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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
IntenuiUonal Bureau 




PCX 

INTERNATIONAL APPLICATION PUBUSHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 ; 
A61K 38/18 



Al 



(11) International PubUcaUon Numbo': WO 97^626 

(43) International Publication Date: 25 September 1997 (25.09.97) 



(21) InUmational Application Number: PCr/US97/04n7 

(22) International FiUng Date: 21 March 1997 (21.03.97) 



(30) Priority Data: 

08/620.444 



22 March 1996 (22.03.96) 



US 



(71) Applicants: CREATIVE BIOMOLECULES, INC. (US/USJ; 

45 South Street. Hopkinion, MA 01748 (US). THE GEN- 
ERAL HOSPITAL CORPORATION [US/US]; 55 Fruit 
Street, Boston. MA 02 U 4 (US). 

(72) Inventors: CHARETTE, Marc. P.; 18 Ellicolt Street, Ncedham, 

MA 02192 (US). HNKLESTEIN, Seth, P.; 8 Greenwood 
Avenue. Needham. MA 02192 (US). 

(74) Agent: FENTON, Gillian. M.; Testa. Hunvitz & Thibeault. 
L.LP., High Street Tower. 125 High Street. Boston. MA 
02110 (US). 



(81) Designated States: AL. AM, AT, AU. AZ, BA. BB. BG. BR, 
BY. CA. CH, OI. CU. CZ. DE. DK, EE, ES. n, GB. GE, 
GH. HU, IL. IS, JP. KE, KG. KP. KR, K2, LC. LK. LR. 
LS. LT. LU, LV. MD. MG. MK, MN. MW, MX. NO, NZ, 
PL, PT, RO, RU, SD. SE, SG. SI, SK, TJ, TM, TR. TT, 
UA. UG. UZ, VN, YU, ARIPO patent (GH, KE, LS. MW, 
SD, SZ. UG), Eurasian patent (AM. AZ, BY. KG. KZ. MD, 
RU. TJ. TM), European patent (AT. BE. CH. DE. DK. ES. 
n. FR. GB. GR, IE, IT. LU, MC. NL. PT. SE). OAPl patent 
(BF, BJ. CF. CG, CI. CM, GA, GN. ML, MR. NE. SN. TD, 
TG). 



Published 

With internationai 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: 



METHODS FOR ENHANCING FUNCTIONAL RECOVERY FOLLOWING CENTRAL NERVOUS SYSTEM ISCHEMIA 
OR TRAUMA 



(57) Abstract 

The present invention provides methods and compositi(xis for treatment of mammals afflicted with an ischemic or traumatic injuiy 
of the central nervous system. The present invention capitalizes in pan upon the discovery that administration of a morphpgen to such a 
mammal provides significant improvement in central nervous system function, even when administered after central nervous system tissue 
has been damaged. The methods involve tht administration of dinneric proteins defined as moiphpgens. inducers of these morphogens, 
or agonists of the conesponding motphogen receptors, or implantation of cells stimulated by exposure to die morphogens.. Tbe proteins 
defined as morphogens comprise a structurally and functionally distinct family within the TGF-^ superfamily. Osteogenic protein- 1 (OP-1) 
is considered to be an exemplary and preferred member of this morphogen family. 



FOR THE PURPOSES OF INFORMATION ONLY 



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



AL 

AM 

AT 

AU 

AZ 

BA 

BB 

BE 

BF 

BG 

Bl 

BR 

BY 

CA 

CF 

CG 

CH 

CI 

CM 

CN 

CU 

CZ 

DE 

DK 

EE 



Albania B5 

Annenia W 

Auitria FR 

Australia GA 

Azcfbaijan GB 

Bofflia and Henesovina GB 

Baitndos GH 

Belgium GN 

Burkina Ftio GR 

Bulgaria HU 
Benin 

Brazn tL 

Belants IS 

Caniida IT 

Centtal African Rqwblic JP 
Congo 

Switzerland KG 

Cba d'lvoirc W* 
CaiQCtoon 

China KR 

Cuba KZ 

Cwch Republic LC 

Germany LI 

DenmariE LK 

Eitonia ^ 



Spain 
Fmtand 
France 
Gabon 

United Kingdom 

Georgia 

Ghana 

Guinea 

GiMce 

Hungary 

Ireland 

Israel 

Iceland 

Italy 

Japan 

Kenya 

Kyrgyzstan 

Democralic Peopk*s 

Republic of Korea 

Republic of Korea 

Kazakstan 

Saint Lucia 

Liechtenstein 

Sri Lanka 

Uberia 



LS 


Lesotho 


SI 


Slovenia 


LT 


Lithuania 


SK 


Slovakia 


LU 


Luxemboorg 


SN 


Senegal 


LV 


Latvia 


SZ 


Swatiland 


MC 


Monaco 


TD 


Chad 


MD 


Republic of Moldova 


TG 


Togo 


MG 


Madagascar 


TJ 


Tajikistan 


MK 


The fofiner Yugoslav 


TM 


Tmknwnisian 




Republic of Macedonia 


TR 


Tuikcy 


ML 


Mali 


TT 


Trinidad and Tobago 


MN 


Mongolia 


UA 


Ukraine 


MR 


Maoritanta 


liG 


Uganda 


MW 


Malawi 


US 


United States of America 


MX 


Mexico 


uz 


Uzbekistan 


NE 


Niger 


VN 


VietNMD 


NL 


Netheriands 


YU 


Yugoslavia 


NO 


Norway 


ZW- 


Zimbabwe 


NZ 


New Zealand 






PL 
PT 


Poland 
Fomtgal 






RO 


Romania 






RU 


Russian Federation 






SD 
SE 


Sudan 
Sweden 






SG 


Singapore 







wo 97/34626 



PCT/US97/04177 



METHODS FOR ENHANCING FUNCTIONAL RECOVERY 
FOLLOWING CENTRAL NERVOUS SYSTEM ISCHEMU OR TRAUMA 



The present application claims priority to U.S. application serial number 08/620,444 filed 
March 22, 1996, the disclosure of which is incorporated herein by reference. 

Field of the Invention 

The present invention generally relates to methods and compositions for the treatment of 
manimals, including humans, following an ischemic or traumatic injury to the central nervous 
system. 

Background of the Invention 

Numerous proteins have now been identified and characterized as morphogenetic or 
growth factors, regulating cell proliferation and/or differentiation of tissues in vertebrates, 
including mammals. Typically these growth factors exert their effects on specific subsets of cells 
and/or tissues. Thus, for example, epidermal growth factors, nerve growth factors, fibroblast 
growth factors, various hormones, and many other proteins inducing or inhibiting cell 
proliferation or differentiation have been identified and shown to affect some subset of cells or 
tissues. 

Neurotrophic factors are polypeptides that are required for the development of the 
nervous system. The first neurotrophic factor discovered, nerve growth factor (NGF), is now 
known to be a part of a large family of growth factors, which also includes BDNF, NTS, and 
NT4/NT5, The dimeric proteins defined in PCT Publication No. WO 94/03200 as raorphogens 
constitute another family of proteins believed to play an important role in neural development 
(Jones, etaL {\99l) Development J J J: 531-542; Ozkaynak, eiaL (1992) J. Biol Chem. 267: 
25220-25227; Lein, etai {mS) Neuron 15: 597-605). 

These proteins, referred to herein as "morphogenic proteins" or **morphogens/' are 
competent to act as true tissue morphogens, able, on their own, to induce the proliferation and 
differentiation of progenitor cells into functional mammalian body tissue. The proteins include 
members of the family of bone morphogenetic proteins (BMPs) which were initially identified by 
their ability to induce ectopic, endochondral bone morphogenesis. 



wo 97/34626 



PCT/US97/04177 



Morphogens generally are classified in the art as a subgroup of the TGF-P superfamily of 
growth factors (Hogan (19%) Genes & Development JO: 1580-1594). Members of the 
morphogen family of proteins include the mammalian osteogenic protein- 1 (OP-1. also know-n as 
BMP-7. and the Drosophila homolog 60A). osteogenic protein-2 (OP-2. also known as BMP-8), 
5 osteogenic protein-3 (OP-3). BMP-2 (also known as BMP-2A or CBMP-2A, and the Drosophila 
homolog DPP), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6 and its 
murine homolog Vgr-1, BMP-9, BMP-10. BMP-1 1, BMP-12. GDF-3 (also known as Vgr2). 
GDF-8, GDF-9, GDF-10, GDF-1 1, GDF-12, BMP-13, BMP-14, BMP-15. GDF-5 (also known 
as CDMP-1 or MP52), GDF.6 (also known as CDMP-2), GDF-7 (also known as CDMP-3). the 
10 Xenopus homolog Vgl and NODAL, UNIVIN, SCREW. ADMP, and NEURAL. The members 
of this family encode secreted polypeptide chains sharing common structural features, including 
processing from a precursor "pro-form" to yield a mature polypeptide chain competent to 
dimerize, and containing a carboxy terminal active domain of approximately 97-106 amino acids. 
All members share a conserved pattern of cysteines in this domain and the active form of these 
15 proteins can be either a disulfide-bonded homodimer of a single family member, or a heterodimer 
of two different members (see. e.g., Massague (1990) ^nm/. Rev. Cell Biol. 6. 597. Sampath. et 
al. (1990) j. Biol Chem. 265: 13198). See also, U.S. 5,01 1.691; U.S. 5.266.683. Ozkaynak et 
al. (]990)EMBOJ. 9: 2085-2093, Wharton a/. (1991)^555: 9214-9218). (Ozkaynak 
(1992) J. Biol. Chem. 267: 25220-25227 and U.S. 5,266,683); (Celeste etal. (1991) PNAS87: 
20 9843-9847); (Lyons et al. (1989 ) PNAS 86: 4554-4558). These disclosures describe the amino 
acid and DNA sequences, as well as the chemical and physical characteristics of these 
morphogcnic proteins. See also Wozney et al (1988) Science 242: 1528-1534); BMP-9 
(WO 93/00432, published January 7. 1993), DPP (Padgett et al. (1987) Nature 325: 81-84; and 
Vg-1 (Weeks (1987) Cell 51: 861-867). 

Morphogens are expressed naturally in a variety of tissues during development, including 
those of the developing newous system (Ozkaynak, et al. (1990) EMBO J. 9: 2085-2093; 
Ozkaynak, etal. {\99\) Biochem. Biophys. Res. Commun. 179: 116-123; Ozkaynak, etal (1992) 
supra). 

