerve Tissue Protection from Chemical 
Trauma 

10 The ability of the morphogens described herein to 

enhance survival of neuronal cells and to induce cell 
aggregation and cell-cell adhesion in redif ferentiated 
cells, indicates that the morphogens will be useful as 
therapeutic agents to maintain neural pathways by 

15 protecting the cells defining the pathway from the 
damage caused by chemical trauma. In particular, the 
morphogens can protect neurons, including developing 
neurons, from the effects of toxins known to inhibit 
the proliferation and migration of neurons and to 

20 interfere with cell-cell adhesion. Examples of such 
toxins include ethanol, one or more of the toxins 
present in cigarette smoke, and a variety of opiates. 
The toxic effects of ethanol on developing neurons 
induces the neurological damage manifested in fetal 

25 alcohol syndrome. The morphogens also may protect 
neurons from the cytoxic effects associated with 
excitatory amino acids such as glutamate. 

For example, ethanol inhibits the cell-cell 
30 adhesion effects induced in morphogen-treated NG108-15 
cells when provided to these cells at a concentration 
of 25-50 mM. Half maximal inhibition can be achieved 
with 5-10 mM ethanol, the concentration of blood 
alcohol in an adult following ingestion of a single 
35 alcoholic beverage. Ethanol likely interferes with the 



WO 94/03200 



PCT/US93/07231 



- 85 - 

hemophilic binding of CAMs between cells, rather than 
their induction, as morphogen- induced N-CAM levels are 
unaffected by ethanol. Moreover, the inhibitory effect 
is inversely proportional to morphogen concentration. 
5 Accordingly, it is envisioned that administration of a 
morphogen or morphogen-stimulating agent to neurons, 
particularly developing neurons, at risk of damage from 
exposure to toxins such as ethanol, may protect these 
cells from nerve tissue damage by overcoming the 
10 toxin's inhibitory effects. The morphogens described 
herein also are useful in therapies to treat damaged 
neural pathways resulting from a neuropathy induced by 
exposure to these toxins. 

15 

Example 6. Morphogen- Induced CAM Expression 

The morphogens described herein induce CAM 
expression, particularly N-CAM expression, as part of 

20 their induction of morphogenesis. CAMs are 

morphoregulatory molecules identified in all tissues as 
an essential step in tissue development. N-CAMs, which 
comprise at least 3 isoforms (N-CAM-180, N-CAM-140 and 
N-CAM-120, where "180", "140" and "120" indicate the 

25 apparent molecular weights of the isoforms as measured 
by polyacrylamide gel electrophoresis) are expressed at 
least transiently in developing tissues, and 
permanently in nerve tissue. Both the N-CAM-180 and N- 
CAM-140 isoforms are expressed in both developing and 

30 adult tissue. The N-CAM-120 isoform is found only in 
adult tissue. Another neural CAM is LI. 

N-CAMs are implicated in appropriate neural 
development, including appropriate nuerulation, 
35 neuronal migration, fasciculation, and synaptogenesis. 



WO 94/03200 PCT/US93/07231 



- 86 - 

Inhibition of N-CAM production, as by complexing the 
molecule with an N-CAM- specific antibody, inhibits 
retina organization, including retinal axon migration, 
and axon regeneration in the peripheral nervous system, 
5 as well as axon synapsis with target muscle cells. In 
addition, significant evidence indicates that physical 
or chemical trauma to neurons, oncogenic transformation 
and some genetic neurological disorders are accompanied 
by changes in CAM expression, which alter the adhesive 
10 or migratory behavior of these cells* Specifically, 
increased N-CAM levels are reported in Huntington's 
disease striatum (e.g., striatal basal ganglia), and 
decreased adhesion is noted in Alzheimer's disease. 

15 The morphogens described herein can stimulate CAM 

production, particularly LI and N-CAM production, 
including all three isoforms of the N-CAM molecule. 
For example, N-CAM expression is stimulated 
significantly in morphogen-treated NG108-15 cells. 

20 Untreated NG108-15 cells exhibit a fibroblastic, or 
minimally differentiated morphology and express only 
the 180 and 140 isoforms of N-CAM normally associated 
with a developing cell. Following morphogen treatment 
these cells exhibit a morphology characteristic of 

25 adult neurons and express enhanced levels of all three 
N-CAM isoforms. Using a similar protocol as described 
in the example below, morphogen treatment of NG108-15 
cells also induced LI expression. 

30 In this example NG108-15 cells were cultured for 

4 days in the presence of increasing concentrations of 
OP-1 and standard Western blots performed on whole 
cells extracts. N-CAM isoforms were detected with an 
antibody which crossreacts with all three isoforms, 

35 mAb B28.123, obtained from Sigma Chemical Co., 



WO 94/03200 



PCT/US93/07231 



- 87 - 

St. Louis, the different isoforms being distinguishable 
by their different mobilities on an electrophoresis 
gel. Control NG108-15 cells (untreated) express both 
the 140 kDa and the 180 kDa isoforms, but not the 120 
5 kDa, as determined by western blot analyses using up to 
100 /jg of protein. Treatment of NG108-15 cells with 
OP-1 resulted in a dose -dependent increase in the 
expression of the 180 kDa and 140 kDa isoforms, as well 
as the induction of the 120 kDa isoform. See Fig. 2A 

10 and 2B. Fig. 2B is a Western blot of OPl-treated 
NG108-15 cell extracts, probed with mAb H28.123, 
showing the induction of all three isoforms. Fig. 2A 
is a dose response curve of N-CAM- 180 and N-CAM- 140 
induction as a function of morphogen concentration. N- 

15 CAM-120 is not shown in the graph as it could not be 
quantitated in control cells. However, as is clearly 
evident from the Western blot in Fig. 2A, N-CAM-120 is 
induced in response to morphogen treatment. The 
differential induction of N-CAM 180 and 140 isoforms 

20 seen may be because constitutive expression of the 140 
isoform is close to maximum. 

The increase in N-CAM expression corresponded in a 
dose -dependent manner with the morphogen induction of 

25 multicellular aggregates. Compare Fig. 2A and Fig 3. 
Fig. 3 graphs the mean number of multilayered 
aggregates (clumps) counted per 20 randomly selected 
fields in 6 independent experiments, versus the 
concentration of morphogen. The induction of the 120 

30 isoform also indicates that morphogen- induced 

redif ferentiation of transformed cells stimulates not 
only redifferentiation of these cells from a 
transformed phenotype, but also differentiation to a 
phenotype corresponding to a developed cell. Standard 

35 immunolocalization studies performed with the mAb 



WO 94/03200 



PCT/US93/07231 



- 88 - 

H28.123 on morphogen-treated cells show N-CAM cluster 
formation associated with the periphery and processes 
of treated cells and no reactivity with untreated 
cells. Moreover, morphogen treatment does not appear 
5 to inhibit cell division as determined by cell counting 
or 3 H-thymidine uptake. Finally, known chemical 
differentiating agents, such as Forskolin and 
dimethylsulf oxide do not induce N-CAM production. 

10 In addition, the cell aggregation effects of OP-1 

on NG108-15 cells can be inhibited with anti-N-CAM 
antibodies or antisense N-CAM oligonucleotides. 
Antisense oligonucleotides can be made synthetically on 
a nucleotide synthesizer, using standard means known in 

15 the art. Preferably, phosphorothioate oligonucleotides 
("S-oligos") are prepared, to enhance transport of the 
nucleotides across cell membranes. Concentrations of 
both N-CAM antibodies and N-CAM antisense 
oliognucleotides sufficient to inhibit N-CAM induction 

20 also inhibited formation of multilayered cell 

aggregates. Specifically, incubation of morphogen- 
treated NG108-115 cells with 0.3-3 pM N-CAM antisense 
S-oligos, 5-500 pM unmodified N-CAM antisense oligos, 
or 10 /ig/ml mAb H28.123 significantly inhibits cell 

25 aggregation. It is likely that morphogen treatment 
also stimulates other CAMS, as inhibition is not 
complete. 

The experiments also have been performed with 
30 soluble morphogen (e.g., mature OP-1 associated with 
its pro domain) which also specifically induced CAM 
expression. 



WO 94/03200 * PCT/US93/07231 



- 89 - 

The morphogens described herein are useful as 
therapeutic agents to treat neurological disorders 
associated with altered CAM levels, particularly N-CAM 
levels, such as Huntington's chorea and Alzheimers' 
5 disease, and the like. In clinical applications, the 
morphogens themselves may be administered or, 
alternatively, a morphogen-stimulating agent may be 
administered. 

10 The efficacy of the morphogens described herein to 

affect N-CAM expression may be assessed in vitro using 
a suitable cell line and the methods described herein. 
In addition to a transformed cell line, N-CAM 
expression can be assayed in a primary cell culture of 

15 neural or glial cells, following the procedures 

described herein. The efficacy of morphogen treatment 
on N-CAM expression in vivo may be evaluated by tissue 
biopsy as described in Example 9, below, and detecting 
N-CAM molecules with an N-CAM-specif ic antibody, such 

20 as mAb H28.123, or using the animal model described in 
Example 11. 

Alternatively, the level of N-CAM proteins or 
protein fragments present in cerebrospinal fluid or 

25 serum also may be detected to evaluate the effect of 
morphogen treatment. N-CAM molecules are known to 
slough off cell surfaces and have been detected in both 
serum and cerebrospinal fluid. In addition, altered 
levels of the soluble form of N-CAM are associated with 

30 normal pressure hydrocephalus and type II 

schizophrenia. N-CAM fluid levels may be detected 
following the procedure described in Example 9 and 
using an N-CAM specific antibody, such as mAb H28.123. 



35 



WO 94/03200 



PCT/US93/07231 



- 90 - 

Example 7. Morphogen-Induced Nerve Gap Repair (PNS) 

The morphogens described herein also stimulate 
peripheral nervous system axonal growth over extended 
5 distances allowing repair and regeneration of damaged 
neural pathways. While neurons of the peripheral 
nervous system can sprout new processes following 
injury, without guidance these sprout ings typically 
fail to connect appropriately and die. Where the break 
10 is extensive, e.g., greater than 5 or 10 mm, 
regeneration is poor or nonexistent. 

In this example morphogen stimulation of nerve 
regeneration was assessed using the rat sciatic nerve 

15 model. The rat sciatic nerve can regenerate 

spontaneously across a 5 mm gap, and occasionally 
across a 10 mm gap, provided that the severed ends are 
inserted in a saline-filled nerve guidance channel. In 
this experiment, nerve regeneration across a 12mm gap 

20 was tested. 

Adult female Sprague-Dawley rats (Charles River 
Laboratories, Inc.) weighing 230-250 g were 
anesthetized with intraperitoneal injections of sodium 

25 pentobarbital 35 mg/kg body weight). A skin incision 
was made parallel and just posterior to the femur. The 
avascular intermuscular plane between vastus lateralis 
and hamstring muscles were entered and followed to the 
loose fibroareolar tissue surrounding the sciatic 

30 nerve. The loose tissue was divided longitudinally 
thereby freeing the sciatic nerve over its full extent 
without devascularizing any portion. Under a surgical 



WO 94/03200 



PCT/US93/07231 



- 91 - 

microscope the sciatic nerves were transected with 
microscissors at mid-thigh and grafted with an OP-1 gel 
graft that separated the nerve stumps by 12 mm. The 
graft region was encased in a silicone tube 20 mm in 
5 length with a 1.5 mm inner diameter, the interior of 
which was filled a morphogen solution. Specif ically, 
The central 12 mm of the tube consisted of an OP-1 gel 
prepared by mixing 1 to 5 pg of substantially pure CHO- 
produced recombinant OP-1 with approximately 100 pi of 

10 MATRIGEL™ (from Collaborative Research, Inc. r Bedford, 
MA), an extracellular matrix extract derived from mouse 
sarcoma tissue, and containing solubilized tissue 
basement membrane, including laminin, type IV collagen, 
heparin sulfate, proteoglycan and entactin, in 

15 phosphate-buffered saline. The OP-l-filled tube was 
implanted directly into the defect site, allowing 4 mm 
on each end to insert the nerve stumps. Each stump was 
abutted against the OP-1 gel and was secured in the 
silicone tube by three stitches of commercially 

20 available surgical 10-0 nylon through the epineurium, 
the fascicle protective sheath. 

In addition to OP-1 gel grafts, empty silicone 
tubes, silicone tubes filled with gel only and 

25 "reverse" autografts, wherein 12 mm transected segments 
of the animal's sciatic nerve were rotated 180° prior 
to suturing, were grafted as controls. All experiments 
were performed in quadruplicate. All wounds were 
closed by wound clips that were removed after 10 days. 

30 All rats were grafted on both legs. At 3 weeks the 
animals were sacrificed, and the grafted segments 
removed and frozen on dry ice immediately. Frozen 



WO 94/03200 



PCT/US93/07231 



- 92 - 

sections then were cut throughout the graft site, and 
examined for axonal regeneration by immunof luorescent 
staining using anti-neurof ilament antibodies labeled 
with flurocein (obtained from Sigma Chemical Co., 
5 St. Louis). 

Regeneration of the sciatic nerve occurred across 
the entire 12 mm distance in all graft sites wherein 
the gap was filled with the OP-1 gel. By contrast, 
10 empty silicone tubes and reverse autografts did not 
show nerve regeneration, and only one graft site 
containing the gel alone showed axon regeneration. 

15 Example 8. Morphoqen- Induced Nerve Gap Repair fCNS) 

Following axonal damage in vivo the CNS neurons are 
unable to resprout processes. Accordingly, trauma to 
CNS nerve tissue, including the spinal cord, optic 

20 nerve and retina, severely damages or destroys the 
neural pathways defined by these cells. Peripheral 
nerve grafts have been used in an effort to bypass CNS 
axonal damage. Successful autologous graft repair to 
date apparently requires that the graft site occur near 

25 the CNS neuronal cell body, and a primary result of CNS 
axotomy is neuronal cell death. The efficacy of 
morphogens described herein on CNS nerve repair, may be 
evaluated using a rat crushed optic nerve model such as 
the one described by Bignami et al., (1979) Exp. Eye 

30 Res. 28 ; 63-69, the disclosure of which is incorporated 
herein by reference. Briefly, and as described 
therein, laboratory rats (e.g., from Charles River 
Laboratories, Wilmington, MA) are anesthesized using 
standard surgical procedures, and the optic nerve 

35 crushed by pulling the eye gently out of the orbit, 



WO 94/03200 



PCT/US93/07231 



- 93 - 

inserting a watchmaker forceps behind the eyeball and 
squeezing the optic nerve with the forceps for 
15 seconds, followed by a 30 second interval and second 
15 second squeeze. Rats are sacrificed at different 
5 time intervals, e.g., at 48 hours, and at 3, 4, 11, 15 
and 18 days after operation. The effect of morphogen 
on optic nerve repair can be assessed by performing the 
experiment in duplicate and providing morphogen or PBS 
(e.g., 25 pi solution, and 25 /ig morphogen) to the 
10 optic nerve, e.g., just prior to the operation, 

concommitant with the operation, or at specific times 
after the operation. 

In the absence of therapy, the surgery induces 
15 glial scarring of the crushed nerve, as determined by 
immunof luoresence staining for glial fibrillary acidic 
protein (GFA), a marker protein for glial scarring, and 
by histology. Indirect immunof luoresence on air-dried 
cryostat sections as described in Bignami et al. (1974) 
20 J. Comp. Neur. 153 : 27-38, using commercially 

available antibodies to GFA (e.g., Sigma Chemical Co., 
St. Louis). Reduced levels of GFA are anticipated in 
animals treated with the morphogen, evidencing the 
ability of morphogens to inhibit glial scar formation 
25 and to stimulate optic nerve regeneration. 