Vascular diseases of the nervous system rank first in frequency amongst all the neurologic 
diseases; they constitute about fifty percem of all neurologic hospital admissions to adult wards. 
The cardinal feature of cerebrovascular disease is the stroke, a term that connotes the sudden and 
dramatic development of a focal neurologic deficit. Obstruction of a nutriem anery supplying a 



25 



30 



wo 97/34626 PCT/US97/04177 

locus of the central nervous system by, for example, a thrombus or an embolus or a failure of the 
systemic circulation and hypotension, if severe and prolonged enough, can deprive brain tissue of 
blood and oxygen, leading to disruption of physiologic function, subsequent death of neurons, and 
necrosis (infarction) of the affected locus. In hemorrhagic infarction, an extravasation of blood 
5 occurs into the brain tissue, the subarachnoid space, or both. Damage results from physical 
disniption of the region directly involved and pressure of the mass of blood on the surrounding 
tissue. 

The neurologic deficit in a stroke reflects both the location and the size of the infarct or 
hemorrhage in the brain. Hemiplegia is the classic sign of vascular disease and occurs with 
10 strokes involving the cerebral hemisphere or the brainstem. Hov^ever, depending on its location, 
a stroke may also give rise to many other manifestations accompanying or independent of 
hemiplegia, including numbness, sensory deficit, dysphasia, blindness, diplopia, dizziness, and 
dysarthria. 

Patients who suffer a "stroke," or any other form of cerebral ischemic or traumatic injury, 
15 usually recover partially, but often remain mildly to severely debilitated. For example, total 
infarction of the middle cerebral artery in a human results in a contralateral hemiplegia, 
hemianesthesia, homonymous hemianopia, global or total sensorimotor aphasia (left hemisphere), 
and apractagnosia (right hemisphere). Once established, the motor, sensory, and language deficits 
usually remain static or very little improved after the passage of months or even years. Seldom 
20 can the patient ever again communicate effectively. Currently, aside from physical therapy, there 
is no treatment that reliably improves the prognosis of a patient who has suffered a stroke or any 
similar injury of the central nervous system. 

Summary of the Invention 

The present invention is directed to methods and compositions for treatment of mammals 
25 afflicted with an ischemic or traumatic injury of the central nervous system. In particular, the 
invention provides treatments for mammals in whom central nervous system tissue has been 
damaged or lost due to stroke or a similar disruption in blood flow, or due to infliction of physical 
{e.g., mechanical) trauma affecting the central nervous system. The methods and compositions 
provided herein capitalize upon the discovery that administration of a morphogen to such a 
30 mammal provides significant improvement in central nervous system function, even when 
administered after central nervous system tissue has been damaged. The methods involve the 



PCT/US97/04177 

WO 97/34626 

administration of dimeric proteins defined as morphogens. inducers of these morphogens, or 
agonists of the corresponding morphogen receptors, or implantation of cells stimulated by 
exposure to the morphogens. 

Accordingly, the invemion features a method for treating a mammal who has suffered an 
5 injure to the central nervous system, such as a stroke or a traumatic injury. The method involves 
administering an effective dose of morphogen to the mammal at least six hours after the onset of 
the injury; for example, twelve, twenty-four, or forty-eight hours or even longer following the 
onset of injury. 

The treatment regimen according to the invention is carried out in terms of administration 
10 mode, timing of the administration, and dosage, so that the functional recovery from impairment 
of the central nervous system is enhanced. The compositions of the presem invention will contain 
therapeutically-effective amounts of the morphogen. morphogen inducers or agonists of 
morphogen receptors. That is. the compositions will contain an amount which provides 
appropriate concentrations of the agent to the affected nervous system tissue for a time sufficient 
15 to stimulate a detectable restoration of central nervous system function, up to and including a 
complete restoration thereof The effective amount of morphogen can be provided in a single 
administration, in two administrations or in a pluraUty of administrations. Where the effective 
amount of morphogen is provided in a plurality of adminirtrations, the morphogen is preferably 
administered to the mammal daily. In an alternative preferred embodiment, the morphogen is 
20 administered to the mammal biweekly (e.g.. every three or four days). In a further alternative 
preferred embodiment, the morphogen is administered to the mammal once a week 

Practice of the invention confers significant clinical benefit on the afflicted mammal, in that 
the invention beneficially confers clinically relevant improvement in at least one of the mammal's 
motor coordination functions (e.g., posture, balance, grasp, gait), sensory perceptions {e.g., 
25 vision, touch, taste, olfaction, proprioception), or speech. Clinically relevant improvement can 
range from a detectable improvemem to a complete restoration of an impaired or lost central 
nervous function. 

The invention can be used to treat adverse consequences of central nervous system injuries 
that resuh from a variety of conditions. Thrombus, embolus, and systemic hypotension are among 
30 themostcommoncausesofstroke. Other injuries may be caused by hypertension, hypertensive 
cerebral vascular disease, rupture of an aneurysm, an angioma, blood dyscrasia. cardiac failure, 



wo 97/34626 



PCT/US97/04177 



cardiac arrest, cardiogenic shock, kidney failure, septic shock, head trauma, spinal cord trauma, 
seizure, bleeding from a tumor, or other loss of blood volume and/or pressure. Administration of 
a morphogen according to the invention confers significant clinical benefit, even when 
administration occurs a significant amount of time following the injury. 

5 Generally, the morphogens usefiil in the methods and compositions of the invention are 

dimeric proteins that induce morphogenesis of one or more eukaryotic (e.g., mammalian) cells, 
tissues or organs. Of particular interest herein are morphogens that induce morphogenesis at least 
of bone or neural tissue. Morphogens comprise a pair of polypeptides that, when folded, adopt a 
configuration sufficient for the resulting dimeric protein to elicit morphogenetic responses in cells 

10 and tissues displaying receptors specific for said morphogen. That is, the morphogens generally 
induce a cascade of events including all of the following in a morphogenically permissive 
environment: stimulating proliferation of progenitor cells; stimulating the differentiation of 
progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth 
and maintenance of differentiated cells. "Progenitor" cells are uncommitted ceDs that are 

15 competent to differentiate into one or more specific types of differentiated cells, depending on 
their genomic repertoire and the tissue specificity of the permissive environment in which 
morphogenesis is induced. Morphogens further can delay or mitigate the onset of senescence- or 
quiescence-associated loss of phenotype and/or tissue fiinction. Morphogens still fijrther can 
stimulate phenotypic expression of differentiated cells, including expression of metabolic and/or 

20 functional, e.g., secretory, properties thereof In addition, morphogens can induce 

redifferentiation of committed cells under appropriate environmental conditions. As noted above, 
morphogens that induce proliferation and/or differentiation at least of neural tissue, and/or 
support the growth, maintenance and/or fimctional properties of neural tissue, are of particular 
interest herein. See, for example, WO 92/15323, WO 93/04692 and WO 94/03200 for more 

25 detailed disclosures as to the tissue morphogenic properties of these proteins. 

As used herein, the terms "morphogen," "bone morphogen," "bone morphogenic 
protein," "BMP," "morphogenic protein" and "morphogenetic protein" all embrace the class 
of proteins typified by human osteogenic protein 1 (hOP-1). Nucleotide and amino acid 
sequences for hOP-1 are provided in SEQ ID NOs: 4 and 5, respectively. For ease of 
30 description, hOP-1 is recited herein below as a representative osteogenic protein. It will be 
appreciated by the artisan of ordinary skill in the art, however, that OP-1 merely is 
representative of the TGF-^ subclass of true tissue morphogens competent to act as 



PCT/13S97/04177 

WO 97/34626 

morphogemc proteins, and is not intended to limit the description. Other known, and useful 

proteins include. BMP-2. BMP.3. BMP-3b. BMP-4, BMP-5. BMP-6. BMP-8. BMP-9. 

BMP-10 BMP-11. BMP-12, BMP-13. BMP-15. GDF-1. GDF-2. GDF-3, GDF-5. GDF-6. 

GDF-7 GDF-8, GDF-9, GDF-10, GDF-11. GDF-12, NODAL. UNIVIN, SCREW. ADMP. 
5 NEURAL and morphogenically active ammo acid variants thereof. Thus, in one embodimem, 
preferred morphogenic proteins, include but are not limited to, GP-1. OP-2, BMP-2, BMP-4, 
BMP-5. and BMP-6. In addition, as will be appreciated by the arusan of ordinary skill m the 
art any one of the morphogenic proteins recited herein also could be used as a reference 
sequence. 

,0 m another preferred embodiment, the proteins useful in the invention include 

biologically active species (pbylogenetic) variants of any of the morphogenic proteins recited 
herein including conservative amino acid sequence variants, proteins encoded by degenerate 
nucleotide sequence variants, and moiphogenically active proteins sharing the conserved seven 
cysteine skeleton as defmed herein and encoded by a DNA sequence competent to hybridize 
,5 under sundard stringency conditions to a DNA sequence encoding a morphogenic protem 
disclosed herein, including, without limiution. OP-1 and BMP-2 or BMP.4. In still another 
embodiment, useful morphogens include those sharing the conserved seven cysteine domam 
and sharing at least 70% amino acid sequence homology (similarity) within the C-termmal 
active domain of a reference morphogen sequence, as defined herein below. Inaprefened 

20 embodiment, the reference sequence is OP-1 . 

m still another embodimem. the morphogens usefbl in the methods and compositions of 
the invention can be defined as morphogenically active proteins having any one of the genenc 
sequences defined herein, including OPXand Generic Sequences7and8(SEQ ID NOs: 1 and2 

respectively), or Generic Sequences 9 and 10 (SEQ ID NOs: 6 and 7. respectively). OPX 
25 accommodates the homologies between the various spec.es of the osteogenic OP-1 and OP-2 
proteins, and is described by the amino acid sequence presented herein below and m SEQ ID 
NO 3 Generic sequence 9 is a 96 amino add sequence containing the six cysteine skeleton 
defined by hOP-1 (residues 335-431 of SEQ ID NO: 5) and wherein the remaining residues 
accommodate the homologies of OP-L OP-2, OP-3. BMP-2. BMP-3, BMP-4. BMP-5. BMP-6. 
30 BMP-8 BMP-9,BMP-10.BMP-n,BMP-15.GDF.l.GDF-3, GDF-5, GDF-6, GDF-7. GDF-8. 

GDF-9' GDF-IO GDF-1 1, UNIVIN, NODAL. DORSALIN, NEURAL. SCREW and ADMP. 



-6- 



wo 97/34626 PCT/US97/04177 

That is. each of the non-cysteine residues is independently selected from the corresponding 
residue in this recited group of proteins. Generic Sequence 10 is a 102 amino acid sequence 
which includes a 5 amino acid sequence added to the N-terminus of the Generic Sequence 9 and 
defines the seven cysteine skeleton defined by hOP- 1 (330-43 1 SEQ ED NO: 5). Generic 
5 Sequences 7 and 8 are 96 and 102 amino acid sequences, respectively, containing either the six 
cysteine skeleton (Generic Sequence 7) or the seven cysteine skeleton (Generic Sequence 8) 
defined by hOP-1 and wherein the remaining residues non-cysteine accommodate the homologies 
of: OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, 60A, DPP, Vgl, BMP-5, BMP-6, Vgr-l, and 
GDF-1 

10 _ As contemplated herein, the family of morphogenic proteins described herein includes 

longer forms of a given protein, as well as phylogenetic, e.g., species and allelic variants, and 
biosynthetic mutants, including C-terminal addition and deletion mutants and variants, such as 
those which may alter the conserved C-terminal cysteine skeleton, provided that the alteration still 
allows the protein to form a dimeric species having a confiDrmation capable of inducing neural 

15 tissue formation in a mammal when provided to a morphogenically permissive site in a mammal. 
In addition, the morphogenic proteins usefiil in the invention may include forms having varying 
glycosylation patterns and varying N-termini, may be naturally occurring or biosynthetically 
derived, and may be produced by expression of recombinant DNA in prokaryotic or eucaryotic 
host cells. The proteins are active as a single species (e.g., as homodimers, including chimeras), 

20 or combined as a mixed species, including heterodimers. 

Of particular interest herein are morphogens which, when provided to neural tissue of a 
. mammal, induce or maintain the normal state of diiferentiation and growth of that tissue. In a 
currently preferred demonstrative embodiment, the present morphogens induce or reinduce a 
developmental cascade of cellular and molecular events that culminates in the formation of 

25 vertebrate central nervous system tissue. In other preferred demonstrative embodiments, the 

present morphogens similarly induce the formation of other vertebrate (e g , avian or mammalian) 
body tissues, such as but not Umited to bone, cartilage, bone marrow, ligament, tooth dentin, 
periodontium, liver, kidney, lung, heart or gastrointestinal lining The present demonstrations can 
be carried out in the context of developing, embryonic tissue, or at an aseptic, unscarred wound 

30 site in post-embryonic tissue. Particularly preferred morphogens induce or trigger a j)attem 

formation cascade in a developing mammalian or avian embryo that culminates in the formation of 
one or more fijnctionally integrated elements of central or peripheral nervous system. Such 



-7- 



wo 97/34626 PCT/US97/04I77 

morphogens can be used to treat a mammal afflicted with ischemic or tfaumatic injury of the 
central nervous system. 

The present invemion alternatively can be practiced with methods and compositions 
comprising a morphogen inducer in lieu of a morphogen. A "morphogen inducer" is a compound 
5 that stimulates the in vivo production {i.e.. transcription, translation, and/or secretion) of a 
therapeutically-effective concentration of an endogenous morphogen in the body of a manunal. 
An "effective" concentration is sufficient to promote the regeneration or maintenance of neural 
tissue and/or to inhibit additional loss thcr«of Such compounds are understood to include 
substances which, when administered to a mammal, act on cells that normally are competent to 
10 produce and/or secrete a morphogen encoded within the genome of the mammal, and which cause 
the endogenous level of the morphogen to be increased. Endogenous or administered 
morphogens can art as endocrine, paracrine or autocrine factors. That is, endogenous 
morphogens can be synthesized by the cells in which the morphogenetic responses are induced, by 
neighboring cells, or by cells of a distant tissue, in which case the secreted endogenous 
15 moiThogen is transported to the site of morphogenesis, e.g.. by the individual's bloodstream. In 
preferred embodiments, the inducer stimulates expression and/or secretion of an endogenous 
morphogen so as to increase amounts thereof available in neural tissue. 

In still other embodiments, an agent which arts as an agonist of a morphogen receptor 
may be administered instead ofthe morphogen itself. An "agonist" of a receptor is a compound 
20 which binds to the receptor, and for which the result of such binding is similar to the result of 
binding the natural, endogenous Ugand ofthe receptor. That is, thecompound must, upon 
interaction with the receptor, produce the same or substantially similar uansraembrane and/or 
inuacellular efferts as the endogenous ligand. Thus, an agonist of a morphogen receptor binds to 
the receptor and such binding has the same or a funrtionally similar result as morphogen binding 
25 {e.g.. induction of morphogenesis). The artivity or potency of an agonist can be less than that of 
the natural ligand, in which case the agonist is said to be a "partial agonist," or it can be equal to 
or greater than that ofthe natural ligand, in which case it is said to be a "foil agonist." Thus, for 
example, a small peptide or other moleoile which can mimic the artivity of a morphogen in 
binding to and activating the morphogen's receptor may be employed as an equivalem ofthe 
30 morphogen. Preferably the agonist is a full agonist, but partial morphogen receptor agonists may 
also be advantageously employed. Methods of identifying such agonists are known in the art and 
include assays for compounds which induce morphogen-mediated responses (e.g., induction of 



■ 8 



wo 97/34626 



PCT/US97/04177 



difFerentiation of metanephric mesenchyme, induction of endochondral bone formation, and the 
like). Such an agonist may also be referred to as a morphogen "mimic," "mimetic." or "analog." 

The morphogens, inducers and agonists of the invention may be administered by any route 
of administration which is compatible with the selected agent, including by intravenous, 
5 subcutaneous, intramuscular, ophthalmic, intraperitoneal, buccal, rectal, vaginal, intraorbital, oral, 
intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracistemal, intracapsular, 
intranasal or by aerosol administration and may be formulated with any pharmaceutically 
acceptable carrier appropriate to the route of administration. In addition, various growth factors, 
hormones, enzymes, therapeutic compositions, antibiotics, or other bioactive agents can be 
10 co-administered with the morphogen. Thus, various known growth factors such as NGF, EGF, 
PDGF, IGF, FGF, TGF-a , and TGF-p, as well as enzymes, enzyme inhibitors and/or 
chemoattractant/ chemotactic factors, can be combined with the morphogen and be delivered to 
the defect locus. 

The method of the invention advantageously stimulates restoration of central nervous 
15 system function even when practiced hours, or even days, following an injury to the central 
nervous system. The invention thus significantly improves on the treatment options available 
when central nervous system injury occurs and is not diagnosed or treated prior to the death of 
involved tissue. 

The preferred methods, material, and examples that will now be described are illustrative 
20 only and are not intended to be limiting. Other features and advantages of the invention will be 
apparent from the following detailed description, and from the claims. 

Brief Description of the Drawings 

FIG. 1 . presents the percent amino acid sequence identity and percent amino acid 
sequence homology C'similarity") that various members of the family of morphogenic proteins as 
25 defined herein share with OP- 1 in the C-terminal seven cysteine domain; 

FIG. 2A-2B are line graphs depicting forelirab placing (2A) and hindlimb placing <2B) 
scores of affected (left) limbs of OP- 1 treated animals (10 ng/intracistemal injection; total OP-1 
delivered in 8 injections=80 |ig/animal; N=7, solid squares) and vehicle treated animals (N=7, 
open squares); 



W0 97/34626 PCT/US97A)4177 

FIGS. 3A.3B are line graphs depicting balance beam (3A) and postural reflex (3B) scores 
in OP-1 treated animals (10 ^g/intracistemal injection; total OP-l delivered in 8 injections=80 
Hg/animal; N=7, solid squares) and vehicle treated animals (N=7 animals, open squares). 

FIG. 4 is a line graph depicting body-weight of OP-1 -treated animals (10 ng/imracisternal 
5 injection; total OP-1 delivered in 8 injections=80 ^g/animal; N=7; solid squares) and 
vehicle-treated animals (N=7; open squares); 

FIGS. 5A-5B are line graphs depirting forelimb placing Scores without (5A) and with 
whisker placing.(5B) of affected (left) limbs of High dose OP-l-treat«l animals 
(10 ng/intracistemal injection; total OP-1 delivered in 2 injections=20 ng/animal; N=9 animals; 
10 solid squares). Low dose OP-l-treated animals (1 ng/intracistemal injection; total OP-1 delivered 
in 2 injections=2 ^g/animal; N=8 animals; open squares), and vehicle-treated animals (N=9. open 
circles); 

FIG. 6 is a line graph depicting hindUmb placing scores of affected (left) Umbs of High 
dose OP-l-treated animals (10 ^g/intracistemal injection, total OP-1 delivered in 2 
15 injectioiis=20 Mg/animal; N=9 animals; solid squares). Low dose OP-l-treated animals 

(1 ug/intracisteraal injection; total OP-1 deUvered in 2 injections=2 ng/animal; N=8 animals; open 
squares), and vehicle-treated animals (N=9, open circles); 

FIG. 7 is a line graph depicting body-weight of High dose OP-l-treated animals 
(10 ng/intracisten»al injection; total OP-1 delivered in 2 injections=20 ng/animal; N=9 animals; 
20 soUd squares). Low dose OP-l-treated animals (1 ^g/inuacistemal injection; total OP-1 

delivered=2 Mg in 2 injections/animal; N=8 animals; open squares), and vehicle-treated animals 
(N=9, open circles); 

FIGS 8A-8B are line graphs depiaing forelimb placing scores without (8A) and with 
whisker placing (8B) of affected (left) limbs of OP-l-treated animals (10 ng/intracisiemal 
25 injection; N=6 animals; solid squares) and vehicle-treated animals (N=8, open squares); 

FIG. 9 is a line graph depicting hindlimb placing scores of affected (left) limbs of affected 
(left) limbs of OP-l-treated animals (10 ng/intracisternal injection; N=6 animals; solid squares) 
and vehicle-treated animals (N=8, open squares); and 



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FIG. 10 is a line graph depicting body-weijght of OP- 1 -treated animals 
(10 |ig/intracistema! injection; N-6 animals; solid squares) and vehicle-treated animals (N=8, 
open squares). 

Detailed Description of Preferred Embodiments 

5 A. General 

The present invention depends, in pan, upon the surprising discovery that functional 
recovery following stroke or traumatic injury of the central nervous system, is . significantly 
enhanced by the administration of a morphogen, even when administered after affected tissue has 
succumbed to the injury and after central nervous system function has been impaired or lost. 

10 Most*surprisingly, practice of the invention does not affect (e.g., reduce) the volume or extent of 
affected (infarcted) tissue. Thus, the invention capitalizes upon the discovery that functional 
central nervous system restoration can be achieved notvwthstanding the loss of tissue originally 
occupying a stroke or traumatic injury loss. Significant (detectable; clinically relevant) restoration 
of CNS funaion can be obtained with even a single administration of a therapeutically-effective 

15 dose of a morphogen. 

The invention features a method for treating a mammal who has suffered an injury to the 
central nervous system, such as stroke or a traumatic injury. The method involves administering a 
morphogen to the affected mammal at least six hours after onset of the injury; for example twelve, 
twenty-four, forty-eight hours, or even longer following injury. No practical end point the 

20 therapeutic window in which the invention can be practiced has yet been established. The 

mvention can be used to treat one or more adverse consequences of central nervous system injury 
that arise from a variety of conditions. Thrombus, embolus, and systemic hypotension are among 
the most common causes of stroke. Other injuries may be caused by hypertension, hypertensive 
cerebral vascular disease, rupture of an aneurysm, an angioma, blood dyscrasia, -cardiac failure, 

25 cardiac arrest, cardiogenic shock, kidney failure, septic shock, head trauma, spinal cord trauma, 
seizure, bleeding from a tumor, or other loss of blood volume or pressure. These injuries lead to 
disruption of physiologic function, subsequent death of neurons, and necrosis (infarction) of the 
affected areas. The term "stroke" connotes the resulting sudden and dramatic neurologic deficits 
associated with any of the foregoing injuries. 