Example 9. Nerve Tissue Diagnostics 

Morphogen localization in nerve tissue can be used 
30 as part of a method for diagnosing a neurological 

disorder or neuropathy. The method may be particularly 
advantageous for diagnosing neuropathies of brain 
tissue. Specifically, a biopsy of brain tissue is 
performed on a patient at risk, using standard 
35 procedures known in the medical art. Morphogen 



WO 94/03200 



PCT/US93/07231 



- 94 - 

expression associated with the biopsied tissue then is 
assessed using standard methodologies, as by 
immunolocalization, using standard immunofluorescence 
techniques in concert with morphogen-specif ic antisera 
5 or monoclonal antibodies. Specifically/ the biopsied 
tissue is thin sectioned using standard methodologies 
known in the art, and f luorescently labelled (or 
otherwise detectable) antibodies incubated with the 
tissue under conditions sufficient to allow specific 

10 antigen-antibody complex formation. The presence and 
quantity of complex formed then is detected and 
compared with a predetermined standard or reference 
value. Detection of altered levels of morphogen 
present in the tissue then may be used as an indicator 

15 of tissue dysfunction. Alternatively, fluctuation in 
morphogen levels may be assessed by monitoring 
morphogen transcription levels, either by standard 
northern blot analysis or in situ hybridization, using 
a labelled probe capable of hybridizing specifically to 

20 morphogen RNA and standard RNA hybridization protocols 
well described in the art. 

Fluctuations in morphogen levels present in the 
cerebrospinal fluid or bloodstream also may be used to 

25 evaluate nerve tissue viability. For example, 

morphogens are detected associated with adendema cells 
which are known to secrete factors into the 
cerebrospinal fluid, and are localized generally 
associated with glial cells, and in the extracellular 

30 matrix, but not with neuronal cell bodies. 



WO94/03200 



PCT/US93/07231 



Accordingly , the cerebrospinal fluid may be a natural 
means of morphogen transport. Alternatively, 
morphogehs may be released from dying cells into 
cerebrospinal fluid. In addition, OP-1 recently has 
5 been identified in human blood, which also may be a 
means of morphogen transport, and/or a repository for 
the contents of dying cells. 

Spinal fluid may be obtained from an individual by 

10 a standard lumbar puncture, using standard 

methodologies known in the medical art. Similarly, 
serum samples may be obtained by standard venipuncture 
and serum prepared by centrifugation at 3,000 RPM for 
ten minutes. The presence of morphogen in the serum or 

15 cerebral spinal fluid then may be assessed by standard 
Western blot (immunoblot) , ELISA or RIA procedures. 
Briefly, for example, with the ELISA, samples may be 
diluted in an appropriate buffer, such as phosphate- 
buffered saline, and 50 pi aliquots allowed to absorb 

20 to flat bottomed wells in microtitre plates pre-coated 
with morphogen-specific antibody, and allowed to 
incubate for 18 hours at 4°C. Plates then may be 
washed with a standard buffer and incubated with 50 pi 
aliquots of a second morphogen-specific antibody 

25 conjugated with a detecting agent, e.g., biotin, in an 
appropriate buffer, for 90 minutes at room temperature. 
Morphogen-antibody complexes then may be detected using 
standard procedures. 

30 Alternatively, a morphogen-specific affinity column 

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

35 eluted. A suitable elution buffer may be determined 



WO 94/03200 



PCT/US93/07231 



- 96 - 

empirically by determining appropriate binding and 
elution conditions first with a control (e.g., 
purified/ recombinantly-produced morphogen. ) Fractions 
then are tested for the presence of the morphogen by 
5 standard immunoblot, and confirmed by N- terminal 

sequencing. Morphogen concentrations in serum or other 
fluid samples then may be determined using standard 
portein quantification techniques, including by 
spectrophotometry absorbance or by quantitation by 
10 ELISA or RIA antibody assays. Using this procedure, 
OP-1 has been identified in serum. 

OP-1 was detected in human serum using the 
following assay. A monoclonal antibody raised against 

15 mammalian, recombinantly produced OP-1 using standard 
immunology techniques well described in the art and 
described generally in Example 13, was immobilized by 
passing the antibody over an activated agarose gel 
(e.g., Affi-Gel™, from Bio-Rad Laboratories, Richmond, 

20 CA, prepared following manufacturer's instructions), 
and used to purify OP-1 from serum. Human serum then 
was passed over the column and eluted with 3M 
K-thiocyanate. K-thiocyanante fractions then were 
dialyzed in 6M urea, 20mM P0 4 , pH 7.0, applied to a CB 

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

acetonitrile/0.1% TFA gradient. Mature, recombinantly 
produced OP-l homodimers elute between 20-22 minutes. 
Fractions then were collected and tested for the 
presence of OP-1 by standard immunoblot. Fig. 4 is an 

30 immunoblot showing OP-1 in human sera under reducing 
and oxidized conditions. In the figure, lanes 1 and 4 
are OP-1 standards, run under oxidized (lane 1) and 



WO 94/03200 



PCT/US93/07231 



- 97 - 

reduced (lane 4) conditions. Lane 5 shows molecular 
weight markers at 17 , 27 and 39 kDa. Lanes 2 and 3 are 
human sera OP-1, run under oxidized (lane 2) and 
reduced (lane 3) conditions. 

5 

Morphogens may be used in diagnostic applications 
by comparing the quantity of morphogen present in a 
body fluid sample with a predetermined reference value , 
with fluctuations in fluid morphogen levels indicating 

10 a change in the status of nerve tissue. Alternatively, 
fluctuations in the level of endogenous morphogen 
antibodies may be detected by this method, most likely 
in serum, using an antibody or other binding protein 
capable of interacting specifically with the endogenous 

15 morphogen antibody. Detected fluctuations in the 
levels of the endogenous antibody may be used as 
indicators of a change in tissue status. 

20 Example 10. Alleviation of Immune Response-Mediated 

Nerve Tissue Damage 

The morphogens described herein may be used to 
alleviate immunologically-related damage to nerve 

25 tissue. Details of this damage and the use of 

morphogens to alleviate this injury are disclosed in 
international application US92/07358 (WO93/04692) . A 
primary source of such damage to nerve tissue follows 
hypoxia or ischemia-reper fusion of a blood supply to a 

30 neural pathway, such as may result from an embolic 

stroke, or may be induced during a surgical procedure. 



WO 94/03200 PCT/US93/07231 



- 98 - 

As described in international application US92/07358 
(WO93/04692) , morphogens have been shown to alleviate 
damage to myocardial tissue following ischemia- 
reperfusion of the blood supply to the tissue. The 
5 effect of morphogens on alleviating immunologically- 
related damage to nerve tissue may be assessed using 
methodologies and models known to those skilled in the 
art and described below. 

10 For example, the rabbit embolic stroke model 

provides a useful method for assessing the effect of 
morphogens on tissue injury following cerebral 
ischemia-reperfusion. The protocol disclosed below is 
essentially that of Phillips et al. (1989) Annals of 

1: Neurology 25: 281-285, the disclosure of which is herein 
ncorporated by reference. Briefly, white New England 
abbits (2-3kg) are anesthetized and placed on a 
respirator. The intracranial circulation then is 
selectively catheterized by the Seldinger technique. 

20 Baseline cerebral angiography then is performed, 
employing a digital substration unit. The distal 
internal carotid artery or its branches then is 
selectively embolized with 0.035 ml of 18-hour-aged 
autologous thrombus. Arterial occlusion is documented 

25 by repeat angiography immediately after embolization. 
After a time sufficient to induce cerebral infarcts (15 
minutes or 90 minutes), reperfusion is induced by 
administering a bolus of a reperfusion agent such as 
the TPA analogue FB-FB-CF (e.g., 0.8 mg/kg over 2 

30 minutes) . 

The effect of morphogen on cerebral infarcts can be 
assessed by administering varying concentrations of 
morphogens, e.g., OP-1, at different times following 
35 embolization and/or reperfusion. The rabbits are 



WO 94/03200 



PCT/US93/07231 



- 99 - 

sacrificed 3-14 days post embolization and their brains 
prepared for neuropathological examination by fixing by 
immersion in 10% neutral buffered formation for at 
least 2 weeks. The brains then are sectioned in a 
5 coronal plane at 2-3 mm intervals, numbered and 
submitted for standard histological processing in 
paraffin, and the degree of nerve tissue necrosis 
determined visually. Morphogen-treated animals are 
anticipated to reduce or significantly inhibit nerve 
10 tissue necrosis following cerebral is chemia-reper fusion 
in the test animals as determined by histology 
comparison with nontreated animals. 

Example 11. Animal Model for Assessing Morphogen 
15 Efficacy In Vivo 

The in vivo activities of the morphogens described 
herein also are assessed readily in an animal model as 
described herein. A suitable animal, preferably 

20 exhibiting nerve tissue damage, for example, 

genetically or environmentally induced, is injected 
intracerebrally with an effective amount of a morphogen 
in a suitable therapeutic formulation, such as 
phosphate-buffered saline, pH 7. The morphogen 

25 preferably is injected within the area of the affected 
neurons. The affected tissue is excised at a 
subsequent time point and the tissue evaluated 
morphologically and/or by evaluation of an appropriate 
biochemical marker (e.g., by morphogen or N-CAM 

30 localization; or by measuring the dose-dependent effect 
on a biochemical marker for CNS neurotrophic activity 
or for CNS tissue damage, using for example, glial 
fibrillary acidic protein as the marker. The dosage 



WO 94/03200 



PCT/US93/07231 



- 100 - 

and incubation time will vary with the animal to be 
tested. Suitable dosage ranges for different species 
may be determined by comparison with established animal 
models. Presented below is an exemplary protocol for 
5 a rat brain stab model. 

Briefly , male Long Evans rats, obtained from 
standard commercial sources, are anesthesized and the 
head area prepared for surgery. The calvariae is 

10 exposed using standard surgical procedures and a hole 
drilled toward the center of each lobe using a 0.035K 
wire, just piercing the calvariae. 25j/l solutions 
containing either morphogen (e.g., OP-1, 25pg) or PBS 
then is provided to each of the holes by Hamilton 

15 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 the animal allowed to 
recover. 

20 

Three days post surgery, rats are sacrificed by 
decapitation and their brains processed for sectioning. 
Scar tissue formation is evaluated by immunof luoresence 
staining for glial fibrillary acidic protein, a marker 

25 protein for glial scarring, to qualitatively determine 
the degree of scar formation. Glial fibrillary acidic 
protein antibodies are available commercially, e.g., 
from Sigma Chemical Co., St. Louis, MO. Sections also 
are probed with anti-OP-1 antibodies to determine the 

30 presence of OP-1. Reduced levels of glial fibrillary 
acidic protein are anticipated in the tissue sections 
of animals treated with the morphogen, evidencing the 
ability of morphogens to inhibit glial scar formation 
and stimulate nerve regeneration. 

35 



WO 94/03200 " PCT/US93/07231 



- 101 - 

Example 12. In Vitro Model for Evaluating Morphogen 
Species Transport Across the Blood-Brain 
Barrier. 



5 Described below is an in vitro method for 

evaluating the facility with which selected morphogen 
species likely will pass across the blood-brain 
barrier. A detailed description of the model and 
protocol are provided by Audus et al. (1987) Ann. N.Y. 
10 Acad. Sci. 507:9-18, the disclosure of which is 
incorporated herein by reference. 

Briefly, microvessel endothelial cells are isolated 
from the cerebral gray matter of fresh bovine brains. 

15 Brains are obtained from a local slaughter house and 
transported to the laboratory in ice cold minimum 
essential medium (MEM) with antibiotics. Under sterile 
conditions the large surface blood vessels and meninges 
are removed using standard dissection procedures. The 

20 cortical gray matter is removed by aspiration, then 

minced into cubes of about 1mm. The minced gray matter 
then is incubated with 0.5% dispase (BMB, Indianapolis, 
IN) for 3 hours at 37° C in a shaking water bath. 
Following the 3 hour digestion, the mixture is 

25 concentrated by centrifugation (1000 x g for 10 min.), 
then resuspended in 13% dextran and centrifuged for 
10 min. at 5800 x g. Supernatant fat, cell debris and 
myelin are discarded and the crude microvessel pellet 
resuspended in 1 mg/ml collagenase/dispase and 

30 incubated in a shaking water bath for 5 hours at 37° C. 
After the 5-hour digestion, the microvessel suspension 
is applied to a pre-established 50% Percoll gradient 
and centrifuged for 10 min at 1000 x g. The band 
containing purified endothelial cells (second band from 

35 the top of the gradient) is removed and washed two 



WO 94/03200 PCT/US93/07231 



- 102 - 

times with culture medium (e.g., 50% MEM/50% F-12 
nutrient mix).l The cells are frozen ( -80° C.) in 
medium containing 20% DMSO and 10% horse serum for 
later use. 

5 

After isolation, approximately 5 x 10 5 cells/cm 2 
are plated on culture dishes or 5*12 mp pore size 
polycarbonate filters that are coated with rat collagen 
and fibronectin. 10-12 days after seeding the cells, 
10 cell monolayers are inspected for confluency by 
microscopy. 

Characterization of the morphological, 
histochemical and biochemical properties of these cells 

15 has shown that these cells possess many of the salient 
features of the blood-brain barrier. These features 
include: tight intercellular junctions, lack of 
membrane fenestrations, low levels of pinocytotic 
activity, and the presence of gamma-glutamyl 

20 transpeptidase, alkaline phosphatase, and Factor VIII 
antigen activities. 

The cultured cells can be used in a wide variety of 
experiments where a model for polarized binding or 

25 transport is required. By plating the cells in 

multi-well plates, receptor and non-receptor binding of 
both large and small molecules can be conducted. In 
order to conduct trans endothelial cell flux 
measurements, the cells are grown on porous 

30 polycarbonate membrane filters (e.g., from Nucleopore, 
Pleasanton, CA). Large pore size filters (5-12 m/j) are 



WO 94/03200 



PCI7US93/07231 



- 103 - 

used to avoid the possibility of the filter becoming 
the rate-limiting barrier to molecular flux. The use 
of these large-pore filters does not permit cell growth 
under the filter and allows visual inspection of the 
5 cell monolayer. 

Once the cells reach confluency, they are placed in 
a side-by-side diffusion cell apparatus (e.g., from 
Crown Glass, Sommerville, NJ). For flux measurements, 

10 the donor chamber of the diffusion cell is pulsed with 
a test substance, then at various times following the 
pulse, an aliquot is removed from the receiver chamber 
for analysis. Radioactive or f luorescently-labelled 
substances permit reliable quantitation of molecular 

15 flux. Monolayer integrity is simultaneously measureid 
by the addition of a non-transportable test substance 
such as sucrose or inulin and replicates of at least 4 
determinations are measured in order to ensure 
statistical significance. 

20 

Example 13. Screening Assay for Candidate Compounds 
which Alter Endogenous Morphoqen Levels 

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

using the following screening assay, in which the level 
of morphogen production by a cell type which produces 
measurable levels of the morphogen is determined with 
and without incubating the cell in culture with the 
30 compound, in order to assess the effects of the 
compound on the cell. This can be accomplished by 
detection of the morphogen either at the protein or RNA 
level. A more detailed description also may be found 
in international application US92/07359 (WO92/05172 ) . 