30 The terms "ischemia" or "ischemic episode," as used herein, mean anyxircumstance that 

resuks in a deficient supply of blood to a tissue. Thus, a central nervous system ischemic episode 

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wo 97/34626 PCT/US97/041 77 

results from an insufficiency or interruption in the blood supply to any locus of the brain such as, 
but not limited to, a locus of the cerebrum, cerebellum or brain stem. The spinal cord, which is 
also a pan of the central nervous system, is equally susceptible to ischemia resulting from 
diminished blood How, An ischemic episode may be caused by a constriction or obstruction of a 
5 blood vessel, as occurs in the case of a thrombus or embolus. Alternatively, the ischemic episode 
may result from any form of compromised cardiac function, including cardiac anest. as described 
above. Where the deficiency is sufRciemly severe and prolonged, it can lead to disruption of 
physiologic function, subsequent death of neurons, and necrosis (infarction) of the affected areas. 
The extent and type of neurologic abnormality resulting from the injury depend on the location 
10 and size of the infirct or the focus of ischemia. Where the ischemia is associated with a stroke, it 
can be either global or focal in extent. 

The term "focal ischemia." as used herein in reference to the central nervous system, 
means the condition that results from the blockage of a single artery that supplies blood to the 
brain or spinal cord, resulting in the death of all celhilar elements (pan-necrosis) in the territory 
15 supplied by that artery. 

The term "global ischemia," as used herein in reference to the central nervous system, 
means the condition that results from a general diminution of blood flow to the entire brain, 
forebrain, or spinal cord, which causes the delayed death of neurons, particularly those in 
metabolically active loci, throughout these tissues. The pathology in each of these cases is quite 
20 different, as are the clinical correlates. Models of focal ischemia apply to patients with focal 
cerebral infarction, while models of global ischemia are analogous to cardiac artest. and other 
causes of systemic hypotension. 

It is expected that the invention will also be useful for treating traumatir ojuries to the 
central nervous system that are caused by mechanical forces, such as a blow to the head. Trauma 
25 can involve a tissue insult selected from abrasion, incision, contusion, puncture, compression, etc., 
such as can arise from traumatic contact of a foreign object with any locus of or appurtenant to 
the mammalian head, neck or vertebral column. Other forms of traumatic injury can arise from 
constriction or compression of mammalian CNS tissue by an inappropriate accumulation of fluid 
{e.g. , a blockade or dysfiinction of normal terebrospinal fluid or vitreous humor fluid production, 
30 turnover or volume regulation, or a subdural or intracranial hematoma or «dema). Similarly, 



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PCT/US97/04177 

traumatic constriction or compression can arise from the presence of a mass of abnormal tissue, 
such as a metastatic or primary tumor. 

B. Biochemical, Structural and Functional Properties of Useful Morohoeenic 
Proteins 

5 As noted above, a protein is morphogenic as defined herein if it induces the developmental 

cascade of cellular and molecular evems that culminate in the formation of new. organ-specific 
tissue. In one preferred embodimem, a morphogen is a dimeric protein comprising a pair of 
polypeptide chains, each chain having a sequence that corresponds to or is functionally equivalent 
to at least the conserved C-terminal six or seven cysteine skeleton of human OP-1, inciiided in 
10 SEQ ID NO: 5, and/or which shares 70% amino acid sequence homology with OP-1 in this 
region. The morphogens generally are competem to induce a cascade of events including all of 
the following, in a morphogenically permissive environment: stimulating proliferation of 
progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of 
differentiated cells; and supporting the growth and maintenance of differentiated cells. Under 
15 appropriate conditions the morphogens also are competent to induce redifferentiation of 
committed cells, particularly of ceUs that have strayed from their "normal" differentiation 
pathway. Details of how the morphogens useful in this invention first were identified, as well as a 
description on how to make, use and test them for morphogenic aaivity are disclosed in 
numerous publications, including U.S. 5,01 1,691. 5,266.683. and the international application 
20 publications WO 92/15323; WO 93/04692; WO 94/03200. As disclosed therein, the morphogens 
can be purified from naturally-sourced material or recombinantly produced from prokaiyotic or 
eukaryotic host cells, using the genetic sequences disclosed therein. Alternatively, novel 
morphogenic sequences can be identified following the procedures disclosed therein. 

Naturally occurring proteins identified and/or appreciated herein to be true tissue 
25 morphogenic proteins and usefiil in the methods and compositions of the invention form a distinct 
subgroup within the loose evolutionary grouping of sequence-related proteins known as the 
TGF-p superfamily or supergene family. The naturally occurring morphogens share substantial 
amino acid sequence homology in their C-terminal regions (domains). Typically, the above- 
mentioned naturally occurring morphogens are translated as a precursor, having an N^erminal 
30 signal peptide sequence, typically less than about 35 residues in length, followed-by a "pro" 
domain that is cleaved to yield the mature protein, which includes the biologically active 
C-terminal domain. The signal peptide is cleaved rapidly upon translation, at a cleavage site that 



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can be predicted in a given sequence using tlie method of Von Heijne (1986) Nucleic Acids 
Research 14: 4683-4691 . The pro domain typically is about three times larger than the folly 
processed mature C-terminal domain. Under native conditions the protein is secreted as a mature 
dimer and the cleaved pro domain can be associated therewith to form a protein complex, 
5 presumably to improve the solubility of the mature dimeric protein. Typically, the complex form 
of a morphogen is more soluble than the mature form under physiological conditions. 

Natural-sourced morphogenic protein in its mature, native form, typically is a glycosylated 
dimer, typically having an apparent molecular wreight of about 30-36 kDa as deteimined by SDS- 
PAGE. When reduced, the 30 kDa protein gives rise to two glycosylated polypeptide subunits 
10 having apparent molecular weights in the range of about 16 kDa and 1 8 kDa. The unglycosylated 
dimeric protein, which also has morphogenic activity, typically has an apparent molecular weight 
in the range of about 27 kDa. When reduced, the 27 kDa protein gives rise to two unglycosylated 
polypeptides having molecular weights typically in the range of about 14 kDa to 16 kDa. 

In preferred embodiments, the each of the polypeptide chains of a dimeric morphogenic 
15 protein as defmed herein comprises an amino acid sequence sharing a defined relationship with an 
amino acid sequence of a reference morphogen. In one embodiment, preferred morphogenic 
polypeptide chains share a defined relationship with a sequence present in morphogenically active 
human OP-1 , SEQ ID NO . 5. However, any one or more of the naturally occurring or 
biosynthetic morphogenic proteins disclosed herein similarly could be used as a reference 
20 sequence. Preferred morphogenic polypeptide chains share a defined relationship with at least the 
C-terminai six cysteine domain of human OP-1. residues 335-431 of SEQ ID NO: 5. Preferably, 
morphogenic polypeptide chains share a defined relationship with at least the C-terminal seven 
cysteine domain of human OP-1, residues 330-431 of SEQ ID NO: 5. That is, preferred 
polypeptide chains in a dimeric protein with tissue morphogenic activity «ach comprise a sequence 
25 that corresponds to a reference sequence or is fijnctionally equivalent thereto. 

Functionally equivalem sequences include fimctionally equivalent arrangements of cysteine 
residues disposed within the reference sequence, including amino acid insertions or deletions 
which alter the linear arrangement of these cysteines, but do not materially impair their 
relationship in the folded structure of the dimeric morphogen protein, including their ability to 
30 form such intra- or inter-chain disulfide bonds as may be necessary Tor morphogenic activity. For 
example naturally occurring morphogens have been described in which at least one internal 



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deletion (of one residue; BMP2) or insenion (of four residues; GDF-1) is present but does not 
abrogate biological activity. Functionally equivalent sequences further include those wherein one 
or more amino acid residues tiiffer from the corresponding residue of a reference sequence, e.g., 
the C-terminal seven cysteine domain (also referred to herein as the conserved seven cysteine 

5 skeleton) of human OP- 1, provided that this difference does not destroy tissue morphogenic 
activity. Accordingly, conservative substitutions of corresponding amino acids in the reference 
sequence are preferred. Amino acid residues that are "conservative substitutions" for 
corresponding residues in a reference sequence are those that are physically or functionally similar 
to the corresponding reference residues, e.g., that have similar size, shape, electric charge, 

10 chemical properties including the ability to form covalent or hydrogen bonds, or the like. 
Particularly preferred conservative substitutions are those fulfilling the criteria defined for an 
accepted point mutation in Dayhoff et aL (1978), 5 Atlas of Protein Sequence and Structure, 
Suppl. 3, ch. 22 (pp. 354-352), Natl. Biomed. Res. Found., Washington, D.C. 20007, the 
teachings of which are incorporated by reference herein. Examples of conservative substitutions 

15 include: Conservative substitutions typically include the substitution of one amino acid for 
another with similar characteristics, e.g., substitutions within the following groups: valine, 
glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, 
glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. The term "conservative 
variation" also includes the use of a substituted amino acid in place of an unsubstituted parent 

20 amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with 
the unsubstituted polypeptide 

As described elsewhere herein, the class of morphogenic proteins useful in the methods 
and compositions of the invention is typified by human osteogenic protein (hOP-l). Other 
morphogenic proteins useful in the practice of the invention include raorphogenically active forms 

25 of OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-9, DPP, Vgl, Vgr, 60A 
protein, GDF-1, GDFO, GDF-5, <jDF-6, GDF-7, BMP-IQ, BMP-1 1, BMP.13, BMP-15, 
UNIVIN, NODAL, SCREW, ADMP or NEURAL and amino acid sequence variants thereof In 
one currently preferred embodiment, osteogenic protein include any one of OP- 1, OP-2, OP-3, 
BMP-2, BMP.4, BMP-5, BMP.6, BMP-9, and amino acid sequence variants and homologs 

30 thereof, including species homologs, thereof 

Publications disclosing these sequences, as well as their chemical and physical properties, 
include: OP-1 andOP-2: U.S. 5,011,691, U.S. 5,266,683, Ozkaynak etal. {\990)EMBOJ. 9: 

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2085-2093; OPS. WO 94/10203 (PCT US93/10520); BMP-2, BMP-3, BMP-4. WO 88/00205, 
Wozney eial. (1988) Science 242: 1528-1534); BMP-5 andBMP-6: Celeste et aL (mi) PNAS 
87: 9843-9847; Vgr-J: Lyons e/ a/. im9)PNAS86: 4554-4558; DPP: Padgett e/ a/. (1987) 
Nature 325: 81-84; Vg-J: Weeks (1987) Cell 51: 861-867; BMP-9: WO 95/33830 
5 (PCT/US95/07084); BMP-10: WO 94/26893 (PCTAJS94/05290); BMP-U: WO 94/26892 
(PCT/US94/05288); WO 95/16035 (PCT/US94/14030); BMP-13: WO 95/16035 

(PCTAJS94/14030); GDF-1: WO 92/00382 (PCT/US9 1/04096) and Lee etal. (\99\)PNAS88: 
4250-4254; GDF-8: WO 94/21681 (PCT/US94/03019); GDF-9: WO 94/15966 
(PCT/US94/00685); GDF-IO: WO 95/10539 (PCT/US94/1 1440), GZ)F-//. WO 96/01845 
10 (PCT/US95/08543); BMP-J5: WO 96/36710 (PCT/US96/06540); MP12J: WO 96/01316 
(PCT/EP95/02552); GDF-5 (CDMP-l, MP52): WO 94/15949 (PCT/US94/00657) and 
WO 96/14335 (PGT/US94/12814) and WO 93/16099 (PCT/EP93/00350); GDF-6 (CDMP-2, 
BMP-13): WO 95/01801 (PCT/US94/07762) and WO 96/14335 and WO 95/10635 
(PCT/US94/14030); GDF-7 (CDMP-3, BMP-12). WO 95/10802 (PCT/US94/07799) and 
15 WO 95/1 0635 (PCT/US94/14030). In another embodiment, useful proteins include biologically 
active biosynthetic constructs, including novel biosynthetic morphogemc proteins and chimeric 
proteins designed using sequences from two or more known morphogens. See also the 
biosynthetic constructs disclosed in U.S. Pat. 5,01 1,691, the disclosure of which is incorporated 
herein by reference (e.g., COP-1, COP-3, COP-4, COP-5, COP-7, and COP.16). 
20 In certain preferred embodiments, useful morphogenic proteins include those in which the 

amino acid sequences comprise a sequence sharing at least 70% amino acid sequence homology 
or "similarity", and preferably 80% homology or similarity vnth a reference morphogenic protein 
selected from the foregoing naturally occurring proteins. Preferably, the reference protein is 
human OP- 1 , and the reference sequence thereof is the C-terminal seven cysteine domain present 
25 in osteogenically active forms of human OP-1, residues 330-431 of SEQ ID NO: 5. Usefiil 

morphogenic proteins accordingly include allelic, phylogenetic counterpart and other variants of 
the preferred reference sequence, whether naturally-occurring or biosynthetically produced (e.g., 
including "muteins" or "mutant proteins"), as well as novel members of the general morphogenic 
family of proteins including those set forth and identified above. Certain particularly preferred 
30 morphogenic polypeptides share at least 60% amino acid identity with the preferred reference 
sequence of human OP-1, still more preferably at least 65% amino acid identity therewith. 



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In certain embodiments, a polypeptide suspected of being functionally equivalent to a 
reference morphocen polypeptide is aligned therewith using the method of Needleman, et ai 
(1970) J. MoL Biol. 48:443-453, implemented conveniently by computer programs such as the 
Align program (DNAstar, Inc.). As noted above, internal gaps and amino acid insertions in the 
5 candidate sequence are ignored for purposes of calculating the defined relationship, 

conventionally expressed as a level of amino acid sequence homology or identity, between the 
candidate and reference sequences. "Amino acid sequence homolog>'" is understood herein to 
include both amino acid sequence identity and similarity. Homologous sequences share identical 
and/or similar amino acid residues, where similar residues are conservation substitutions for, or 
10 "allowed point mutations", of, con-esponding amino acid residues in an aligned reference 

sequence. Thus, a candidate polypeptide sequence that shares 70% amino acid homology with a 
reference sequence is one in which any 70% of the aligned residues are either identical to, or are 
conservative substitutions of, the corresponding residues in a reference sequence. In a currently 
preferred embodiment, the reference sequence is OP- 1 . 

FJG. 1 recites the percent amino acid sequence homology (similarity) and percent identity 
within the C-terminal seven cysteine domain of various representative members of the TGF-D 
family, using OP-1 as the reference sequence. The percent homologies recited in the figure are 
calculated with the sequences aligned essentially following the method of Needleman, et ai 
( 1 970) J. Mol, Biol. , 48: 443-453, calculated using the Align Program (DNAstar, Inc.). 
20 Insertions and deletions from the reference morphogen sequence, here the C-terminai, biologically 
active seven-cysteine domain or skeleton of hOP-1, are ignored for purposes of calculation. 

As is apparent to one of ordinary skill in the art revievinng the sequences for the proteins 
listed in FIG 1, significant amino acid changes can be made from the reference sequence while 
retaining morphogenic activity. For example, while the GDF-1 protein sequence shares only 

25 about 50% amino acid identity with the hOP-1 sequence described herein, the <H)F.l -sequence 
shares greater than 70% amino acid sequence homology with the hOP-1 sequence, where 
"homology" is as defined above. Moreover, GDF-1 contains a four amino acid insert XGly-Gly- 
Pro-Pro) between the two residues corresponding to residue 372 and 373 of OP-1 (SEQ ID NO: 
5). Similarly, BMP-3 has a "extra" residue, a valine, inserted between the two residues 

30 corresponding to residues 385 and 386 of hOP-1 (SEQ ID NO: 5). Also, fiMP-2 and BMP-4 
both are "missing" the amino acid residue corresponding to residue 389 of OP-1 <SEQ ID NO: 5) 



wo 97/34626 PCT/US97/04I77 

None of these "deviations" from the reference sequence appear to interfere with biological 
activity. 

In other preferred embodiments, the family of morphogenic polypeptides useful in the 
presem invention, and members thereof, are defined by a generic amino acid sequence. For 
5 example. Generic Sequence 7 (SEQ ID NO: 1) and Generic Sequence 8 (SEQ ID NO: 2) 

disclosed below, accommodate the homologies shared among preferred protein family members 
identified to date, including at least OP-1. OP-2, OP.3. CBMP-2A, CBMP-2B, BMP-3. 60A, 
DPP. Vgl, BMP-5, BMP-6, Vgr-1, and GDF-1 . The amino acid sequences for these proteins are 
described herein and/or in the art, as summarized above. The generic sequences include both the 
10 amino acid identity shared by these sequences in the C-terminal domain, defined by the six and 
seven cysteine skeletons (Generic Sequences 7 and 8, respeaively), as well as alternative residues 
for the variable positions within the sequence. The generic sequences provide an appropriate 
cysteine skeleton where inter- or intramolecular disulfide bonds can form, and contain certain 
critical amino acids likely to influence the tertiary structure of the folded proteins. In addition, the 
,5 generic sequences allow for an additional cysteine at position 36 (Generic Sequence 7) or position 
41 (Generic Sequence 8). thereby encompassing the morphogenically active sequences of OP-2 
andOP-3. 

Generic Sequence 7 (SEQ ID NO: 1) 



Leu Xaa Xm Xaa Phe Xaa Xaa 

5 

Xaa Xaa Xaa Xaa Pro 

Xaa Xaa Xaa Xaa Ala 



1 5 
Xaa Gly Tip Xaa Xaa 
10 

Xaa Tyr Cys Xaa Gly 

20 2* 
Xaa cys Xaa X.. ft" xaa X«. xaa X«. Xaa 

Xaa Xaa Xa. Asn His Ala Xaa xaa Xaa Xaa 
40 

X«. Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

50 

Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa 

60 

xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

70 

xaa X«> Xaa Val Xaa Uu Xaa Xaa Xaa Xaa 

80 

Xaa Me, Xaa Val Xaa Xaa Cys Xaa Cys Xaa 
90 



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wherein each Xaa independently is selected from a group of one or more specified amino acids 
defined as follows: "Res. " means "residue" and Xaa at res. 2 = (Tyr or Lys); Xaa at res. 3 = Val 
or He); Xaa at res. 4 = (Ser, Asp or Glu); Xaa at res. 6 = {Arg, Gin, Ser, Lys or Ala), Xaa at 
res. 7 - (Asp or Glu); Xaa at res. 8 = (Leu, Val or He), Xaa at res. 1 1 = (Gin, Leu, Asp, His, Asn 

5 or Ser); Xaa at res. 12 = (Asp, -\rg, Asn or Glu); Xaa at res. 13 = (Trp or Ser); Xaa at res. 14 = 
(He or Val); Xaa at res. 1 5 - (He or Val); Xaa at res. 16 (Ala or Ser); Xaa at res. 1 8 = (Glu, Gin. 
Leu, Lys, Pro or Arg); Xaa at res. 19 = (Gly or Ser); Xaa at res. 20 = (Tyr or Phe); Xaa at 
res. 21 - (Ala, Ser, Asp, Met, His, Gin, Leu or Gly); Xaa at res. 23 = (Tyr, Asn or Phe); Xaa at 
res. 26 (Glu, His, Tyr, Asp, Gin, Ala or Ser); Xaa at res. 28 = (Glu, Lys, Asp, Gin or Ala); Xaa 

10 at res. 30 = (Ala, Ser, Pro. Gin, He 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 Ue); Xaa at res. 37 = (Met, Phe, Gly or 
Leu); Xaa at res. 38 = (Asn, Ser or Lys); Xaa at res. 39 = (Ala, Ser, Gly or Pro); Xaa at res. 40 = 
(Thr, Leu or Ser); Xaa at res. 44 = (lie, Val or Thr); Xaa at res. 45 = (Val, Leu, Met or lie); Xaa 

15 at res. 46 = (Gin or Arg); Xaa at res. 47 = (Thr, Ala or Ser); Xaa at res. 48 = <Leu or He); Xaa at 
res. 49 = (Val or Met); Xaa at res. 50 = (His, Asn or Arg); Xaa at res. 5 1 = (Phe, Leu, Asn, Ser, 
Ala or Val); Xaa at res. 52 = (He, Met, Asn, Ala, Val, Gly or Leu); Xaa at res. 53 = (Asn, Lys, 
Ala, Glu, Gly or Phe), Xaa at res. 54 = (Pro, Ser or Val); Xaa at res. 55 = (Glu, Asp, Asn, Gly, 
Val, Pro or Lys); Xaa at res. 56 = (Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, He or His); Xaa at 

20 res. 57 ^ (Val, Ala or He), Xaa at res. 58 = (Pro or Asp), Xaa at res. 59 = (Lys, Leu or Glu); Xaa 
at res. 60 = (Pro, Val or Ala); Xaa at res. 63 = (Ala or Val); Xaa at res. 65 = (Thr, Ala or Glu); 
Xaa at res. 66 = (Gin, Lys, Arg or Glu); Xaa at res. 67 = (Leu, Met or Val); Xaa at res. 68 = . 
(Asn, Ser, Asp or Gly); Xaa at res. 69 = (Ala, Pro or Ser); Xaa at res. 70 = (He, Thr, Val or Leu), 
Xaa at res. 71 = (Ser, Ala or Pro); Xaa at r^s. 72 = (Val, Leu, Met or He); Xaa at res. 74 = (Tyr 

25 or Phe); Xaa at res. 75 = (Phe, Tyr, Leu or His), Xaa at r«s. 76 = (Asp, Asn or Leu); Xaa at 
res. 77 - (Asp, Glu, Asn, Arg or Ser); Xaa at res, 78 = (Ser, Gin, Asn, Tyr or Asp); Xaa at 
res. 79 = (Ser, Asn, Asp, Glu or Lys); Xaa at res. 80 = (Asn, Thr or Lys); Xaa at res. 82 = (He, 
Val or Asn); Xaa at res. 84 = (Lys or Arg); Xaa at res. 85 = (Lys, Asn, Gin, His, Arg or Val); Xaa 
at res. 86 = (Tyr, Glu or His); Xaa at res. 87 = (Arg, Gin, Glu or Pro), Xaa at res. 88 = (Asn, Glu, 

30 Trp or Asp); Xaa at res. 90 = (Val, Thr, Ala or He); Xaa at res. 92 = (Arg, Lys, Val, Asp, Gin or 
Glu); Xaa at res. 93 = (Ala, Gly, Glu or Ser); Xaa at res. 95 = (Gly or Ala) and Xaa at res. 97 = 
(His or Arg). 