35 



WO 94/03200 ' PCT/US93/07231 



- 104 - 

13.1 Growth of Cells in Culture 

Cell cultures of kidney, adrenals, urinary bladder, 
brain, or other organs, may be prepared as described 
5 widely in the literature. For example, kidneys may be 
explanted from neonatal or new born or young or adult 
rodents (mouse or rat) and used in organ culture as 
whole or sliced (1-4 mm) tissues. Primary tissue 
cultures and established cell lines, also derived from 

10 kidney, adrenals, urinary, bladder, brain, mammary, or 
other tissues may be established in multiwell plates (6 
well or 24 well) according to conventional cell culture 
techniques, and are cultured in the absence or presence 
of serum for a period of time (1-7 days). Cells may be 

15 cultured, for example, in Dulbecco's Modified Eagle 
medium (Gibco, Long Island, NY) containing serum (e.g., 
fetal calf serum at 1%-10%, Gibco) or in serum-deprived 
medium, as desired, or in defined medium (e.g., 
containing insulin, transferrin, glucose, albumin, or 

20 other growth factors). 

Samples for testing the level of morphogen 
production includes culture supernatants or cell 
lysates, collected periodically and evaluated for OP-1 

25 production by immunoblot analysis (Sambrook et al«, 
eds., 1989, Molecular Cloning, Cold Spring Harbor 
Press, Cold Spring Harbor, NY), or a portion of the 
cell culture itself, collected periodically and used to 
prepare polyA+ RNA for RNA analysis. To monitor de 

30 novo OP-1 synthesis, some cultures are labeled 
according to conventional procedures with an 
3 5 S-methionine/ 3 5 S-cysteine mixture for 6-24 hours and 
then evaluated to OP-1 synthesis by conventional 
immunoprecipitation methods • 



35 



WO 94/03200 



PCT/US93/07231 



- 105 - 

13.2 Determination of Level of Morphogenic Protein 

In order to quantitate the production of a 
morphogenic protein by a cell type, an immunoassay may 
5 be performed to detect the morphogen using a polyclonal 
or monoclonal antibody specific for that protein. For 
example, OP-1 may be detected using a polyclonal 
antibody specific for OP-1 in an ELISA, as follows. 

10 1 pg/100 fjl of affinity-purified polyclonal rabbit 

IgG specific for OP-1 is added to each well of a 
96-well plate and incubated at 37 °C for an hour. The 
wells are washed four times with 0.167M sodium borate 
buffer with 0.15 M NaCl (BSB), pH 8.2, containing 0.1% 

15 Tween 20. To minimize non-specific binding, the wells 
are blocked by filling completely with 1% bovine serum 
albumin (BSA) in BSB and incubating for 1 hour at 37 °C. 
The wells are then washed four times with BSB 
containing 0.1% Tween 20. A 100 pi aliquot of an 

20 appropriate dilution of each of the test samples of 
cell culture supernatant is added to each well in 
triplicate and incubated at 37 °C for 30 min. After 
incubation, 100 pi biotinylated rabbit anti-OP-1 serum 
(stock solution is about 1 mg/ml and diluted 1:400 in 

25 BSB containing 1% BSA before use) is added to each well 
and incubated at 37 °C for 30 min. The wells are then 
washed four times with BSB containing 0.1% Tween 20. 
100 pi strepavidin-alkaline (Southern Biotechnology 
Associates, Inc. Birmingham, Alabama, diluted 1:2000 in 

30 BSB containing 0.1% Tween 20 before use) is added to 

each well and incubated at 37 °C for 30 min. The plates 
are washed four times with 0.5M Tris buffered Saline 



WO 94/03200 PCT/US93/07231 



- 106 - 

(TBS), pH 7.2. 50pl substrate (ELISA Amplification 
System Kit, Life Technologies, Inc., Bethesda, MD) is 
added to each well incubated at room temperature for 15 
min. Then, 50 pi amplifier (from the same 
5 amplification system kit) is added and incubated for 
another 15 min at room temperature. The reaction is 
stopped by the addition of 50 pi 0.3 M sulphuric acid. 
The OD at 490 nm of the solution in each well is 
recorded. To guantitate OP-1 in culture media, a OP-1 
10 standard curve is performed in parallel with the test 
samples. 

Polyclonal antibody may be prepared as follows. 
Each rabbit is given a primary immunization of 100 

15 ug/500 pi E. coli produced OP-1 monomer ( amino acids 
328-431 in SEQ ID NO: 5) in 0.1% SDS mixed with 500 pi 
Complete Freund's Adjuvant. The antigen is injected 
subcutaneous ly at multiple sites on the back and flanks 
of the animal. The rabbit is boosted after a month in 

20 the same manner using incomplete Freund's Adjuvant. 
Test bleeds are taken from the ear vein seven days 
later. Two additional boosts and test bleeds are 
performed at monthly intervals until antibody against 
OP-1 is detected in the serum using an ELISA assay. 

25 Then, the rabbit is boosted monthly with 100 pg of 
antigen and bled (15 ml per bleed) at days seven and 
ten after boosting. 

Monoclonal antibody specific for a given morphogen 
30 may be prepared as follows. A mouse is given two 

injections of E. coli produced OP-1 monomer. The first 
injection contains lOOpg of OP-1 in complete Freund's 
adjuvant and is given subcutaneously. The second 
injection contains 50 pg of OP-1 in incomplete adjuvant 
35 and is given intraperitoneal ly. The mouse then 



WO 94/03200 



PCT/US93/07231 



- 107 - 

receives a total of 230 fjg of OP-1 (amino acids 307-431 
in SEQ ID NO: 5) in four intraperitoneal injections at 
various times over an eight month period . One week 
prior to fusion, both mice are boosted 
5 intraperitoneally with 100 pg of OP-1 (307-431) and 30 
//g of the N-tenninal peptide (Ser 293 -Asn 3Q9 -Cys) 
conjugated through the added cysteine to bovine serum 
albumin with SMCC crosslinking agent. This boost was 
repeated five days (IP), four days (IP), three days 

10 (IP) and one day (IV) prior to fusion. The mouse 
spleen cells are then fused to myeloma (e.g., 653) 
cells at a ratio of 1:1 using PEG 1500 (Boeringer 
Mannheim), and the cell fusion is plated and screened 
for OP-l-specific antibodies using OP-1 (307-431) as 

15 antigen. The cell fusion and monoclonal screening then 
are according to standard procedures well described in 
standard texts widely available in the art. 

The invention may be embodied in other specific 
20 forms without departing from the spirit or essential 
characteristics thereof. The present embodiments are 
therefore to be considered in all respects as 
illustrative and not restrictive, the scope of the 
invention being indicated by the appended claims rather 
25 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 embraced 
therein. 



WO 94/03200 



PCI7US93/07231 



- 108 - 



SEQUENCE LISTING 



(1) GENERAL INFORMATION: 

5 

(i) APPLICANT: 

(A) NAME: CREATIVE BIOMOLECULES, INC. 

(B) STREET: 35 SOUTH STREET 

(C) CITY: HOPKINTON 

10 (D) STATE: MASSACHUSETTS 

(E) COUNTRY: USA 

(F) POSTAL CODE (ZIP): 01748 

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

(H) TELEFAX: 1-508-435-0454 
15 (I) TELEX: 

(ii) TITLE OF INVENTION: MORPHOGEN- INDUCED NERVE REGENERATION AND 
REPAIR 

20 (iii) NUMBER OF SEQUENCES :. 33 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: CREATIVE BIOMOLECULES , INC. 

(B) STREET: 35 SOUTH STREET 
25 (C) CITY: HOPKINTON 

(D) STATE: MASSACHUSETTS 

(E) COUNTRY: USA 

(F) ZIP: 01748 

30 (V) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

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

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

35 

(viii) ATTORNEY/AGENT INFORMATION: 

(A) NAME: KELLEY, ROBIN D. 

(B) REGISTRATION NUMBER: 34,637 

(C) REFERENCE/DOCKET NUMBER: CRP-070 

40 

(ix) TELECOMMUNICATION INFORMATION: 

(A) TELEPHONE: 617/248-7000 

(B) TELEFAX: 617/248-7100 

45 

(2) INFORMATION FOR SEQ ID NO:l: 

(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 97 amino acids 
50 (B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



WO 94/03200 



PCI7US93/07231 



- 109 - 



(ii) MOLECULE TYPE: protein 



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

(B) LOCATION: 1..97 

(D) OTHER INFORMATION: /label» GENERIC- SEQ1 

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

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

15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

15 10 15 



20 



35 



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

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



Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa 
25 50 55 60 

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

30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 

85 90 95 



Xaa 

(2) INFORMATION FOR SEQ ID NO: 2: 



(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 97 amino acids 
40 (B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

45 



WO 94/03200 



PCT/US93/07231 



- 110 - 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..97 

(D) OTHER INFORMATION: /label= GENERIC-SEQ2 
5 /note= "WHEREIN EACH XAA INDEPENDENTLY INDICATES 

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

10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
1 5 10 15 

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

20 25 30 



20 



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

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



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

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

30 Xaa 



(2) INFORMATION FOR SEQ ID NO: 3: 

35 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



40 



(ii) MOLECULE TYPE: protein 



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

(B) LOCATION: 1. .97 

(D) OTHER INFORMATION: /label- GENERIC- SEQ3 

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



WO 94/03200 



PCI7US93/07231 



- Ill - 

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

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

5 

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

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Leu 
10 35 40 45 

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

15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

65 70 75 80 



20 



25 



45 



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

Xaa 



(2) INFORMATION FOR SEQ ID NO: 4: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

(C) STRAND EDNESS : single 
30 (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

35 (ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

(D) OTHER INFORMATION: /label* GENERIC-SEQ4 

/note* "VHEREIN EACH XAA IS INDEPENDENTLY SELECTED 
40 FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS 

AS DEFINED IN THE SPECIFICATION." 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 

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



Xaa Trp Xaa Xaa Ala Pro Xaa Gly Xaa Xaa Ala Xaa Tyr Cys Xaa Gly 
50 20 25 30 



WO 94/03200 



PCT/US93/07231 



- 112 - 

Xaa Cys laa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala 
35 40 45 

Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
5 50 55 60 

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

10 Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Het Xaa Val 

85 90 95 



15 



Xaa Xaa Cys Gly Cys Xaa 
100 

(2) INFORMATION FOB SEQ ID NO: 5: 



(i) SEQUENCE CHARACTERISTICS : 

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

(C) STRAND EDNESS: single 

(D) TOPOLOGY: linear 



25 



(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

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

30 (ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1. .139 

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

35 

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

Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro Lys 
15 10 15 

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

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

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

50 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 

65 70 75 80 



40 



WO 94/03200. 



PCT/US93/07231 



- 113 - 

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

Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala He 
5 100 105 HO 

Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val He Leu Lys Lys Tyr 
115 120 125 

10 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 

130 135 

(2) INFORMATION FOR SEQ ID N0:6: 

15 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acid 

(C) STRANDEONESS: single 

(D) TOPOLOGY: linear 



20 



40 



(ii) MOLECULE TYPE: protein 



(vi) ORIGINAL SOURCE: 

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

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1- .139 

30 (D) OTHER INFORMATION: /label- MOP 1 -MATURE 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 

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

15 10 15 



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

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



Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly Tyr Ala Ala 
45 50 55 60 

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

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

85 90 95 



WO 94/03200 



PCT/US93/07231 



- 114 - 



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

Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val He Leu Lys Lys Tyr 
5 115 120 125 

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

10 (2) INFORMATION FOR SEQ ID NO: 7: 

(i) SEQUENCE CHARACTERISTICS : 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acid 

15 (C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

20 (vi) ORIGINAL SOURCE: 

(A) ORGANISM: HOMO SAPIENS 
(F) TISSUE TYPE: HIPPOCAHPUS 

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

(B) LOCATION: 1..139 

(D) OTHER INFORMATION: /label= HOP 2 -MATURE 

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

Ala Val Arg Pro Leu Arg Arg Arg Gin Pro Lys Lys Ser Asn Glu Leu 
1 5 10 15 

35 Pro Gin Ala Asn Arg Leu Pro Gly He Phe Asp Asp Val His Gly Ser 

20 25 30 



40 



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

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



Tyr Tyr Cys Glu Gly Glu Cys Ser Phe Pro Leu Asp Ser Cys Met Asn 
45 65 70 75 80 

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

50 Asn Ala Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr 

100 105 110 



WO 94/03200 



PCT/US93/07231 



• 115 - 



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

Arg Asn Met Val Val Lys Ala Cys Gly Cys His 
5 130 135 

(2) INFORMATION FOR SEQ ID N0:8: 

(i) SEQUENCE CHARACTERISTICS: 
10 (A) LENGTH: 139 amino acids 

(B) TYPE: amino acid 

(C) STRAND EDNESS: single 

(D) TOPOLOGY: linear 

15 (ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

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

20 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..139 

(D) OTHER INFORMATION: /label* MOP2 -MATURE 

25 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 

Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn Glu Leu 
30 1 5 10 15 

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

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

35 AO 45 

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

40 

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

Ala Thr Asn His Ala He Leu Gin Ser Leu Val His Leu Met Lys Pro 
45 85 90 95 



WO 94/03200 PCI7US93/07231 



- 116 - 

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

Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val He Leu Arg Lys His 
5 115 120 125 

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

10 (2) INFORMATION FOR SEQ ID NO: 9: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 101 amino acids 

(B) TYPE: amino acid 

15 (C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

20 (vi) ORIGINAL SOURCE: 

(A) ORGANISM: bovinae 

(ix) FEATURE: 

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

(0) OTHER INFORMATION: /label* CBMP-2A-FX 



30 



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

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



Asp Trp He Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly 
35 20 25 30 

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

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

50 55 60 



45 



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



WO 94/03200 PCT/US93/07231 



- 117 - 

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

Gly Cys Gly Cys Arg 
5 100 

(2) INFORMATION FOR SEQ ID NO: 10: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

15 (ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HOMO SAPIENS 
(F) TISSUE TYPE: hippocampus 

20 

(ix) FEATURE: 

(A) NAME /KEY: Protein 

(B) LOCATION: 1..101 

(D) OTHER INFORMATION: /label** CBMP-2B-FX 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 

Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn 
30 1 5 10 15 

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

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

35 40 45 



25 



40 



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

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



Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr Gin Glu Met Val Val Glu 
45 85 90 95 

Gly Cys Gly Cys Arg 
100 



WO 94/03200 PCT/US93/07231 



10 



30 



- 118 - 



(2) INFORMATION FOR SEQ ID NO: 11: 

(1) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: DROSOPHILA HELANOGASTER 



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

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

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

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

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

20 25 30 



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

Val Val Gin Thr Leu Val Asn Asn Asn Asn Pro Gly Lys Val Pro Lys 
50 55 60 



Ala Cys Cys Val Pro Thr Gin Leu Asp Ser Val Ala Met Leu Tyr Leu 
35 65 70 75 80 

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

40 Val Gly Cys Gly Cys Arg 

100 

(2) INFORMATION FOR SEQ ID NO: 12: 

45 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

50 



WO 94/03200 



PCT/US93/07231 



10 



25 



- 119 - 



(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: XENOPUS 

(ix) FEATURE: 

(A) NAME /KEY: Protein 

(B) LOCATION: 1..102 

(D) OTHER INFORMATION: /label- VGL-FZ 



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



Cys Lys Lys Arg His Leu Tyr Val Glu Pbe Lys Asp Val Gly Trp Gin 
15 1 5 10 15 

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

20 Glu Cys Pro Tyr Pro Leu Thr Glu lie Leu Asn Gly Ser Asn His Ala 

35 40 45 



lie Leu Gin Thr Leu Val His Ser lie Glu Pro Glu Asp He Pro Leu 
50 55 60 

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



Asp Asn Asn Asp Asn Val Val Leu Arg His Tyr Glu Asn Met Ala Val 
30 85 90 95 

Asp Glu Cys Gly Cys Arg 
100 

35 (2) INFORMATION FOR SEQ ID NO: 13: 

(i) SEQUENCE CHARACTERISTICS : 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

40 (C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

45 (vi) ORIGINAL SOURCE: 

(A) ORGANISM: HURIDAE 

(ix) FEATURE: 

(A) NAME/KEY: Protein 
50 (B) LOCATION: 1..102 

(D) OTHER INFORMATION: /label- VGR-l-FX 



WO 94/03200 



PCT/US93/07231 



- 120 - 

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

Cys Lys Lys Bis Glu Leu Tyr Val Ser Phe Gin Asp Val Gly Trp Gin 
15 10 15 

5 

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

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

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

15 Pro Cys Cys Ala Pro Thr Lys Val Asn Ala He Ser Val Leu Tyr Phe 

65 70 75 80 



20 



25 



35 



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

Arg Ala Cys Gly Cys His 
100 

(2) INFORMATION FOR SEQ ID NO: 14: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 106 amino acids 

(B) TYPE: amino acid 

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



(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 



(Vi) ORIGINAL SOURCE: 

(A) ORGANISM: Homo sapiens 
40 (F) TISSUE TYPE: brain 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..106 

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 

50 Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp His 

15 10 15 



WO 94/03200 



PCT/US93/07231 



- 121 - 



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

Gin Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala 
5 35 40 45 

Leu Asn His Ala Val Leu Arg Ala Leu Met His Ala Ala Ala Pro Gly 
50 55 60 

10 Ala Ala Asp Leu Pro Cys Cys Val Pro Ala Arg Leu Ser Pro He Ser 

65 70 75 80 

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

15 

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

(2) INFORMATION FOR SEQ ID NO: 15: 

20 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 5 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: peptide 



30 

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

Cys Xaa Xaa Xaa Xaa 
1 5 

35 

(2) INFORMATION FOR SEQ ID NO: 16: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1822 base pairs 
40 (B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

45 

(iii) HYPOTHETICAL: NO 

(iv) ANTI-SENSE: NO 

50 (vi) ORIGINAL SOURCE: 

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



WO 94/03200 



« 



PCTYUS93/07231 



- 122 - 



10 



(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 49.. 1341 

(C) IDENTIFICATION METHOD: experimental 

(D) OTHER INFORMATION: /function- "OSTEOGENIC PROTEIN" 

/product* "0P1" 
/evidence^ EXPERIMENTAL 
/standard name= "OP1" 



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



GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG 57 

Met His Val 

15 1 

CGC TCA CTG CGA GCT GCG GCG CCG CAC AGC TTC GTG GCG CTC TGG GCA 105 

Arg Ser Leu Arg Ala Ala Ala Pro His Ser Pbe Val Ala Leu Trp Ala 

5 10 15 

20 

CCC CTG TTC CTG CTG CGC TCC GCC CTG GCC GAC TTC AGC CTG GAC AAC 153 

Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn 

20 25 30 35 

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

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

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

CTG GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG GGC GGC GGG CCC GGC 345 
Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly Gly Pro Gly 
B5 90 95 

40 

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

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

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



WO 94/03200 



PCT/US93/07231 



- 123 - 



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



537 



5 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 
10 Tyr 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 
Gin Val Leu Gin Glu His Leu Gly Arg Glu Ser Asp Leu Phe Leu Leu 
15 200 205 210 



681 



20 



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 



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

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



825 



873 



35 



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



921 



40 



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 Het Ala Asn Val Ala Glu Asn Ser Ser 
310 315 320 



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

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



WO 94/03200 



PCT/US93/07231 



- 124 - 

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 Fro Leu Asn Ser Tyr Met 
360 365 370 

5 AAC GCC ACC AAC CAC GCC ATC GIG CAG ACG CTG GTC CAC TTC ATC AAC 1209 * 

Asn Ala Thr Asn His Ala lie 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 
10 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 
15 405 410 415 

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 

20 

GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT CCATTGCTCG CCTTGGCCAG 1411 

GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA ACTTTAAAGG 1471 

25 TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CAGTGGCAGC 1531 

ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AAAAAACAAC 1591 

GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TGCACGGACT 1651 

30 

CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GGAGGAAGGG 1711 

GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTGAC CCGGAAGTTC 1771 

35 CTGTAATAAA TGTCACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A 1822 

(2) INFORMATION FOR SEQ ID NO: 17: 

40 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 431 amino acids 

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

45 (ii) MOLECULE TYPE: protein 

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

Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 
50 1 5 10 15 



WO 94/03200 



PCT/US93/07231 



- 125 - 



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 
5 35 AO 45 

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

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



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

Gly Pro Gly Gly Gin Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser 
100 105 HO 

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

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

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



15 



20 



30 



35 



45 



50 



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

Tyr Lys Asp Tyr He Arg Glu Arg Phe Asp Asn Glu 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 



40 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 

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 



WO 94/03200 



PCT/US93/07231 



- 126 - 

Arg Ser lie Arg Ser Thr Gly Ser Lys Gin Arg Sex Gin Asn Arg Ser 
290 295 300 

Lys Thr Pro Lys Asn Gin Glu Ala Leu Arg Met Ala Asn Val Ala Glu 
5 305 310 315 320 

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

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

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

15 

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 Thr Gin 
20 385 390 395 400 

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

25 Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
420 425 430 

(2) INFORMATION FOR SEQ ZD NO: 18: 

30 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1873 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



35 



(ii) MOLECULE TYPE: cDNA 

(iii) HYPOTHETICAL: NO 

40 (iv) ANTI-SENSE: NO 

(vi) ORIGINAL SOURCE: 

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

45 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 104.. 1393 

(D) OTHER INFORMATION: /function* "OSTEOGENIC PROTEIN" 
50 /product- "M0P1" 

/note= "M0P1 (CDNA)" 



WO 94/03200 



PCT/US93/07231 



- 127 - 

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

CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CCACCTGGGG 

5 CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGGC GCG ATG CAC GTG CGC 

Met His Val Arg 
1 

TCG CTG CGC GCT GCG GCG CCA CAC AGC TTC GTG GCG CTC TGG GCG CCT 
10 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 
Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn Glu 
15 25 30 35 

GTG CAC TCC AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG CGG 

Val His Ser Ser Phe He His Arg Arg Leu Arg Ser Gin Glu Arg Arg 

40 45 50 

20 

GAG ATG CAG CGG GAG ATC CTG TCC ATC TTA GGG TTG CCC CAT CGC CCG 

Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu Pro His Arg Pro 

55 60 65 

25 CGC CCG CAC CTC CAG GGA AAG CAT AAT TCG GCG CCC ATG TTC ATG TTG 
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 
30 Asp Leu Tyr 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 
Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gin Gly Pro Pro 
35 105 110 115 

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



40 



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



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

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



WO 94/03200 PCI7US93/07231 



- 128 - 

CGG GAG CGA TTT GAC AAC GAG ACC TTC CAG ATC ACA GTC TAT CAG GTG 691 
Arg Glu Arg Phe Asp Asn Glu Thr Phe Gin lie Thr Val Tyr Gin Val 
185 190 195 

5 CTC CAG GAG CAC TCA GGC AGG GAG TCG GAC CTC TTC TTG CTG GAC AGC 739 
Leu Gin Glu His Ser Gly Arg Glu Ser Asp Leu Phe Leu Leu Asp Ser 
200 205 210 

CGC ACC ATC TGG GCT TCT GAG GAG GGC TGG TTG GTG TTT GAT ATC ACA 787 
10 Arg Thr lie 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 
15 230 235 240 



20 



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 Het 
265 270 275 

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

ACG GGG GGC AAG CAG CGC AGC CAG AAT CGC TCC AAG ACG CCA AAG AAC 1027 
30 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 
35 310 315 320 

CAG AGG CAG GCC TGC AAG AAA CAT GAG CTG TAC GTC AGC TTC CGA GAC 1123 
Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp 
325 330 335 340 

40 

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 

45 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 Het Asn Ala 
360 365 370 

ACC AAC CAC GCC ATC GTC CAG ACA CTG GTT CAC TTC ATC AAC CCA GAC 1267 
50 Thr Asn His Ala He Val Gin Thr Leu Val His Phe He Asn Pro Asp 
375 380 385 



WO 94/03200 



PCT/US93/07231 



- 129 - 



ACA GTA CCC AAG CCC TGC TGT GCG CCC ACC CAG CTC AAC GCC ATC TCT 1315 
Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala lie Ser 
390 395 400 

5 GTC CTC TAC TTC GAC GAC AGC TCT AAT GTC GAC CTG AAG AAG TAC AGA 1363 
Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Asp Leu Lys Lys Tyr Arg 
405 410 415 420 

AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCTTCC TGAGACCCTG 1413 
10 Asn Met Val Val Arg Ala Cys Gly Cys His 

425 430 

ACCTTTGCGG GGCCACACCT TTCCAAATCT TCGATGTCTC ACCATCTAAG TCTCTCACTG 1473 

15 CCCACCTTGG CGAGGAGAAC AGACCAACCT CTCCTGAGCC TTCCCTCACC TCCCAACCGG 1533 

AAGCATGTAA GGGTTCCAGA AACCTGAGCG TGCAGCAGCT GATGAGCGCC CTTTCCTTCT 1593 

GGCACGTGAC GGACAAGATC CTACCAGCTA CCACAGCAAA CGCCTAAGAG CAGGAAAAAT 1653 

20 

GTCTGCCAGG AAAGTGTCCA GTGTCCACAT GGCCCCTGGC GCTCTGAGTC TTTGAGGAGT 1713 

AATCGCAAGC CTCGTTCAGC TGCAGCAGAA GGAAGGGCTT AGCCAGGGTG GGCGCTGGCG 1773 

25 TCTGTGTTGA AGGGAAACCA AGCAGAAGCC ACTGTAATGA TATGTCACAA TAAAACCCAT 1833 

GAATGAAAAA AAAAAAAAAA AAAAAAAAAA AAAAGAATTC 1873 



30 (2) INFORMATION FOR SEQ ID NO: 19: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 430 amino acids 

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

(ii) MOLECULE TYPE: protein 

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

40 

Het His Val Arg Ser Leu Arg Ala Ala Ala Fro 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 
45 20 25 30 

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

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



WO 94/03200 



PCT/US93/07231 



- 130 - 

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

Met Fhe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Ser Gly 
5 85 90 95 

Fro Asp Gly Gin Gly Fhe Ser Tyr Fro Tyr Lys Ala Val Fhe Ser Thr 
100 105 110 

10 Gin Gly Pro Fro Leu Ala Ser Leu Gin Asp Ser His Fhe Leu Thr Asp 
115 120 125 



15 



Ala Asp Met Val Met 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 



20 



Lys He Pro Glu Gly Glu Arg 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 

25 Val Tyr Gin Val Leu Gin Glu His Ser Gly Arg Glu Ser Asp Leu Phe 
195 200 205 



30 



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 
35 245 250 255 

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

40 Gin Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg 
275 280 285 



45 



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

Thr Fro Lys Asn Gin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn 
305 310 315 320 



50 



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



WO 94/03200 



PCT/US93/07231 



- 131 - 

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

Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser 
5 355 360 365 

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

10 He Asn Pro Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu 
385 390 395 400 



15 



20 



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

Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
420 425 430 

(2) INFORMATION FOR SEQ ZD NO: 20: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1723 base pairs 

(B) TYPE: nucleic acid 

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

(ii) MOLECULE TYPE: cDNA 

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

(F) TISSUE TYPE: HIPPOCAMPUS 

(ix) FEATURE: 

(A) NAME /KEY: CDS 
35 (B) LOCATION: 490.. 1696 

(D) OTHER INFORMATION: /function* "OSTEOGENIC PROTEIN" 

/product* "hOP2-PP" 
/note= "hOP2 (cDNA)" 

40 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
GGCGCCGGCA GAGCAGGAGT GGCTGGAGGA GCTGTGGTTG GAGCAGGAGG TGGCACGGCA 60 
45 GGGCTGGAGG GCTCCCTATG AGTGGCGGAG ACGGCCCAGG AGGCGCTGGA GCAACAGCTC 120 
CCACACCGCA CCAAGCGGTG GCTGCAGGAG CTCGCCCATC GCCCCTGCGC TGCTCGGACC 180 
GCGGCCACAG CCGGACTGGC GGGTACGGCG GCGACAGAGG CATTGGCCGA GAGTCCCAGT 240 

50 



WO 94/03200 



PCT/US93/07231 



- 132 - 

CCGCAGAGTA GCCCCGGCCT CGAGGCGGTG GCGTCCCGGT CCTCTCCGTC CAGGAGCCAG 300 

GACAGGTGTC GCGCGGCGGG GCTCCAGGGA CCGCGCCTGA GGCCGGCTGC CCGCCCGTCC 360 

5 CGCCCCGCCC CGCCGCCCGC CGCCCGCCGA GCCCAGCCTC CTTGCCGTCG GGGCGTCCCC 420 

AGGCCCTGGG TCGGCCGCGG AGCCGATGCG CGCCCGCTGA GCGCCCCAGC TGAGCGCCCC 480 

CGGCCTGCC ATG ACC GCG CTC CCC GGC CCG CTC TGG CTC CTG GGC CTG 528 
10 Met 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 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 



20 



40 



CGC GAG ATC CTG GCG GTG CTC GGG CTG CCT GGG CGG CCC CGG CCC CGC 672 
Arg Glu He Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg 
50 55 60 



25 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 Met 
65 70 75 

CTG GAC CTG TAC CAC GCC ATG GCC GGC GAC GAC GAC GAG GAC GGC GCG 768 
30 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 
35 95 100 105 

AAC ATG GTG GAG CGA GAC CGT GCC CTG GGC CAC CAG GAG CCC CAT TGG 864 
Asn Met 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 GAG GCG GTC 912 
Lys Glu Phe Arg Phe Asp Leu Thr Gin He Pro Ala Gly Glu Ala Val 
130 135 140 



45 ACA GCT GCG GAG TTC CGG ATT TAC AAG GTG CCC AGC ATC CAC CTG CTC 960 

Thr Ala Ala Glu Phe Arg He lyr Lys Val Pro Ser He His Leu Leu 
145 150 155 

AAC AGG ACC CTC CAC GTC AGC ATG TTC CAG GTG GTC CAG GAG CAG TCC 1008 

50 Asn Arg Thr Leu His Val Ser Met Phe Gin Val Val Gin Glu Gin Ser 
160 165 170 



WO 94/03200 



PCT/US93/07231 



- 133 - 



AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT CTT CAG ACG CTC CGA GCT 
Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gin Thr Leu Arg Ala 
175 180 185 

5 GGA GAC GAG GGC TGG CTG GTG CTG GAT 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 

TGG TTG CTG AAG CGT CAC AAG GAC CTG GGA CTC CGC CTC TAT GTG GAG 
10 Trp Leu Leu Lys Arg His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu 

210 215 220 



15 



20 



25 



ACT GAG GAC GGG CAC AGC GTG GAT CCT GGC CTG GCC GGC CTG CTG GGT 
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 
Gin Arg Ala Pro Arg Ser Gin Gin Pro Phe Val Val Thr Phe Phe Arg 
240 245 250 

GCC AGT CCG AGT CCC ATC CGC ACC CCT CGG GCA GTG AGG CCA CTG AGG 
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 
Arg Arg Gin Pro Lys Lys Ser Asn Glu Leu Pro Gin Ala Asn Arg Leu 
270 275 280 285 



1056 



1104 



1152 



1200 



1248 



1296 



1344 



CCA GGG ATC TTT GAT GAC GTC CAC GGC TCC CAC GGC CGG CAG GTC TGC 
30 Pro Gly lie Phe Asp Asp Val His 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 
Arg Arg His Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Leu Asp 
35 305 310 315 

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

40 

TGC TCC TTC CCA CTG GAC TCC TGC ATG AAT GCC ACC AAC CAC GCC ATC 
Cys Ser Phe Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His Ala He 
335 340 345 