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Generic Sequence 8 (SEQ ID NO: 2) includes all of Generic Sequence 7 (SEQ ID NO: 1 ) 
and in addition includes the following sequence (SEQ ID NO: 8) at its N-terniinus: 

SEQ ID NO: 8 

Cys Xaa Xaa Xaa Xaa 

1 5 

Accordingly, beginning with residue 7, each "Xaa" in Generic Sequence 8 is a specified amino 

5 acid defined as for Generic Sequence 7, with the distinction that each residue number described 
for Generic Sequence 7 is shifted by five in Generic Sequence 8. Thus, "Xaa at res. 2 =(Tyr or 
Lys)" in Generic Sequence 7 refers to Xaa at res. 7 in Generic Sequence 8. In Generic Sequence 
8, Xaa at res. 2 = (Lys, Arg, Ala or Gin); Xaa at res. 3 = (Lys, Arg or Met); Xaa at res. 4 = (His. 
Arg or Gin); and Xaa at res. 5 = (Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr). 

10 In another embodiment, usefiil osteogenic proteins include those defined by Generic 

Sequences 9 and 10 (SEQ ID NOs: 6 and 7, respectively), described herein above. Specifically, 
Generic Sequences 9 and 10 are composite amino acid sequences of the following proteins: 
human OP-1 , human OP.2, human OP-3, human BMP-2. human BMP-3, human BMP-4. human 
BMP-5, human BMP-6, human BMP-8, human BMP-9, human BMP-10, human BMP-1 1 , 

15 Drosophila 60A, Xenopus Vg-1 , sea urchin UNIVIN, human CDMP-1 (mouse GDF-5), human 
CDMP-2 (mouse GDF-6, human BMP-13), human CDMP-3 (mouse GDF-7. human BMP-12), 
mouse GDF-3. human GDF-1, mouse GDF-1. chicken DORSALIN. Drosophila dpp, Drosophila 
SCREW, mouse NODAL, mouse GDF-8. human GDF.8. mouse GDF-9, mouse GDF-10, human 
GDF-l 1, mouse GDF-1 1, human BMP-15, and rat BMP-3b. Like Generic Sequence 7, Generic 

20 Sequence 9 accommodates the C-tenninal she cysteine skeleton and, like Generic Sequence 8, 
Generic Sequence 1 0 accommodates the seven cysteine skeleton. 

Generic Sequence 9 (SEQ ID NO: 6) 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

1 5 ; 10 

Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa 

15 20 



Xaa Xaa Xaa 



Xaa Cys Xaa Gly Xaa Cys Xaa 

25 • ^0 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

35 ^0 



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wo 97/34626 PCT/US97/04177 



Xaa 


Xaa 


Xaa 


Xaa 


Xaa 
45 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 

50 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 

55 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 
60 


Xaa 


Cys 


Xaa 


Pro 


Xaa 

65 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 

70 


Xaa 


Xaa 


Leu 


Xaa 


Xaa 
75 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 
SO 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 

85 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 
90 


Xaa 


Xaa 


Xaa 


Cys 


Xaa 


Cys 


Xaa 









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. 1 = (Phe, Leu or Glu); Xaa at res. 2 = 
(Tyr, Phe, His, Arg, Thr, Lys, Gin, Val or Glu); Xaa at res. 3 = (Val, He, Uu or Asp); Xaa at 
res, 4 = (Ser, Asp, Glu, Asn or Phe); Xaa at res. 5 = (Phe or Glu); Xaa at res. 6 = (Arg, Gin, Lys, 
5 Ser, Glu, Ala or Asn); Xaa at res. 7 = (Asp, Glu, Leu, Ala or Gin); Xaa at res 8 = (Leu, Val, 
Met, De or Phe); Xaa at res. 9 = (Gly, His or Lys); Xaa at res. 10 = (Trp or Met); Xaa at res. 1 1 = 
(Gin, Leu, His, Glu, Asn, Asp, Ser or Gly); Xaa at res. 12 - (Asp, Asn, Ser, Lys, Arg, Glu or 
His); Xaa at res. 13 = (Trp or Ser); Xaa at res. 14 = (He or Val); Xaa at res. 15 ^ (He or Val); 
Xaa at res. 16 = (Ala, Ser, Tyr or Trp); Xaa at res. 18 = (Glu, Lys, Gin, Met, Pro, Leu, Arg, His 

10 or Lys); Xaa at res. 1 9 = (Gly, Glu, Asp, Lys, Ser, Gin, Arg or Phe); Xaa at res. 20 = (Tyr or 
Phe); Xaa at res 21 = (Ala, Ser, Gly, Met, Gin, His, Glu, Asp, Leu, Asn, Lys or Thr); Xaa at 
res. 22 = (Ala or Pro); Xaa at res. 23 = (Tyr, Phe, Asn, Ala or Arg); Xaa at res 24 = (Tyr, His, 
Glu, Phe or Arg), Xaa at res, 26 = (Glu, Asp, Ala, Ser, Tyr, His, Lys, Arg, Gin or Gly); Xaa at 
res, 28 = (Glu, Asp, Leu, Val, Lys, Gly, Thr, Ala or Gin); Xaa at res. 30 - (Ala, Ser, He, Asn, 

15 Pro, Glu, Asp, Phe, Gin or Leu); Xaa at res. 3 1 = (Phe, Tyr, Leu, Asn, Gly or Arg); Xaa at 
res. 32 = (Pro, Ser, Ala or Val); Xaa at res 33 = (Leu, Met, Glu, Phe or Val); Xaa at s. 34 = 
(Asn, Asp, Thr, Gly, Ala, Arg, Leu or Pro); Xaa at res. 35 = (Ser, Ala, Glu, Asp, Thr, Leu, Lys, 
Gin or His); Xaa at res. 36 = (Tyr, His, Cys, He, Arg, Asp, Asn, Lys, Ser, Glu or Gly); Xaa at 
res. 37 = (Met, Leu, Phe, Val, Gly or Tyr), Xaa at res. 38 = (Asn, Glu, Thr, Pro, Lys, His, Gly, 

20 Met, Val or Arg); Xaa at res. 39 = (Ala. Ser, Gly, Pro or Phe); Xaa at res. 40 - (Thr, Ser, Leu, 
Pro, His or Met); Xaa at res. 41 = (Asn, Lys, Val, Thr or Gin); Xaa atr^s. 42 = (His, Tyr or Lys); 
Xaa at res. 43 = (Ala, Thr, Leu or Tyr); Xaa at res. 44 = (He, Thr, Val, Phe, Tyr, Met or Pro); 
Xaa at res. 45 = (Val Leu. Met, He or His); Xaa at res. 46 = (Gin, Arg or Thr); Xaa at res. 47 = 



W0 97«4626 PCT/US97/04I77 

(Thr, Ser. Ala, Asn or His), Xaa at res. 48 = (Leu, Asn or He); Xaa at res. 49 = (Val, Met, Leu. 
Pro or He); Xaa at res. 50 = (His, Asn, Arg, Lys. Tyr or Gin); Xaa at res. 51 = (Phe, Leu. Ser, 
Asn, Met. Ala, Arg, Glu, Gly or Gin); Xaa at res. 52 = (He, Met. Leu. Val, Lys. Gin, Ala or Tyr); 
Xaa'at res. 53 = (Asn. Phe. Lys, Glu. Asp, Ala, Gin. Gly, Leu or Val); Xaa at res. 54 = (Pro. Asn. 
5 Ser. Val or Asp). Xaa at res. 5 5 = (Glu. Asp. Asn, Lys, .Arg. Ser. Gly, Thr. Gin, Pro or His); Xaa 
at res. 56 = (Thr. His. Tyr. Ala, He, Lys. Asp. Ser. Gly or Arg); Xaa at res 57 = (Val, He, Thr, 
Ala, Leu or Ser); Xaa at res. 58 = (Pro. Gly, Ser, Asp or Ala); Xaa at res. 59 = (Lys, Leu, Pro, 
Ala, Ser. Glu, Arg or Gly); Xaa at res. 60 = (Pro, Ala, Val, Thr or Ser); Xaa at res. 61 = (Cys, 
Val or Ser); Xaa at res. 63 = (Ala, Val or Thr); Xaa at res. 65 = (Thr. Ala. Glu. Val, Gly. Asp or 
10 Tyr); Xaa at res. 66 = (Gin, Lys. Glu, Arg or Val); Xaa at res. 67 = (Leu, Met, Thr or Tyr); Xaa 
at res. 68 = (Asn. Ser, Gly, Thr, Asp. Glu, Lys or Val); Xaa at res. 69 = (Ala, Pro. Gly or Ser); 
Xaa at res. 70 = (He. Thr, Leu or Val); Xaa at res. 71 = (Ser. Pro. Ala, Thr, Asn or Gly); Xaa at 
res. 2 = (Val, He, Leu or Met); Xaa at res. 74 = (Tyr. Phe, Arg, Thr. Tyr or Met); Xaa at 
res. 75 = (Phe. Tyr. His. Uu. He. Lys. Gin or Val); Xaa at res. 76 = (Asp. Leu, Asn or Glu); Xaa 
15 at res. 77 = (Asp, Ser. Ar^. Asn, Glu, Ala, Lys, Gly or Pro); Xaa at res. 78 = (Ser, Asn, Asp, Tyr. 
Ala, Gly. Gin, Met. Glu, Asn or Lys); Xaa at res. 79 = (Ser, Asn, Glu. Asp. Val, Lys. Gly. Gin or 
Arg); Xaa at res. 80 = (Asn, Lys. Thr. Pro, Val, He. Arg, Ser or Gin); Xaa at res. 81 = (Val. He, 
Thr or Ala); Xaa at res. 82 = (He, Asn. Val. Leu. Tyr. Asp or Ala); Xaa at res. 83 = (Leu. Tyr, 
Lys or He); Xaa at res. 84 = (Lys. Arg, Asn, Tyr, Phe, Thr. Glu or Gly); Xaa at res. 85 = (Lys, 
20 Arg. His. Gin. Asn, Glu or Val); Xaa at res. 86 = (Tyr. His. Glu or He); Xaa at r«. 87 = (Arg. 
. Glu, Gin, Pro or Lys); Xaa at res. 88 = (Asn. Asp. Ala, Glu. Gly or Lys); Xaa at res. 89 = (Met or 
Ala); Xaa at res. 90 = (Val, He. Ala, Thr, Ser or Lys); Xaa at res. 91 = (Val or Ala); Xaa at 
res. 92 = (Arg, Lys. Gin. Asp. Glu. Val, Ala. Ser or Thr); Xaa at res. 93 = (Ala. Ser, Glu. Gly, 
Arg or Thr); Xaa at res. 95 = (Gly, Ala or Thr); Xaa at res. 97 -(His, Arg. Gly, Leu or Ser). 
25 Further, after res. 53 in rBMP-3b and mGDF-10 there is an He; after res. 54 in GDF-1 there is a 
T; after res. 54 in BMP-3 there is a V; after res. 78 in BMP-8 and Dorsalin there is a G; after 
res. 37 in hGDF-l there is Pro. Gly, Gly, Pro. 