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



1392 



1440 



1488 



1536 



1584 



WO 94/03200 PCT/US93/07231 



- 134 - 

TGC TGT GCA CCC ACC AAG CT6 AGC GCC ACC TCT GTG CTC TAC TAT GAC 1632 
Cys Cys Ala Fro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp 
370 375 380 

5 AGC AGC AAC AAC GTC ATC CTG CGC AAA GCC CGC AAC ATG GTG GTC AAG 1680 * 

Ser Ser Asn Asn Val lie Leu Arg Lys Ala Arg Asn Met Val Val Lys 
385 390 395 

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

(2) INFORMATION FOR SEQ ID NO: 21: 

15 

(i) SEQUENCE CHARACTERISTICS : 

(A) LENGTH: 402 amino acids 

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

20 

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: 

25 Met Thr Ala Leu Fro Gly Fro Leu Trp Leu Leu Gly Leu Ala Leu Cys 
15 10 15 

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

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 Fro Pro 
35 50 55 60 

Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro Leu Phe Met Leu Asp Leu 
65 70 75 80 

40 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 

45 

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 lie Pro Ala Gly Glu Ala Val Thr Ala Ala 
50 130 135 140 



WO 94/03200 



PCT/US93/07231 



- 135 - 

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

Leu His Val Ser Het Phe Gin Val Val Gin Glu Gin Ser Asn Arg Glu 
5 165 170 175 

Ser Asp Leu Phe Phe Leu Asp Leu Gin Thr Leu Arg Ala Gly Asp Glu 
180 185 190 

10 Gly Trp Leu Val Leu Asp Val Thr Ala Ala Ser Asp Cys Trp Leu Leu 
195 200 205 



15 



20 



Lys Arg 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 
225 230 235 240 

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 



25 Pro Lys Lys Ser Asn Glu Leu Pro Gin Ala Asn Arg Leu Pro Gly He 
275 280 285 



30 



35 



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 Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ser Phe 
325 330 335 

Pro Leu Asp Ser Cys Het Asn Ala Thr Asn His Ala He Leu Gin Ser 
340 345 350 



40 Leu Val His Leu Het Lys Pro Asn Ala Val Pro Lys Ala Cys Cys Ala 
355 360 365 



45 



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

Asn Val He Leu Arg Lys Ala Arg Asn Het Val Val Lys Ala Cys Gly 

385 390 395 400 



50 



Cys His 



WO 94/03200 



PCT/US93/07231 



- 136 - 

(2) INFORMATION FOR SEQ ID NO: 22: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1926 base pairs 
5 (B) TYPE: nucleic acid 

(C) STRAND EDNESS: single 

(D) TOPOLOGY: linear 

(Vi) ORIGINAL SOURCE: 
10 (A) ORGANISM: HURIDAE 

(F) TISSUE TYPE: EMBRYO 

(ix) FEATURE: 

(A) NAME/KEY: CDS 
15 (B) LOCATION: 93. .1289 

(D) OTHER INFORMATION: /function* "OSTEOGENIC PROTEIN" 
/products "mOP2-PP" 
/note= "mOP2 cDNA" 

20 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: 

GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC CCGACCAGCT 60 

25 ACCAGTGGAT GCGCGCCGGC TGAAAGTCCG AG ATG GCT ATG CGT CCC GGG CCA 113 

Met Ala Met Arg Pro Gly Pro 
1 5 

CTC TGG CTA TTG GGC CTT GCT CTG TGC GCG CTG GGA GGC GGC CAC GGT 161 
30 Leu Trp Leu Leu Gly Leu Ala Leu Cys Ala Leu Gly Gly Gly His Gly 
10 15 20 

CCG CGT CCC CCG CAC ACC TGT CCC CAG CGT CGC CTG GGA GCG CGC GAG 209 
Pro Arg Pro Pro His Thr Cys Pro Gin Arg Arg Leu Gly Ala Arg Glu 
35 25 30 35 

CGC CGC GAC ATG CAG CGT GAA ATC CTG GCG GTG CTC GGG CTA CCG GGA 257 
Arg Arg Asp Met Gin Arg Glu He Leu Ala Val Leu Gly Leu Pro Gly 
40 45 50 55 

40 

CGG CCC CGA CCC CGT GCA CAA CCC GCC GCT GCC CGG CAG CCA GCG TCC 305 
Arg Pro Arg Pro Arg Ala Gin Pro Ala Ala Ala Arg Gin Pro Ala Ser 
60 65 70 

45 GCG CCC CTC TTC ATG TTG GAC CTA TAC CAC GCC ATG ACC GAT GAC GAC 353 
Ala Pro Leu Phe Met Leu Asp Leu Tyr His Ala Met Thr Asp Asp Asp 
75 80 85 

GAC GGC GGG CCA CCA CAG GCT CAC TTA GGC CGT GCC GAC CTG GTC ATG 401 
50 Asp Gly Gly Pro Pro Gin Ala His Leu Gly Arg Ala Asp Leu Val Met 
90 95 100 



WO 94/03200 



PCI7US93/07231 



- 137 - 



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



449 



CCA CAC TGG AAG GAA TTC CAC TIT GAC CTA ACC CAG ATC CCT GCT GGG 
Fro His Trp Lys Glu Phe His Phe Asp Leu Thr Gin He Pro Ala Gly 
120 125 130 135 



497 



GAG GCT GTC ACA GCT GCT GAG TTC CGG ATC TAC AAA GAA CCC AGC ACC 
10 Glu Ala Val Thr Ala Ala Glu Phe Arg He Tyr Lys Glu Pro Ser Thr 

140 145 150 



545 



CAC CCG CTC AAC ACA ACC CTC CAC ATC AGC ATG TTC GAA GTG GTC CAA 
His Pro Leu Asn Thr Thr Leu His He Ser Het Phe Glu Val Val Gin 
15 155 160 165 



593 



20 



GAG CAC TCC AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT CTT CAG ACG 641 
Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gin Thr 
170 175 180 

CTC CGA TCT GGG GAC GAG GGC TGG CTG GTG CTG GAC ATC ACA GCA GCC 689 
Leu Arg Ser Gly Asp Glu Gly Trp Leu Val Leu Asp He Thr Ala Ala 
185 190 195 



25 AGT GAC CGA TGG CTG CTG AAC CAT CAC AAG GAC CTG GGA CTC CGC CTC 
Ser Asp Arg Trp Leu Leu Asn Bis His Lys Asp Leu Gly Leu Arg Leu 
200 205 210 215 



737 



TAT GTG GAA ACC GCG GAT GGG CAC AGC ATG GAT CCT GGC CTG GCT GGT 
30 Tyr Val Glu Thr Ala Asp Gly His Ser Het Asp Pro Gly Leu Ala Gly 

220 225 230 



785 



CTG CTT GGA CGA CAA GCA CCA CGC TCC AGA CAG CCT TTC ATG GTA ACC 
Leu Leu Gly Arg Gin Ala Pro Arg Ser Arg Gin Pro Phe Het Val Thr 
35 235 240 245 

TTC TTC AGG GCC AGC CAG AGT CCT GTG CGG GCC CCT CGG £CA GCG AGA 
Phe Phe Arg Ala Ser Gin Ser Pro Val Arg Ala Pro Arg Ala Ala Arg 
250 255 260 



40 



CCA CTG AAG AGG AGG CAG CCA AAG AAA ACG AAC GAG CTT CCG CAC CCC 
Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn Glu Leu Pro His Pro 
265 270 275 



833 



881 



929 



45 AAC AAA CTC CCA GGG ATC TTT GAT GAT GGC CAC GGT TCC CGC GGC AGA 
Asn Lys Leu Pro Gly He Phe Asp Asp Gly His Gly Ser Arg Gly Arg 
280 285 290 295 



977 



GAG GTT TGC CGC AGG CAT GAG CTC TAC GTC AGC TTC CGT GAC CTT GGC 
50 Glu Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly 

300 305 310 



1025 



WO 94/03200 PCT/US93/07231 



- 138 - 



TGG CTG GAC TGG GTC ATC GCC CCC CAG GGC TAC TCT GCC TAT TAC TGT 1073 
Trp Leu Asp Trp Val lie Ala Pro Gin Gly Tyr Ser Ala Tyr Tyr Cys 
315 320 325 

5 GAG GGG GAG TGT GCT TTC CCA CTG GAC TCC TGT ATG AAC GCC ACC AAC 1121 
Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys Met Asn Ala Thr Asn 
330 335 340 

CAT GCC ATC TTG CAG TCT CTG GTG CAC CTG ATG AAG CCA GAT GTT GTC 1169 
10 His Ala He Leu Gin Ser Leu Val His Leu Met Lys Pro Asp Val Val 
345 350 355 

CCC AAG GCA TGC TGT GCA CCC ACC AAA CTG AGT GCC ACC TCT GTG CTG 1217 
Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu 
15 360 365 370 375 

TAC TAT GAC AGC AGC AAC AAT GTC ATC CTG CGT AAA CAC CGT AAC ATG 1265 
Tyr Tyr Asp Ser Ser Asn Asn Val He Leu Arg Lys His Arg Asn Met 
380 385 390 

20 

GTG GTC AAG GCC TGT GGC TGC CAC TGAGGCCCCG CCCAGCATCC TGCTTCTACT 1319 
Val Val Lys Ala Cys Gly Cys His 
395 



25 


ACCTTACCAT 


CTGGCCGGGC 


CCCTCTCCAG 


AGGCAGAAAC 


CCTTCTATGT 


TATCATAGCT 


1379 




CAGACAGGGG 


CAATGGGAGG 


CCCTTCACTT 


CCCCTGGCCA 


CTTCCTGCTA 


AAATTCTGGT 


1439 


30 


CTTTCCCAGT 


TCCTCTGTCC 


TTCATGGGGT 


TTCGGGGCTA 


TCACCCCGCC 


CTCTCCATCC 


1499 


TCCTACCCCA 


AGCATAGACT 


GAATGCACAC 


AGCATCCCAG 


AGCTATGCTA 


ACTGAGAGGT 


1559 




CTGGGGTCAG 


CACTGAAGGC 


CCACATGAGG 


AAGACTGATC 


CTTGGCCATC 


CTCAGCCCAC 


1619 


35 


AATGGCAAAT 


TCTGGATGGT 


CTAAGAAGGC 


CCTGGAATTC 


TAAACTAGAT 


GATCTGGGCT 


1679 




CTCTGCACCA 


TTCATTGTGG 


CAGTTGGGAC 


ATTTTTAGGT 


ATAACAGACA 


CATACACTTA 


1739 


40 


GATCAATGCA 


TCGCTGTACT 


CCTTGAAATC 


AGAGCTAGCT 


TGTTAGAAAA 


AGAATCAGAG 


1799 


CCAGGTATAG 


CGGTGCATGT 


CATTAATCCC 


AGCGCTAAAG 


AGACAGAGAC 


AGGAGAATCT 


1859 




CTGTGAGTTC 


AAGGCCACAT 


AGAAAGAGCC 


TGTCTCGGGA 


GCAGGAAAAA 


AAAAAAAAAC 


1919 


45 


GGAATTC 












1926 



WO 94/03200 



PCT/US93/07231 



- 139 - 

(2) INFORMATION FOR SEQ ID NO: 23: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 399 amino acids 
5 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: 

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

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 lie Leu 
35 40 45 

20 

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

Ala Ala Arg Gin Pro Ala Ser Ala Pro Leu Phe Met Leu Asp Leu Tyr 
25 65 70 75 80 

His Ala Met Thr Asp Asp Asp Asp Gly Gly Pro Pro Gin Ala His Leu 
85 90 95 

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

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

35 

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

He Tyr Lys Glu Pro Ser Thr His Pro Leu Asn Thr Thr Leu His He 
40 145 150 155 160 

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

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

Val Leu Asp He Thr Ala Ala Ser Asp Arg Trp Leu Leu Asn His His 
195 200 205 

50 

Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Ala Asp Gly His Ser 
.210 215 220 



WO 94/03200 



PCT/US93/07231 



- 140 - 

Met Asp Pro Gly Leu Ala Gly Leu Leu Gly Arg Gin Ala Pro Arg Ser 
225 230 235 240 

Arg Gin Pro Pbe Het Val Thr Phe Phe Arg Ala Ser Gin Ser Pro Val 
5 245 250 255 

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

10 Thr Asn Glu Leu Pro His Pro Asn Lys Leu Pro Gly lie Phe Asp Asp 
275 280 285 

Gly His Gly Ser Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr 
290 295 300 

15 

Val Ser Phe Arg Asp Leu Gly Trp Leu Asp Trp Val He Ala Pro Gin 
305 310 315 320 

Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp 
20 325 330 335 

Ser Cys Het Asn Ala Thr Asn His Ala He Leu Gin Ser Leu Val His 
340 345 350 

25 Leu Het Lys Pro Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys 
355 360 365 

Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val He 
370 375 380 

30 

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

(2) INFORMATION FOR SEQ ID NO: 24: 

35 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1368 base pairs 

(B) TYPE: nucleic acid 

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

(ii) MOLECULE TYPE: cDNA 



45 (ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 1..1368 



50 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: 



WO 94/03200! 



PCT/US93/07231 



- 141 - 



ATG TCG GGA CTG CGA AAC ACC TCG GAG GCC GTT GCA GTG CTC GCC TCC 
Met Ser Gly Leu Arg Asn Thr Ser Glu Ala Val Ala Val Leu Ala Ser 
1 5 10 15 

5 CTG GGA CTC GGA ATG GTT CTG CTC ATG TTC GTG GCG ACC ACG CCG CCG 
Leu Gly Leu Gly Bet Val Leu Leu Met Phe Val Ala Thr Thr Pro Pro 
20 25 30 

GCC GTT GAG GCC ACC CAG TCG GGG ATT TAC ATA GAC AAC GGC AAG GAC 
10 Ala Val Glu Ala Thr Gin Ser Gly He Tyr He Asp Asn Gly Lys Asp 
35 40 45 



15 



20 



40 



CAG ACG ATC ATG CAC AGA GTG CTG AGC GAG GAC GAC AAG CTG GAC GTC 
Gin Thr He Met His Arg Val Leu Ser Glu Asp Asp Lys Leu Asp Val 
50 55 60 

TCG TAC GAG ATC CTC GAG TTC CTG GGC ATC GCC GAA CGG CCG ACG CAC 
Ser Tyr Glu He Leu Glu Phe Leu Gly He Ala Glu Arg Pro Thr His 
65 70 75 80 

CTG AGC AGC CAC CAG TIG TCG CTG AGG AAG TCG GCT CCC AAG TTC CTG 
Leu Ser Ser His Gin Leu Ser Leu Arg Lys Ser Ala Pro Lys Phe Leu 
85 90 95 



25 CTG GAC GTC TAC CAC CGC ATC ACG GCG GAG GAG GGT CTC AGC GAT CAG 
Leu Asp Val Tyr His Arg He Thr Ala Glu Glu Gly Leu Ser Asp Gin 
100 105 HO 

GAT GAG GAC GAC GAC TAC GAA CGC GGC CAT CGG TCC AGG AGG AGC GCC 
30 Asp Glu Asp Asp Asp Tyr Glu Arg Gly His Arg Ser Arg Arg Ser Ala 
115 120 125 

GAC CTC GAG GAG GAT GAG GGC GAG CAG CAG AAG AAC TTC ATC ACC GAC 
Asp Leu Glu Glu Asp Glu Gly Glu Gin Gin Lys Asn Phe He Thr Asp 
35 130 135 140 

CTG GAC AAG CGG GCC ATC GAC GAG AGC GAC ATC ATC ATG ACC TTC CTG 
Leu Asp Lys Are Ala He Asp Glu Ser Asp He He Met Thr Phe Leu 
145 150 155 160 



AAC AAG CGC CAC CAC AAT GTG GAC GAA CTG CGT CAC GAG CAC GGC CGT 
Asn Lys Arg His His Asn Val Asp Glu Leu Arg His Glu His Gly Arg 
165 170 175 



45 CGC CTG TGG TTC GAC GTC TCC AAC GTG CCC AAC GAC AAC TAC CTG GTG 
Arc Leu Trp Phe Asp Val Ser Asn Val Pro Asn Asp Asn Tyr Leu Val 
180 185 190 

ATG GCC GAG CTG CGC ATC TAT CAG AAC GCC AAC GAG GGC AAG TGG CTG 
50 Met Ala Glu Leu Arg He Tyr Gin Asn Ala Asn Glu Gly Lys Trp Leu 
195 200 205 



48 



96 



144 



192 



240 



288 



336 



384 



432 



480 



528 



576 



624 



WO 94/03200 



PCI7US93/07231 



- 142 - 



ACC GCC AAC AGG GAG TTC ACC ATC ACG GTA TAC GCC ATT GGC ACC GGC 
Thr Ala Asn Arg Glu Fhe Thr lie Thr Val Tyr Ala He Gly Thr Gly 
210 215 220 



672 



ACG CTG GGC CAG CAC ACC ATG GAG CCG CTG TCC TCG GTG AAC ACC ACC 
Thr Leu Gly Gin His Thr Met Glu Pro Leu Ser Ser Val Asn Thr Thr 
225 230 235 240 



720 » 



GGG GAC TAC GTG GGC TGG TTG GAG CTC AAC GTG ACC GAG GGC CTG CAC 
10 Gly Asp Tyr Val Gly Trp Leu Glu Leu Asn Val Thr Glu Gly Leu His 

245 250 255 



768 



GAG TGG CTG GTC AAG TCG AAG GAC AAT CAT GGC ATC TAC ATT GGA GCA 
Glu Trp Leu Val Lys Ser Lys Asp Asn His Gly He Tyr He Gly Ala 
15 260 265 270 



816 



20 



CAC GCT GTC AAC CGA CCC GAC CGC GAG GTG AAG CTG GAC GAC ATT GGA 864 
His Ala Val Asn Arg Pro Asp Arg Glu Val Lys Leu Asp Asp He Gly 
275 280 285 

CTG ATC CAC CGC AAG GTG GAC GAC GAG TTC CAG CCC TTC ATG ATC GGC 912 
Leu He His Arg Lys Val Asp Asp Glu Phe Gin Fro Phe Net He Gly 
290 295 300 



25 TTC TTC CGC GGA CCG GAG CTG ATC AAG GCG ACG GCC CAC AGC AGC CAC 
Phe Phe Arg Gly Pro Glu Leu He Lys Ala Thr Ala His Ser Ser His 
305 310 315 320 



960 



CAC AGG AGC AAG CGA AGC GCC AGC CAT CCA CGC AAG CGC AAG AAG TCG 
30 His Arg Ser Lys Arg Ser Ala Ser His Pro Arg Lys Arg Lys Lys Ser 

325 330 335 



1008 



35 



GTG TCG CCC AAC AAC GTG CCG CTG CTG GAA CCG ATG GAG AGC ACG CGC 
Val Ser Pro Asn Asn Val Pro Leu Leu Glu Pro Met Glu Ser Thr Arg 
340 345 350 



1056 



40 



AGC TGC CAG ATG CAG ACC CTG TAC ATA GAC TTC AAG GAT CTG GGC TGG 1104 
Ser Cys Gin Met Gin Thr Leu Tyr He Asp Phe Lys Asp Leu Gly Trp 
355 360 . 365 

CAT GAC TGG ATC ATC GCA CCA GAG GGC TAT GGC GCC TTC TAC TGC AGC 1152 
His Asp Trp He He Ala Pro Glu Gly Tyr Gly Ala Phe Tyr Cys Ser 
370 375 380 



45 GGC GAG TGC AAT TTC CCG CTC AAT GCG CAC ATG AAC GCC ACG AAC CAT 
Gly Glu Cys Asn Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 
385 390 395 400 



1200 



GCG ATC GTC CAG ACC CTG GTC CAC CTG CTG GAG CCC AAG AAG -GTG CCC 
50 Ala He Val Gin Thr Leu Val His Leu Leu Glu Pro Lys Lys Val Pro 

405 410 415 



1248 



WO 94/03200 



PCT/US93/07231 



- 143 - 

AAG CCC TGC TGC GCT CCG ACC AGG CTG GGA GCA CTA CCC GTT CTG XAC 1296 
Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Ala Leu Pro Val Leu Tyr 
420 425 430 

5 CAC CTG AAC GAC GAG AAT GTG AAC CTG AAA AAG TAT AGA AAC ATG ATT 1344 
His Leu Asn Asp Glu Asn Val Asn Leu Lys Lys Tyr Arg Asn Met lie 
435 440 445 

GTG AAA TCC TGC GGG TGC CAT TGA 1368 
10 Val Lys Ser Cys Gly Cys His 
450 455 

(2) INFORMATION FOB SEQ ID NO: 25: 

15 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 455 amino acids 

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

20 

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: 

25 Met Ser Gly Leu Arg Asn Thr Ser Glu Ala Val Ala Val Leu Ala Ser 
1 5 10 15 

Leu Gly Leu Gly Met Val Leu Leu Met Phe Val Ala Thr Thr Pro Pro 
20 25 30 

30 

Ala Val Glu Ala Thr Gin Ser Gly lie Tyr lie Asp Asn Gly Lys Asp 
35 40 45 

Gin Thr He Met His Arg Val Leu Ser Glu Asp Asp Lys Leu Asp Val 
35 50 55 60 

Ser Tyr Glu He Leu Glu Phe Leu Gly He Ala Glu Arg Pro Thr His 
65 70 75 60 

40 Leu Ser Ser His Gin Leu Ser Leu Arg Lys Ser Ala Pro Lys Phe Leu 

85 90 95 

Leu Asp Val Tyr His Arg He Thr Ala Glu Glu Gly Leu Ser Asp Gin 
100 105 110 

45 

Asp Glu Asp Asp Asp Tyr Glu Arg Gly His Arg Ser Arg Arg Ser Ala 
115 120 125 

Asp Leu Glu Glu Asp Glu Gly Glu Gin Gin Lys Asn Phe He Thr Asp 
50 130 135 140 



WO 94/03200 



PCI7US93/07231 



- 144 - 

Leu Asp Lys Arg Ala lie Asp Glu Ser Asp He He Met Tbr Phe Leu 
145 150 155 160 

Asn Lys Arg His His Asn Val Asp Glu Leu Arg His Glu His Gly Arg 
5 165 170 175 

Arg Leu Trp Phe Asp Val Ser Asn Val Pro Asn Asp Asn Tyr Leu Val 
180 185 190 

10 Het Ala Glu Leu Arg He Tyr Gin Asn Ala Asn Glu Gly Lys Trp Leu 
195 200 205 



15 



Thr Ala Asn Arg Glu Phe Thr He Thr Val Tyr Ala He Gly Thr Gly 
210 215 220 

Thr Leu Gly Gin His Thr Het Glu Pro Leu Ser Ser Val Asn Thr Thr 
225 230 235 240 



20 



Gly Asp Tyr Val Gly Trp Leu Glu Leu Asn Val Thr Glu Gly Leu His 
245 250 255 



Glu Trp Leu Val Lys Ser Lys Asp Asn His Gly He Tyr He Gly Ala 
260 265 270 

25 His Ala Val Asn Arg Pro Asp Arg Glu Val Lys Leu Asp Asp He Gly 
275 280 285 



30 



Leu He His Arg Lys Val Asp Asp Glu Phe Gin Pro Phe Met He Gly 
290 295 300 

Phe Phe Arg Gly Pro Glu Leu He Lys Ala Thr Ala His Ser Ser His 

305 310 315 320 



His Arg Ser Lys Arg Ser Ala Ser His ?ro Arg Lys Arg Lys Lys Ser 
35 325 330 335 

Val Ser Pro Asn Asn Val Pro Leu Leu Glu Pro Met Glu Ser Thr Arg 
340 345 350 

40 Ser Cys Gin Met Gin Thr Leu Tyr He Asp Phe Lys Asp Leu Gly Trp 
355 360 365 



45 



His Asp Trp He He Ala Pro Glu Gly Tyr Gly Ala Phe Tyr Cys Ser 
370 375 380 

Gly Glu Cys Asn Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 
385 390 395 400 



50 



Ala He Val Gin Thr Leu Val His Leu Leu Glu Pro Lys Lys Val Pro 
405 410 415 



WO 94/03200 



PCT/US93/07231 



- 145 - 

Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Ala Leu Pro Val Leu Tyr 
420 425 430 

Bis Leu Asn Asp Glu Asn Val Asn Leu Lys Lys Tyr Arg Asn Met lie 
5 435 440 445 

Val Lys Ser Cys Gly Cys His 
450 455 

10 

(2) INFORMATION FOR SEQ ID NO: 26: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 104 amino acids 
15 (B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



20 



35 



(ii) MOLECULE TYPE: protein 



(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..104 

25 (D) OTHER INFORMATION: /notes "BHP3" 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26: 

30 Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp lie Gly Trp Ser 

1 5 10 15 



Glu Trp lie lie Ser Pro Lys Ser Pbe Asp Ala Tyr Tyr Cys Ser Gly 
20 25 30 

Ala Cys Gin Phe Pro Het Pro Lys Ser Leu Lys Pro Ser Asn His Ala 
35 40 45 



Thr lie Gin Ser He Val Ala Arg Ala Val Gly Val Val Pro Gly He 
40 50 55 60 

Pro Glu Pro Cys Cys Val Pro Glu Lys Het Ser Ser Leu Ser He Leu 
65 70 75 80 

45 Phe Phe Asp Glu Asn Lys Asn Val Val Leu Lys Val Tyr Pro Asn Het 

85 90 95 



Thr Val Glu Ser Cys Ala Cys Arg 
100 



50 



WO 94/03200 PCT/US93/07231 



10 



30 



- 146 - 



(2) INFORMATION FOR SEQ ID NO: 27: 

(i) SEQUENCE CHARACTERISTICS : 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HOMO SAPIENS 



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

(B) LOCATION: 1..102 
(D) OTHER INFORMATION: /note= "BMP5" 

20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: 

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

25 Asp Trp He He Ala Pro Glu Gly Tyr Ala Ala Phe Tyr Cys Asp Gly 

20 25 30 



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

He Val Gin Thr Leu Val His Leu Met Phe Pro Asp His Val Pro Lys 
50 55 -60 

Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala lie Ser Val Leu Tyr Phe 
35 65 70 75 80 

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

40 Arg Ser Cys Gly Cys His 

100 

(2) INFORMATION FOR SEQ ID NO: 28: 

45 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



50 



(ii) MOLECULE TYPE: protein 



WO 94/03200 



PCT/US93/07231 



- 147 - 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HOMO SAPIENS 

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

(B) LOCATION: 1..102 

(0) OTHER INFORMATION: /note= "BMP6" 

10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: 

Cys Are Lys His Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Gin 
15 10 15 

15 Asp Trp He He Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly 

20 25 30 



20 



Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 
35 40 45. 

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



Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala He Ser Val Leu Tyr Phe 

25 65 70 75 80 

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



30 Arg Ala Cys Gly Cys His 

100 

(2) INFORMATION FOR SEQ ID NO: 29: 

35 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

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

40 (ii) MOLECULE TYPE: protein 



(ix) FEATURE: 

(A) NAME/KEY: Protein 
45 (B) LOCATION: 1..102 

(D) OTHER INFORMATION: /label* OPX 

/note= "WHEREIN EACH XAA IS INDEPENDENTLY SELECTED 
FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS 
AS DEFINED IN THE SPECIFICATION (SECTION II.B.2.)" 



WO 94/03200 PCI7US93/07231 



- 148 - 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: 

Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xaa Asp Leu Gly Trp Xaa 
1 5 10 15 

5 Asp Trp Xaa lie Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly 

20 25 30 

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

He Xaa Gin Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys 
50 55 60 

15 Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 

65 70 75 80 



20 



25 



45 



Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xaa Arg Asn Met Val Val 
85 90 95 

Xaa Ala Cys Gly Cys His 
100 

(2) INFORMATION FOR SEQ ID N0:30: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 

35 (ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..97 

(D) OTHER INFORMATION: /label* GENERIC-SEQ5 

/note= "WHEREIN EACH XAA IS INDEPENDENTLY SELECTED 
40 FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS 

AS DEFINED IN THE SPECIFICATION." 



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

Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa Xaa Xaa 
15 10 15 



Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro 
50 20 25 30 



WO 94/03200 PCI7US93/07231 



- 149 - 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa 
35 40 45 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro 
5 50 55 60 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
65 70 75 80 

10 Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys 

85 90 95 



15 



25 



Xaa 



(2) INFORMATION FOR SEQ ID N0:31: 



(i) SEQUENCE CHARACTERISTICS: 

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

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

30 (D) OTHER INFORMATION: /label- GENERIC-SEQ6 

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

35 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31: 

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

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

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

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

50 Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 

65 70 75 80 



40 



WO 94/03200 PCT/US93/07231 



- 150 - 

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

Xaa Xaa Cys Xaa Cys Xaa 
5 100 

(2) INFORMATION FOB SEQ ID NO: 32: 

(i) SEQUENCE CHARACTERISTICS: 
10 (A) LENGTH: 1247 base pairs 

(B) TYPE: nucleic acid 

(C) STRAND EDNESS: single 

(D) TOPOLOGY: linear 

15 (ii) MOLECULE TYPE: cDNA 

(Vi) ORIGINAL SOURCE: 

(A) ORGANISM: HOMO SAPIENS 
(F) TISSUE TYPE: BRAIN 

20 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 84.. 1199 

(D) OTHER INFORMATION: /product- "GDF-1" 
25 /note= "GDF-1 CDNA" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: 

30 GGGGACACCG GCCCCGCCCT CAGCCCACTG GTCCCGGGCC GCCGCGGACC CTGCGCACTC 60 

TCTGGTCATC GCCTGGGAGG AAG ATG CCA CCG CCG CAG CAA GGT CCC TGC 110 

Met Pro Pro Pro Gin Gin Gly Pro Cys 
1 5 

35 

GGC CAC CAC CTC CTC CTC CTC CTG GCC CTG CTG CTG CCC TCG CTG CCC 158 
Gly His His Leu Leu Leu Leu Leu Ala Leu Leu Leu Pro Ser Leu Pro 
10 15 20 25 

40 CTG ACC CGC GCC CCC GTG CCC CCA GGC CCA GCC GCC GCC CTG CTC CAG 206 
Leu Thr Arg Ala Pro Val Pro Pro Gly Pro Ala Ala Ala Leu Leu Gin 
30 35 40 

GCT CTA GGA CTG CGC GAT GAG CCC CAG GGT GCC CCC AGG CTC CGG CCG 254 
45 Ala Leu Gly Leu Arg Asp Glu Pro Gin Gly Ala Pro Arg Leu Arg Pro 
45 50 55 

GTT CCC CCG GTC ATG TGG CGC CTG TTT CGA CGC CGG GAC CCC CAG GAG 302 
Val Pro Pro Val Met Trp Arg Leu Phe Arg Arg Arg Asp Pro Gin Ulu 
50 60 65 70 