Generic Sequence 10 (SEQ ID NO: 7) includes all of Generic Sequence 9 (SEQ ID NO: 
6) and in addition includes the following sequence (SEQ ID NO: 9) at its N-temunus: 

SEQ ID NO: 9 



30 

Cys xaa xaa xaa Xaa 

1 ^ . ' 



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Accordingly, beginning with residue 6, each "Xaa" in Generic Sequence 10 is a specified amino 
acid defined as for Generic Sequence 9, with the distinction that each residue number described 
for Generic Sequence 9 is shifted by five in Generic Sequence 10. Thus, "Xaa at res. I =( Tyr, 
Phe, His, Arg, Thr, Lys, Gin, Vai or Glu)" in Generic Sequence 9 refers to Xaa at res. 6 in 
5 Generic Sequence 10. In Generic Sequence 10, Xaa at res. 2 = (Lys, Arg, Gin, Ser, His, Glu, 
Ala, or Cys); Xaa at res. 3 = (Lys, Arg, Met, Lys, Thr, Leu, Tyr, or Ala); Xaa at res. A = (His, 
Gin, Arg, Lys, Thr, Leu, Val, Pro, or Tyr); and Xaa at res. 5 = (Gin, Thr, His, Arg, Pro, Ser, Ala, 
Gin, Asn, Tyr, Lys, Asp, or Leu). 

Based upon alignment of the naturally occurring morphogens within the definition of 
10 Generic Sequence 10, it should be clear that gaps and/or insertions of one or more amino acid 
residues can be tolerated (without abrogating biological activity) at least between or invol\ang 
residues 1 M2, 42-43, 59-60, 68-69 and 83-84. 

As noted above, certain currently preferred morphogenic polypeptide sequences useful in 
this invention have greater than 60% identity, preferably greater than 65% identity, with the 

15 amino acid sequence defining the preferred reference sequence of hOP- 1 . These particularly 
preferred sequences include allelic and phylogenetic counterpart variants of the OP-1 and OP-2 
proteins, including the DrosopMla 60 A protein, as well as the closely related proteins BMP-5, 
BMP-6 and Vgr-1. Accordingly, in certain particularly preferred embodiments, useful 
morphogenic proteins include active proteins comprising pairs of polypeptide chains within the 

20 generic amino acid sequence herein referred to as "OPX" (SEQ ID NO: 3), which defines the 
seven cysteine skeleton and accommodates the homologies between several identified variants of 
OP-1 and OP-2. Accordingly, each "Xaa" at a given position in OPX independently is selected 
firom the residues occurring at the corresponding position in the C-terminal sequence of mouse or 
human OP-1 or OP.2. Specifically, each "Xaa" is independently selected fi-om a group of one or 

25 more specified amino acids as defined below: 



wo 97/34626 



PCTAJS97/04177 



Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp Leu Gly Trp Xaa Asp Trp 

1 5 10 15 

Xaa lie Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly Glu Cys Xaa Phe Pro 
20 2S 30 35 

5 Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala lie Xaa Gin Xaa Leu Val His Xaa 
40 45 50 56 

Xaa Xaa Pro Xaa Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala 
60 . 65 ^0 

■ Xaa Ser Val Leu Tyr Xaa Asp Xaa Ser Xaa Asn Val He Leu Xaa Lys Xaa Arg 
10 75 80 85 .90 

Asn Met Val Val Xaa Ala Cys Gly Cys His 
95 100 

wherein Xaa at res. 2 = (Lys or Arg); Xaa at res. 3 = (Lys or Arg); Xaa at res 1 1 = (Arg or Gin); 

Xaa at res. 1 6 = (Gin or Leu); Xaa at res. 1 9 = (Ue or Val); Xaa at res. 23 = (Glu or Gin); Xaa at 
15 res. 26 = (Ala or Ser), Xaa at res. 35 = (Ala or Ser); Xaa at res. 39 = (Asn or Asp); Xaa at 

res. 4 1 = (Tyr or Cys); Xaa at res. 50 = (Val or Leu); Xaa at res. 52 = (Ser or Thr); Xaa at 

res. 56 = (Phe or Leu); Xaa at res. 57 = (He or Met); Xaa at res. 58 = (Asn or Lys); Xaa at 

res. 60 = (Glu, Asp or Asn); Xaa at res. 61 = (Thr, Ala or Val); Xaa at res 65 = (Pro or Aia); 

Xaa at res. 71 = (Gin or Lys); Xaa at res. 73 = (Asn or Ser); Xaa at res. 75 = (lie or Thr); Xaa at 
20 res. 80 = (Phe or Tyr); Xaa at res. 82 = (Asp or Ser); Xaa at res. 84 = (Ser or Asn); Xaa at 

res. 89 = (Lys or Arg); Xaa at res. 91 = (Tyr or His); and Xaa at res. 97 = (Arg or Lys). 

In still another preferred embodiment, useful morphogenically active proteins have 
polypeptide chains with amino acid sequences comprising a sequence encoded by a nucldc acid 
that hybridizes, under low, medium or high stringency hybridization conditions, to DNA or RNA 
25 encoding reference morphogen sequences, e.g. , C-terminal sequences defining the conserved 
seven cysteine domains of OP-1, OP-2. BMP-2, BMP-4, BMP.5, BMP-6, 60A, GDF.3. GDF-5. 
GDF-6, GDF-7 and the like. As used herein, high stringency hybridization conditions are defined 
as hybridization according to known techniques in 40% formamide, 5 X SSPE, 5 X Denhardt's 
Solution, and 0.1% SDS at 37°C overnight, and washing in 0.1 X SSPE, 0.1% SDS at 50°C. 
30 Standard stringency conditions are well characterized in standard molecular biology cloning texts. 
See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed. , ed. by Sambrbok, Fritsch 
and Maniatis (Cold Spring Harbor Laboratory Press; 1"989); DNA Cloning, Volumes I and II 
(D.N. Glover ed , 1 985); Oligonucleotide Synthesis (M.J. Gait ed., 1 984). Nucleic Acid 
Hybridization (B. D. Hames & S J. Higgins eds 1984); and B Perbal, A Practical Guide To 
35 Molecular Cloning {inA). 



wo 97/34626 



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Accordingly, the morphogenic proteins useful in the materials and methods of this 
invention can include proteins comprising any of the polypeptide chains described above, whether 
isolated from naturally-occurring sources, or produced by recombinant DNA or other synthetic 
techniques, and includes allelic and phylogenetic counterpan variants of these proteins, as well as 
5 biosynthetic variants (muteins) thereof, and various truncated and fusion constructs. Deletion or 
addition mutants also are envisioned to be active, including those which may alter the conserved 
C-terminal six or seven cysteine domain, provided that the alteration does not functionally disrupt 
the relationship of these cysteines in the folded structure. Accordingly, such active forms are 
considered the equivalent of the specifically described constructs disclosed herein. The proteins 
1 0 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 native or biosynthetic proteins, produced by expression of recombinant DNA in host cells. 

The bone morphogenic proteins contemplated herein can be expressed from intact or 
truncated cDNA or from synthetic DNAs in prokaryotic or eukaryotic host cells, and purified, 

15 cleaved, refolded, and dimerized to form morphogenically active compositions. Currently 
preferred host cells include, without limitation, prokaryotes, including £. coli, and eukaryotes, 
including yeast, and manmiaUan cells, such as CHO, COS or BSC cells. One of ordinary skill in 
the art will appreciate that other host cells can be used to advantage. Detailed descriptions of the 
morphogenic proteins useful in the practice of this invention, including how to make, use and test 

20 them for aaivity, are disclosed in numerous publications, including those recited herein, the 

disclosures of which are incorporated by reference herein. Accordingly, using standard molecular 
biology texts and procedures, and the knowledge available in the art, the skilled genetic 
engineer/molecular biologist can isolate genes from cDNA or genomic libraries of various 
different biological species, which encode appropriate amino acid sequences, or construct DNAs 

25 from oligonucleotides, and then can express them in various types of host cells, including both 
prokaryotes and eukaryotes, to produce large quantities of active proteins capable of stimulating 
neural tissue morphogenesis in a mammal. 

In other embodiments, as an alternative to the administration of a morphogenic protein, an 
effective amount of an agent competent to stimulate or induce increased endogenous morphogen 
30 expression in a mammal may be administered by any of the routes described herein. Such an 
inducer of a morphogen may be provided to a mammal, eg., by systemic administration to the 
mammal or by direct administration to the neural tissue. A method for identifying and testing 

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inducers (stimulating agents) competent to modulate the levels of endogenous morphogens in a 
given tissue .s described in detail in published applications WO 93/05172 and WO 93/0575 1, the 
teachings of which are incorporated herein by reference. Briefly, candidate compounds can be 
identified and tested by incubation in v.tro with a test tissue or cells thereof, or a cultured cell line 
5 derived therefrom, for a time sufficient to allow the compound to affect the production. ,.e., the 
expression and/or secretion, ofamorphogen produced by the cells of that tissue. Suitable tissue. 

or cultured cells of a suitable tissue, preferably can be selected from renal epithelium, ovarian 
tissue, fibroblasts, and osteoblasts. 