WO 94/03200 



PCT/US93/07231 



- 151 - 

ACC AGG TCT GGC TCG CGG CGG ACG TCC CCA GGG GTC ACC CTG CAA CCG 350 

Thr Arg Ser Gly Ser Arg Arg Thr Ser Pro Gly Val Thr Leu Gin Pro 
75 80 85 

TGC CAC GTG GAG GAG CTG GGG GTC GCC GGA AAC ATC GTG CGC CAC ATC 398 

Cys His Val Glu Glu Leu Gly Val Ala Gly Asn lie Val Arg His lie 
90 95 100 105 



CCG GAC CGC GGT GCG CCC ACC CGG GCC TCG GAG CCT GTC TCG GCC GCG 
10 Pro Asp Arg Gly Ala Pro Thr Arg Ala Ser Glu Pro Val Ser Ala Ala 

110 115 120 



446 



GGG CAT TGC CCT GAG TGG ACA GTC GTC TTC GAC CTG TCG GCT GTG GAA 
Gly His Cys Pro Glu Trp Thr Val Val Phe Asp Leu Ser Ala Val Glu 
15 125 130 135 



494 



20 



CCC GCT GAG CGC CCG AGC CGG GCC CGC CTG GAG CTG CGT TTC GCG GCG 542 
Pro Ala Glu Arg Pro Ser Arg Ala Arg Leu Glu Leu Arg Phe Ala Ala 
140 145 150 

GCG GCG GCG GCA GCC CCG GAG GGC GGC TGG GAG CTG AGC GTG GCG CAA 590 
Ala Ala Ala Ala Ala Pro Glu Gly Gly Trp Glu Leu Ser Val Ala Gin 
155 160 165 



25 GCG GGC CAG GGC GCG GGC GCG GAC CCC GGG CCG GTG CTG CTC CGC CAG 
Ala Gly Gin Gly Ala Gly Ala Asp Pro Gly Pro Val Leu Leu Arg Gin 
170 175 180 185 



638 



TTG GTG CCC GCC CTG GGG CCG CCA GTG CGC GCG GAG CTG CTG GGC GCC 
30 Leu Val Pro Ala Leu Gly Pro Pro Val Arg Ala Glu Leu Leu Gly Ala 

190 195 200 



35 



GCT TGG GCT CGC AAC GCC TCA TGG CCG CGC AGC CTC CGC CTG GCG CTG 
Ala Trp Ala Arg Asn Ala Ser Trp Pro Arg Ser Leu Arg Leu Ala Leu 
205 210 215 



686 



734 



40 



GCG CTA CGC CCC CGG GCC CCT GCC GCC TGC GCG CGC CTG GCC GAG GCC 782 
Ala Leu Arg Pro Arg Ala Pro Ala Ala Cys Ala Arg Leu Ala Glu Ala 
220 225 230 

TCG CTG CTG CTG GTG ACC CTC GAC CCG CGC CTG TGC CAC CCC CTG GCC 830 
Ser Leu Leu Leu Val Thr Leu Asp Pro Arg Leu Cys His Pro Leu Ala 
235 240 245 



45 CGG CCG CGG CGC GAC GCC GAA CCC GTG TTG GGC GGC GGC CCC <XG GGC 
Arg Pro Arg Arg Asp Ala Glu Pro Val Leu Gly Gly Gly Pro Gly Gly 
250 255 260 265 



878 



GCT TGT CGC GCG CGG CGG CTG TAC GTG AGC TTC CGC GAG GTG GGC TGG 
50 Ala Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp 

270 275 280 



926 



WO 94/03200 



PCT/US93/07231 



- 152 - 

CAC CGC TGG GTC ATC GCG CCG CGC GGC TTC CTG GCC AAC TAC TGC CAG 974 
His Are Trp Val He Ala Pro Arg Gly Phe Leu Ala Asn Tyr Cys Gin 
285 290 295 

5 GGT CAG TGC GCG CTG CCC GTC GCG CTG TCG GGG TCC GGG GGG CCG CCG 1022 
Gly Gin Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly Gly Pro Pro 
300 305 310 

GCG CTC AAC CAC GCT GTG CTG CGC GCG CTC ATG CAC GCG GCC GCC CCG 1070 
10 Ala Leu Asn His Ala Val Leu Arg Ala Leu Met His Ala Ala Ala Pro 
315 320 325 

GGA GCC GCC GAC CTG CCC TGC TGC GTG CCC GCG CGC CTG TCG CCC ATC 1118 
Gly Ala Ala Asp Leu Pro Cys Cys Val Pro Ala Arg Leu Ser Pro He 
15 330 335 340 345 

TCC GTG CTC TTC TTT GAC AAC AGC GAC AAC GTG GTG CTG CGG CAG TAT 1166 
Ser Val Leu Phe Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gin Tyr 
350 355 360 

20 

GAG GAC ATG GTG GTG GAC GAG TGC GGC TGC CGC TAACCCGGGG CGGGCAGGGA 1219 
Glu Asp Met Val Val Asp Glu Cys Gly Cys Arg 
365 370 

25 CCCGGGCCCA ACAATAAATG CCGCGTGG 1247 

(2) INFORMATION FOR SEQ ID NO: 33: 

30 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 372 amino acids 

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

35 (ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33: 

Het Pro Pro Pro Gin Gin Gly Pro Cys Gly His His Leu Leu Leu Leu 
40 1 5 10 15 

Leu Ala Leu Leu Leu Pro Ser Leu Pro Leu Thr Arg Ala Pro Val Pro 
20 25 30 

45 Pro Gly Pro Ala Ala Ala Leu Leu Gin Ala Leu Gly Leu Arg Asp Glu 
35 40 45 



50 



Pro Gin Gly Ala Pro Arg Leu Arg Pro Val Pro Pro Val Met Trp Arg 
50 55 60 

Leu Phe Arg Arg Arg Asp Pro Gin Glu Thr Arg Ser Gly Ser Arg Arg 
65 70 75 80 



WO 94/03200 



PCT/US93/07231 



- 153 - 

Tbr Ser Pro Gly Val Thr Leu Gin Pro Cys His Val Glu Glu Leu Gly 
85 90 95 

Val Ala Gly Asn He Val Arg His He Pro Asp Arg Gly Ala Pro Thr 
5 100 105 110 

Arg Ala Ser Glu Pro Val Ser Ala Ala Gly His Cys Pro Glu Trp Thr 
115 120 125 

10 Val Val Phe Asp Leu Ser Ala Val Glu Pro Ala Glu Arg Pro Ser Arg 
130 135 140 



15 



20 



Ala Arg Leu Glu Leu Arg Phe Ala Ala Ala Ala Ala Ala Ala Pro Glu 
145 150 155 160 

Gly Gly Trp Glu Leu Ser Val Ala Gin Ala Gly Gin Gly Ala Gly Ala 
165 170 175 

Asp Pro Gly Pro Val Leu Leu Arg Gin Leu Val Pro Ala Leu Gly Pro 
180 185 190 

Pro Val Arg Ala Glu Leu Leu Gly Ala Ala Trp Ala Arg Asn Ala Ser 
195 200 205 



25 Trp Pro Arg Ser Leu Arg Leu Ala Leu Ala Leu Arg Pro Arg Ala Pro 
210 215 220 



30 



35 



Ala Ala Cys Ala Arg Leu Ala Glu Ala Ser Leu Leu Leu Val Thr Leu 
225 230 235 240 

Asp Pro Arg Leu Cys His Pro Leu Ala Arg Pro Arg Arg Asp Ala Clu 
245 250 255 

Pro Val Leu Gly Gly Gly Pro Gly Gly Ala Cys Arg Ala Arg Arg Leu 
260 265 270 

Tyr Val Ser Phe Arg Glu Val Gly Trp His Arg Trp Val He Ala Pro 
275 280 285 



40 Arg Gly Phe Leu Ala Asn Tyr Cys Gin Gly Gin Cys Ala Leu Pro Val 
290 295 300 



45 



Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala Leu Asn His Ala Val Leu 
305 310 315 320 



WO 94/03200 



PCI7US93/07231 



- 154 - 

Arg Ala Leu Met His Ala Ala Ala Pro Gly Ala Ala Asp Leu Pro Cys 
325 330 335 

Cys Val Pro Ala Arg Leu Ser Pro lie Ser Val Leu Phe Phe Asp Asn 
5 340 345 350 

Ser Asp Asn Val Val Leu Arg Gin Tyr Glu Asp Met Val Val Asp Glu 
355 360 365 

10 Cys Gly Cys Arg 
370 



PCT/US93/07231 



- 155 - 

What is claimed is: 

The use of a morphogen in the manufacture of a 
pharmaceutical for enhancing survival of neural 
cells at risk of dying. 

A method for enhancing survival of neural cells at 
risk of dying, the method comprising providing a 
morphogen to said cells at a concentration and for 
a time sufficient to enhance survival of said 
cells. 

The invention of claim 1 or 2 wherein said cells 
are at risk of dying due to chemical or mechanical 
trauma to nerve tissue comprising said cells. 

The invention of claim 3 wherein said trauma 
comprises a transected nerve. 

The invention of claim 3 wherein said morphogen is 
provided to said cells prior to said trauma. 

The invention of claim 3 wherein said trauma 
results in demyelination of said cells. 

The invention of claim 3 wherein said trauma 
results from exposure of said cells to a cellular 
toxin. 

The invention of claim 7 wherein said toxin 
comprises ethanol. 



WO 94/03200 PCT/US93/07231 



- 156 - 

9. The invention of claim 1 or 2 wherein said cells 
are at risk of dying due to a neuropathy. 

10. The invention of claim 9 wherein the etiology of 
5 said neuropathy is metabolic , infectious, toxic, 

autoimmune, nutritional, or ischemic. 

11. The invention of claim 10 wherein said neuropathy 
comprises Parkinson's disease, Huntington's chorea, 

10 amyotrophic lateral sclerosis, multiple sclerosis 

or Alzheimer's disease. 

12. The invention of .claim 1 or 2 wherein said cells 
are at risk of dying due a neoplastic lesion 

15 associated with nerve tissue comprising said cells. 

13. The invention of claim 12 wherein said lesion 
results from a neoplasm comprising cells of 
neuronal origin. 

20 

14 . The invention of claim 13 wherein said neoplasm 
comprises a neuroblastoma or a retinoblastoma. 

15. The invention of claim 12 wherein said lesion 
25 results from a neoplasm comprising glial cells. 

16. The invention of claim 1 or 2 wherein said neural 
cells at risk of dying comprise part of the central 
nervous system. 



30 



17. The invention of claim 16 wherein said cells 
comprise striatal basal ganglia neurons. 



WO 94/03200 



PCT/US93/07231 



- 157 - 

18. The invention of claim 16 wherein said cells 
comprise neurons of the substantia nigra. 

19. The invention of claim 1 or 2 wherein said cells at 
5 risk of dying comprise part of the peripheral 

nervous system. 

20. The invention of claim 1 or 2 wherein said 
morphogen stimulates cell adhesion molecule 

10 production in said cells. 

21. The invention of claim 20 wherein said cell 
adhesion molecule is a nerve cell adhesion 
molecule* 



15 



22. The invention of claim 21 wherein nerve cell 
adhesion molecule is selected from the group 
consisting of N-CAM-120, N-CAM-140 and N-CAM-180, 



20 23. The invention of claim 1 or 2 wherein said 

morphogen comprises an amino acid sequence sharing 
at least 70% homology with one of the sequences 
selected from the group consisting of: OP-1, OP-2, 
CBMP2 , Vgl(fx), Vgr(fx), DPP(fx), GDF-l(fx) and 

25 60A(fx). 



24. The invention of claim 23 wherein said morphogen 
comprises an amino acid sequence sharing at least 
80% homology with one of the sequences selected 
30 from the group consisting of: OP-1, OP-2, CBMP2, 

Vgl(fx), Vgr(fx), DPP(fx), GDF-l(fx), and 60A (fx). 



WO 94/03200 PCT/US93/07231 



- 158 - 

25. The invention of claim 24 wherein said morphogen 
comprises an amino acid sequence having greater 
than 60% amino acid identity with the sequence 
defined by residues 43-139 of Seq. ID No. 5 (hOPl.) 

5 

26. The invention of claim 25 wherein said morphogen 
comprises an amino acid sequence having greater 
than 65% amino acid identity with the sequence 
defined by residues 43-139 of Seq. ID No. 5 (hOPl.) 

10 

27. The invention of claim 22 wherein said morphogen 
comprises an amino acid sequence defined by 
residues 43-139 of Seq. ID No. 5 (hOPl), including 
allelic and species variants thereof. 

15 

28. A method for enhancing the survival of neural cells 
at risk of dying in a mammal, the method comprising 
the step of administering to said mammal an 
effective amount of an agent capable of stimulating 

20 production of an endogenous morphogen. 

29. The method of claim 28 wherein said agent 
stimulates production of an endogenous morphogen in 
the tissue comprising said neural cells. 

25 

30. A method for maintaining a neural pathway in a 
mammal, comprising: 

providing a morphogen to the neurons defining 
said pathway at a concentration and for a time 
30 sufficient to maintain said pathway. 



31. 



The method of claim 30 wherein said morphogen is 
provided prior to injury to said pathway. 



WO 94/03200 PCT/US93/07231 



- 159 - 

32. The method of claim 30 wherein said morphogen is 
sufficient to stimulate repair of a damaged neural 
pathway . 

5 33. The method of claim 32 wherein said damaged neural 
pathway, results from mechanical or chemical trauma 
to said pathway. 

34. The method of claim 33 wherein said trauma 
10 comprises a severed nerve. 

35. The method of claim 33 wherein said trauma 
comprises demy elinat ion of the neurons defining 
said pathway. 

15 

36. The method of claim 33 wherein said trauma results 
from exposure of the cells defining said pathway to 
a cellular toxin. 

20 37. The method of claim 36 wherein said toxin comprises 
ethanol • 

38. The method of claim 30 wherein said damaged neural 
pathway results from a neuropathy of the cells 

25 defining said pathway. 

39. The method of claim 38 wherein the etiology of said 
neuropathy is metabolic, infectious, toxic, 
autoimmune, nutritional, or ischemic. 



30 



40. The method of claim 39 wherein said neuropathy 

comprises Parkinson's disease, Huntington's chorea, 
amyotrophic lateral sclerosis, multiple sclerosis, 
or Alzheimer's disease. 



35 



WO 94/03200 PCT/US93/07231 



- 160 - 

41. The method of claim 38 wherein said neuropathy 
comprises axonal degeneration* 

42. The method of claim 38 wherein said neuropathy 
5 comprises a demyelinating neuropathy. 

43. The method of claim 30 wherein said damaged neural 
pathway results from a neoplastic lesion. 

10 44. The method of claim 43 wherein said neoplastic 

lesion is caused by a neuroblastoma or a glioma. 

45. The method of claim 30 wherein said morphogen 
stimulates cell adhesion molecule production in a 

15 cell defining said pathway. 

46. The method of claim 45 wherein said cell adhesion 
molecule is a nerve cell adhesion molecule. 

20 47. The method of claim 46 wherein nerve cell adhesion 
molecule is selected from the group consisting of 
N-CAM-120, N-CAM-140 and N- CAM- 180. 

48. The method of claim 30 or 45 wherein said morphogen 
25 comprises an amino acid sequence sharing at least 

70% homology with one of the sequences selected 
from the group consisting of: OP-1, OP-2, CBMP2, 
Vgl(fx), Vgr(fx), DPP(fx), GDF-l(fx) and 60A(fx). 



WO 94/03200 PCT/US93/07231 



- 161 - 

49. The method of claim 48 wherein said morphogen 
comprises an amino acid sequence sharing at least 
80% homology with one of the sequences selected 
from the group consisting of: OP-1, OP-2 / CBMP2, 

5 Vgl(fx), Vgr(fx), DPP(fx), GDF-l(fx), and 60A (fx). 

50. The method of claim 49 wherein said morphogen 
comprises an amino acid sequence having greater 
than 60% amino acid identity with the sequence 

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

51. The method of claim 50 wherein said morphogen 
comprises an amino acid sequence having greater 
than 65% amino acid identity with the sequence 

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

52. The method of claim 51 wherein said morphogen 
comprises an amino acid sequence defined by 
residues 43-139 of Seq. ID No. 5 (hOPl), including 

20 allelic and species variants thereof. 

53. The invention of claims 1, 2 f 30 or 46 wherein said 
morphogen comprises a polypeptide chain encoded by 
a nucleic acid that hybridizes under stringent 

25 conditions with the DNA sequence defined by 

nucleotides 1036-1341 of Seq. Id No. 16 or 
nucleotides 1390-1695 of Seq. ID No. 20. 

54. The invention of claims 1, 2, 26, 30, 45 or 51 

30 wherein said morphogen comprises a dimeric protein 

species complexed with a peptide comprising a pro 
region of a member of the morphogen family, or an 
allelic, species or other sequence variant thereof. 



4 



WO 94/03200 PCT/US93/07231 



- 162 - 

55. The invention of claim 54 wherein said dimeric 

morphogen species is noncovalently complexed with 
said peptide. 

5 56. The invention of claims 54 or 55 wherein said 

dimeric morphogen species is complexed with two 
said peptides. 

57. The invention of claims 54 or 55 wherein said 

10 peptide comprises at least the first 18 amino acids 

of a sequence defining said pro region. 

58. The invention of claim 57 wherein said peptide 
comprises the full length form of said pro region. 

15 

59. The invention of claims 54 or 55 wherein said 
peptide comprises a nucleic acid that hybridizes 
under stringent conditions with a DNA defined by 
nucleotides 136-192 of Seq. ID No. 16 , or 

20 nucleotides 157-211 of Seq. ID No. 20. 

60. The invention of claims 54 or 55 wherein said 
complex is further stabilized by exposure to a 
basic amino acid, a detergent or a carrier protein. 

25 

61. A method of maintaining a neural pathway in a 
mammal comprising: 

administering said mammal an effective amount 
of an agent capable of stimulating production of an 
30 endogenons morphogen in a cell defining said 

pathway • 



WO 94/03200 



PCI7US93/07231 



- 163 - 

62. A composition for promoting regeneration of a 
neural pathway at a site of injury in a mammal, 
comprising: 

a biocompatible, in vivo bioresorbable carrier 
5 suitable for maintaining a protein at a site in 

vivo, and 

a morphogen, such that said morphogen, when 
dispersed in said carrier and provided to said site 
of injury, is capable of stimulating neural pathway 
10 regeneration at said site. 



63. The composition of claim 62 wherein said carrier is 
structurally sufficient to assist direction of 
axonal growth. 

15 

64. The composition of claim 63 wherein said carrier 
comprises a polymeric material. 

65. The composition of claim 63 wherein said carrier 
20 comprises laminin or collagen. 

66. A device for repairing a break in a neural pathway, 
the device comprising: 

a biocompatible tubular casing comprising an 
25 exterior and an interior surface and defining a 

channel through which a neural process may 
regenerate, 

said device having a shape and dimension 
sufficient to span a break in a neural pathway, and 
30 having openings adapted to receive the ends of a 

severed nerve, and 



WO 94/03200 



PCT/US93/07231 



• 164 - 

a morphogen disposed within the channel 
defined by said tubular casing and accessible to 
severed nerve ends defining a break in a neural 
pathway, such that said morphogen stimulates neural 
5 pathway regeneration when disposed in said channel 

and accessible to said nerve ends* 

67. The device of claim 66 wherein said morphogen is 
disposed in said channel together with a 

10 biocompatible, bioresorbable carrier suitable for 

maintaining a protein at a site in vivo . 

68. The device of claim 67 wherein said carrier 
comprises sufficient structure to assist direction 

15 of axonal growth within said channel. 

69. The device of claim 67 wherein the outer surface of 
said casing is substantially impermeable. 

20 70. The device of claim 66 wherein said carrier 
comprises a polymer. 

71. The device of claim 67 wherein said carrier 
comprises laminin or collagen. 

25 

72. A method for inducing the redif ferentiation of 
transformed cells of neural origin, the method 
comprising the step of: 

contacting said transformed cells with a 
30 morphogen composition at a concentration and for a 

time sufficient to induce redif ferentiation of said 
cells to a morphology characteristic of 
untrans formed neuronal cells. 



WO 94/03200 



PCIYUS93/07231 



- 165 - 

73. The method of claim 72 wherein said morphology 
characteristic of untransf ormed nerve cells 
includes formation of neurite outgrowths. 

5 74. The method of claim 72 wherein said morphology 
characteristic of untransf ormed nerve cells 
includes cell aggregation and cell adhesion. 

75. The method of claim 72 wherein said morphogen 

10 composition induces nerve cell adhesion molecule 

production in said cells. 

76. The method of claim 72 wherein said induced nerve 
cell adhesion molecules include N-CAM-180, N-CAM- 

15 140 and N- CAM- 120. 

77. The method of claim 72 wherein said transformed 
cells comprise neuroblastoma cells. 



20 78. A kit for detecting a neuropathy in a mammal or for 
evaluating the efficacy of a therapy for treating a 
neuropathy in a mammal, the kit comprising: 
c) means for capturing a cell or body fluid 
sample obtained from a mammal; 

25 b) a binding protein that interacts specifically 

with a morphogen in said sample so as to form a 
binding protein-morphogen complex; 
c) means for detecting said complex. 



30 



79. 



The kit of claim 78 which said binding protein has 
specificity for an epitope defined by part or all 
of the pro region of a morphogen. 



- 166 - 



80. A method for detecting a neuropathy in a mammal, 
the method comprising the step of: 

detecting fluctuations in the physiological 
concentration of a morphogen present in the serum 
or cerebrospinal fluid of said mammal, said 
fluctuations being indicative of an increase in 
neuronal cell death. 

81. A method for detecting a neuropathy in a mammal, 
the method comprising the step of: 

detecting fluctuations in the physiological 
concentration of a morphogen antibody titer present 
in the serum or cerebrospinal fluid of said mammal, 
said fluctuations being indicative of an increase 
in neuronal cell death. 

82. The invention of claims 78, 80 or 81 wherein said 
neuropathy results from a neurodegenerative 
disease, nerve demyelineation, myelin dysfunction, 
neuronal neoplasias, or nerve trauma. 

83. A method of stimulating production of cell adhesion 
molecules in a tissue comprising the step of: 

providing a morphogen to said tissue for a 
time and at a concentration sufficient to induce 
production of cell adhesion molecules in cells of 
said tissue. 

84. The method of claim 83 wherein said cell adhesion 
molecules comprises nerve cell adhesion molecules. 

85. The method of claim 84 wherein said cells comprise 
neurons • 



WO 94/03200 



PCT/US93/07231 



- 167 - 

86. The method of claim 78, 80 or 81 wherein said 
morphogen comprises an amino acid sequence sharing 
at least 70% homology with one of the sequences 
selected from the group consisting of: OP-1, OP-2, 

5 CBMP2, Vgl(fx), Vgr(fx), DPP(fx), GDF-l(fx) and 

60A(fx). 

87. The method of claim 86 wherein said morphogen 
comprises an amino acid sequence sharing at least 

10 80% homology with one of the sequences selected 

from the group consisting of: OP-1, OP-2, CBMP2, 
Vgl(fx), Vgr(fx), DPP(fx), GDF-l(fx) and 60A (fx). 



88. The method of claim 87 wherein said morphogen 
15 comprises an amino acid sequence having greater 

than 60% amino acid identity with the sequence 
defined by residues 43-139 of Seq. ID No* 5 (hOPl.) 

89* The method of claim 88 wherein said morphogen 
20 comprises an amino acid sequence having greater 

than 65% amino acid identity with the sequence 
defined by residues 43-139 of Seq. ID No. 5 (hOPl.) 

90. The method of claim 89 wherein said morphogen 
25 comprises an amino acid sequence defined by 

residues 43-139 of Seq. ID No. 5 (hOPl), including 
allelic and species variants thereof. 



91. The method of claim 78, 80 or 81 wherein said 
30 morphogen comprises an amino acid sequence encoded 

by a nucleic acid that hydridizes under stringent 
conditions with the sequence defined by nucleotides 
1036-1341 of Seq. ID No. 16 or nucleotides 1390- 
1695 of Seq. ID No. 20. 



35 



WO 94/03200 



PCT/US93/07231 



- 168 - 

92. A composition for enhancing survival of neuronal 
cells at risk of dying comprising a morphogen in 
association with a molecule capable of enhancing 
the transport of said morphogen across the 
5 blood-brain barrier. 



93. 



The invention of claims 62 or 67 wherein said 
carrier comprises brain tissue derived 
extracellular matrix. 



WO 94/03200 



PCI7USW/07231 




Fig. W 



WO 94/03200 



PCT/US93/07231 





WO 94/03200 * PCT/US93/0723J 

3/3 




0 0.1 1 10 40 100 

OP-1, ng/ml 

Fig. 2B 



I 2 3 4 5 




Fig. 4 



INTERNATIONAL SEARCH REPORT 



Internal Application No 

PCT/US 93/07231 



. CUVSSinCATlONgFSUBIECTMATjnER 

PC 5 A61K37/02 G01N33/68 



tn international Patent gasification (IPC) or to both national tlurificalian and IPC 



Minimum documentation .tanned (clamlicalion system followed by clarification aymbob) 

PC 5 A61K C07K 



Documentation searched other 



than miiumum <locumtniaticm to the extent thai such documents are included in the fields searched 



Electronic data base 



consulted during the international search (name of data base and, where practical, search t 



C DOCUMENTS CONSIDERED TO BE RELEVANT 



Category * Citation of document, with indication, where appropriate, of the relevant passages 



Relevant to daim No. 



x.p 



x,p 



WO, A, 92 00382 (CARNEGIE INSTITUTION OF 
WASHINGTON) 9 January 1992 

see page 9, line 15 - page 15, line 29 

WO, A, 92 15323 (CREATIVE BIOMOLECULES, 

INC.) 17 September 1992 

cited in the application 

see page 6, line 1 - page 26, line 18 

PROCEEDINGS OF THE NATIONAL ACADEMY OF 
SCIENCES OF USA. 

vol . 89 , November 1992 , WASHINGTON US 
pages 10326 - 10330 

GEORGE PERIDES ET AL. 1 INDUCTION OF THE 
NEURAL CELL ADHESION MOLECULE AND NEURONAL 
AGGREGATION BY OSTEOGENIC PROTEIN V 
THE WHOLE ARTICLE 



1-24,78, 

79,82, 

86,87 



1-93 



1,20-27, 
53 



j y| Further document! arc listed in the continuation of box C 



a 



Patent family members arc listed in annex. 



* Special categories of dted documents : 

*A* document defining the general state of the art which is not 

considered to be of particular relevance 
'E* earlier document but published on or after the international 

filing date 

•L* document which may throw doubts on priority daimfs) or 
^^Tdutf to <^ the publication date of another 

citation or other special reason (as specified) 
"O* document referring to an oral disdojure, use, exhibition or 

other means 

•p- document published prior to the international filing date but 
later than me priority date claimed 



T later document published after the international filing date 
or priority date and not in conflict with the application but 
dted to understand the prindple or theory underlying the 



*X* document of particular relevance; the claimed invention 
cannot be considered novd or cannot be cctnsdercd to 
involve an inventive step when the document is taken alone 

•Y* document of particular relevance; the daimed mvention 
cannot be considered to involve an inventive step when the 
document is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
in the art. 

"ft* document member of the same patent family 



Date of the actual completion of the intcnaiional search 

8 November 1993 



Date of mailing of the international starch report 

07.12.93 



Name and 



_ig address of the ISA 
European Patent Office, P.B. 5818 Patentiaan 2 
NL-2280HVRijswijk 
Td. (+31-70) 340-2040, Tx 31 651 epo nl. 
Far (+ 31-70) 340-3016 



Authorized officer 



REMPP, 6 



Form PCT/iSAtflO (second thmi) (July WW) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT bun ui Appucrtm No 

PCT/US 93/07231 


C .(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT 


Category " 


Citation of document, with indication, where appropriate, of the relevant passages 


Relevant to claim No. 


A 


BIOLOGICAL ABSTRACTS vol . 91 

1991, Philadelphia, PA, US; 

abstract no. 106862, 

JONES, C. ET AL. 'INVOLVEMENT OF BONE 

MORPHOGENETIC PROTEIN-4 (BMP-4) AND VGR-1 

IN MORPHOGENESIS AND NEUROGENESIS IN THE 

MOUSE' 

see abstract 
& DEVELOPMENT (CAMB) 
vol. Ill, no. 2 , 1991 
pages 531 - 542 


i 



Form PCT/ISA/210 (continuation of uamU thaat) (July 1993) 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 



rnaitonai application No. 

PCT/ US 93/ 07231 



Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) 



This international search rcpor 



ch report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: 



because^y relate to subject matter not required to be searched by this Authority, namely. 

Remark: Although claims 2,28-52,61,72-77,80,81,83,85 are directed to a 
method of treatment of the human/animal body the search has been carried 
out and based on the alleged effects of the compound/composition. 



tacwv Uiey' relate to parts of the international application that do not comply with the prescribed requirements to such 
an extent that no meaningful international search can be carried out, specifically: 



□ 



£™l*ta£ arc dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). 



Box II Ob.%cr vatimm where unity of invention is lacking (Continuation of item 2 of first sheet) 



This Internauonal Seai Jung Authority found multiple invenuuns in this international application, as follows: 



1. Q As all requited addiuonal search fees were umely paid by the applicant, this international search report covers all 
searchable claims. 

2. ("I As all searchable claims could be searches without effort justifying an additional fee, this Authority did not invite payment 
of any additional fee. 



3 I I As only some of the required additional search fees were timely paid by iht 
1 — 1 covers only those daims for which fees were paid, specifically claims Nosu 



the applicant, this international search report 



4 r~1 No required additional search fees were umely paid by the applicant. Consequently, this international search report is 
restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 



Kcitidfk un Prole** 



| j The addiuonal search fees were accompanied by the applicant's protest. 
| j No protest accompanied the payment of addiuonal search fees. 



Form PCT.ISA.210 (continuation of first sheel (1)) (July 1992) 



INTERNATIONAL SEARCH REPORT 

~«lonnatioa on patent family members * 


Intern al Application No 

PCT/US 93/07231 


Patent document 
cited in search report 


Publication 
date 


Patent family 
member(s) 


Publication 
date 



WO-A-9200382 09-01-92 AU-A- 8496491 23-01-92 



W0-A-9215323 17-09-92 AU-A- 1754392 06-10-92 



Fmu PCT/KA/J10 (ptuet runOy tnaw) (July IMS) 



This Page is Inserted by IFW Indexing and Scanning 
Operations and is not part of the Official Record 

BEST AVAILABLE IMAGES 

Defective images within this document are accurate representations of the original 
documents submitted by the applicant. 

Defects in the images include but are not limited to the items checked: 

□ BLACK BORDERS 

□ IMAGE CUT OFF AT TOP, BOTTOM OR SIDES 
□TfADED TEXT OR DRAWING 

\j& BLURRED OR ILLEGIBLE TEXT OR DRAWING 

□ SKEWED/SLANTED IMAGES 

□ COLOR OR BLACK AND WHITE PHOTOGRAPHS 

□ GRAY SCALE DOCUMENTS 

□ LINES OR MARKS ON ORIGINAL DOCUMENT 

□ REFERENCE(S) OR EXHIBIT(S) SUBMITTED ARE POOR QUALITY 

□ OTHER: 

IMAGES ARE BEST AVAILABLE COPY. 
As rescanning these documents will not correct the image 
problems checked, please do not report these problems to 
the IFW Image Problem Mailbox.