In yet other embodiments, an agent which acts as an agonist of a morphogen receptor may 
,0 be administered instead of the morphogen itself Such an agent may also be referred to an a 
morphogen "mimic," -mimetic," or "analog." Thus, for example, a small peptide or other 
molecule which can mimic the activity of a morphogen in binding to and activating the 
morpbogen's receptor may be employed as an equivalent of the morphogen. Preferably the 
agonist is a full agonist, but partial morphogen receptor agonists may also be advantageously 
15 employed. Methods of identifying such agonists are known in the art and include assays for 
compounds which induce morphogen-mediated responses (e.g:. induction of differentiation of 
metanephric mesenchyme, induction of endochondral bone formation). For example, methods of 
identifying morphogen inducers or agonists of morphogen receptors may be found in U.S. Ser. 
No. 08/478,097 filed June 7, 1995 and U.S. Ser. No. 08/507,598 filed July 26, 1995, the 
20 disclosures ofwhich are incorporated herein by reference. 

Finally, as described below, in other embodiments, cells may be implanted into the subject 
afflicted with an ischemic or traumatic injury of the central nervous system in order to serve as a 
source of morphogen and/or to provide a source of additional functional neural tissue. Such cells 
may be host or donor cells wWch normally express morphogens, which have been uansformed so 
25 as to express morphogens, or which have been ueated with morphogens to induce differentiation. 
C. Mammals Eligible for Treatment 

As a general matter, the methods of the present invention may be applied to the treatment 
of any mammalian subject afflicted with stroke or a traumatic injury of the central nervous system. 
The method can be practiced with mammals in whom the stroke or other traumatic injury arose at 
30 least 6 hours before the stan of treatment, for example as twelve, twenty-four or forty-eight hours 
or lonaer before treatment. Practice of the invention confers a.ignificant clinical benelit on the 



-26- 



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afflicted mammal, in that the invention beneficially confers a detectable, clinically significant 
restoration of a central nervous system fijnction as defined herein. The invention is suitable for 
the treatment of any primate, preferably a higher primate such as a human. In addition, however, 
the invention may be employed in the treatment of domesticated mammals which are maintained 

5 as human companions (e.g., dogs, cats, horses), which have significant commercial value (e.g., 
goats, pigs, sheep, cattle, sporting or draft animals), which have significant scientific value {e.g., 
captive or free specimens of endangered species, or inbred or engineered animal strains), or which 
otherwise have value. One of ordinary skill in the medical or veterinary arts is trained to 
recognize whether a mammal is afflicted with an ischemic or traumatic injury of the central 

10 nervous system. For example, routine testing and/or clinical or veterinary diagnostic evaluation 
will reveal whether the mammal has acquired an impairment or loss of central nervous system 
(e.g., neurologic) fijnction. Clinical and non-clinical indications, as well as accumulated 
experience, relating to the presently disclosed and other methods of treatment, should 
appropriately inform the skilled practitioner in deciding whether a given individual is afflicted with 

15 an ischemic or traumatic injury of the central nervous system and whether any particular treatment 
is best suited to the subject's needs, including treatment according to the present invention. 

D. Formulations and Methods of Treatment 

The morphogens, morphogen inducers, or agonists of morphogen receptors of the present 
invention may be administered by any route which is compatible with the particular morphogen, 

20 inducer, or agonist employed. Thus, as appropriate, administration may be oral or parenteral, 
including intravenous and intraperitoneal routes of administration. In addition, administration 
may be by periodic injections of a bolus of the morphogen, inducer or agonist, or may be made 
more continuous by intravenous or intraperitoneal administration fi'om a reservoir which is 
external (e.g., an i.v. bag) or internal (e.g., a bioerodable implant, or a colony of implanted, 

25 morphogen-producing cells). 

The therapeutic agents of the invention {i.e., morphogens, morphogen inducers or 
agonists of morphogen receptors) may be provided to an individual by any suitable means, tlirectly 
(e.g., locally, as by injection, implantation or topical administration to a tissue locus) or 
systeraically (e.g., parenterally or orally). Where the agent is to be provided parenterally, such as 
30 by intravenous, subcutaneous, intramolecular, ophthalmic, intraperitoneal, intramuscular, buccal, 
rectal vaginal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, 

-27- 



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intracistemal, intracapsular, intranasal or by aerosol administration the agent preferably comprises 
part of an aqueous solution. The solution is physiologically acceptable so that in addition to 
delivery of the desired agent to the patient, the solution does not otherwise adversely affect the 
patient's electrolyte and/or volume balance. The aqueous medium for the agent thus can comprise 
5 normal physiologic saline {e,g., 9.85% NaCl, 0. 1 5M, pH 7-7.4). 

If desired, a given morphogen or other agent may be made more soluble by association 
with a suitable molecule. For example, association of the mature morphogen dimer with the pro 
domain results in the pro form of the morphogen which typically is more soluble or dispersible in 
physiological solutions than the corresponding mature form. In fact, endogenous morphogens are 

10 thought to be transported (e.g., secreted and circulated) in the mammalian body in this form. This 
soluble form of the protein can be obtained from culture medium of morphogen-secreting 
mammalian cells, e.g., cells transfected with nucleic acid encoding and competent to express the 
morphogen. Alternatively, a soluble species can be formulated by complexing the mature, 
morphogenically active polypeptide dimer <or an active fragment thereof) with a morphogen pro 

15 domain or a solubility-enhancing fragment thereof Solubility-enhancing pro domain fragments 
can be any N-terminal, C-terminal or internal fragment of the pro region of a member of the 
morphogen family that complexes with the mature polypeptide dimer to enhance stability and/or 
dissolubility of the resulting noncovalent or convalent complex. Typically, useful fragments are 
those cleaved at the proteolytic site Arg-Xaa-Xaa-Arg. A detailed description of soluble complex 

20 forms of morphogenic proteins, including how to make, test and use them, is described in 

WO 94/03600 (PCT/US93/07 1 89). In the case of OP- 1 , useful pro domain fragments include the 
intact pro domain (residues 30-292) and fragments 48-292 or 1 58-292, all of Se. ID No. 5. 
Another molecule capable of enhancing solubility and particularly useful for ora[ administrations, 
is casein. For example, addition of 0.2% casein increases solubility of the mature active form of 

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

Useful solutions for parenteral administration may be prepared by any of the methods well 
known in the pharmaceutical art, described, for example, in Reminpon's Pharmaceutical Sciences 
(Gennaro, A., ed.), Mack Pub., 1990. Formulations of the therapeutic agents of the invention 
may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable 
30 origin, hydrdgenated naphthalenes, and the like. Formulations for direct administration, in 
particular, may include glycerol and other compositions of high viscosity io help maintain the 
agent at the desired locus. Biocompatible, preferably bioresorbable, polymers, including, for 

- 28 - 



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PCTAJS97/04177 



example, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, lactide, and glycoiide 
polymers and lactide/glycolide copolymers, may be useful excipienls to control the release of the 
agent in vivo. Other potentially useful parenteral delivery systems for these agents include 
ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and 
5 liposomes. Formulations for inhalation administration contain as excipienls, for example, lactose, 
or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, 
glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or 
as a gel to be applied intranasally. Formulations for parenteral administration may also include 
glycocholate for buccal administration, methoxysalicylate for rectal administration, or cutric acid 
10 for vaginal administration. Suppositories for rectal administration also may be prepared by mixing 
the morphogen, inducer or agonist with a non-irritating excipient such as cocoa butter or other 
compositions which are solid at room temperature and liquid at body temperatures. 

Formulations for topical administration to the skin surface may be prepared by dispersing 
the morphogen, inducer or agonist with a dermatologically acceptable carrier such as a lotion, 

15 cream, ointment or soap. Particularly useful are carriers capable of forming a film or layer over 
the skin to localize apphcation and inhibit removal. For topical administration to internal tissue 
surfaces, the agent may be dispersed in a liquid tissue adhesive or other substance knov^ to 
enhance adsorption to a tissue surface. For example, hydroxypropylcellulose or 
fibrinogen/thrombin solutions may be used to advantage. Alternatively, tissue-coating solutions, 

20 such as pectin-containing formulations may be used. 

Alternatively, the agents described herein may be administered orally. Oral administration 
of proteins as therapeutics generally is not practiced as most proteins are readily degraded by 
digestive enzymes and acids in the mammalian digestive system before they can be absorbed into 
the bloodstream. However, the morphogens described herein typically are acid stable and 

25 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 fi-om manunary gland extract is morphogenically active and also is 
detected in the bloodstream. Maternal administration, via ingested milk, may be a natural delivery 
route ofTGF-P superfamily proteins. Letterio, etaL (1994), Science 264: 1936-1938, report that 

30 TGF-p is present in murine milk, and that radiolabelled TGF-P is absorbed by gastrointestinal 
mucosa of suckling juveniles. Labeled, ingested TGF-P appears rapidly in intact form in the 
juveniles* body tissues, including lung, heart and liver. Finally, soluble form morphogen, e.g., 

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wo 97/34626 PCT/US97/04I77 

mature morphogen associated with the pro domain, is morphogenically active: These findings, as 
well as those disclosed in the examples below, indicate that oral and parenteral administration are. 
viable means for administering TGF-p superfamily proteins, including the morphogens, to an 
individual. In addition, while the mature forms of certain morphogens described herein typically 
5 are sparingly soluble, the morphogen form found in milk (and mammary gland extract and 

colostrum) is readily soluble, probably by association of the mature, morphogenically active form 
with pan or all of the pro domain of the expressed, full length polypeptide sequence and/or by 
association with one or more milk components. Accordingly, the compounds provided herein 
also may be associated with molecules capable of enhancing their solubility /« vitro or invito 
10 The compounds provided herein also may be associated with molecules capable of 

targeting the morphogen, inducer or agonist to the desired tissue. For example, an antibody, 
antibody fragmcm, or other binding protein that interacts specifically with a surface molecule on 
cells of the desired tissue, may be used. Useful targeting molecules may be designed, for example, 
using the single chain binding site technology disclosed, for example, in U.S. Pat. No. 5.091,513. 
15 Targeting molecules can be covalently or non-covalently associated with the morphogen, inducer 
or agonist. 

As will be appreciated by one of ordinary skill in the art, the formulated compositions 
contain therapeutically-effective amounts of the morphogen. morphogen inducers or agonists of 
morphogen receptors. That is. they contain an amoum which provides appropriate concentrations 
20 of the agent to the affected nervous system tissue for a time sufficient to stimulate a detecuble 
restoration of central nen^ous system fiinction, up to and including a complete restoration thereof 
As will be appreciated by those skilled in the art, these concentrations will vary depending upon a 
number of factors, including the biological efficacy of the selected agent, the chemical 
characteristics {e.g., hydrophobicity) of the specific agem, the formulation thereof, including a 
25 mixture with one or more excipients. the administration route, and the treatment envisioned, 
including whether the active ingrediem will be administered directly into a tissue site, or whether 
it will be administered systemically. The preferred dosage to be administered also is likely to 
depend on such variables such as the condition of the diseased or damaged tissues, and the overall 
health status of the particular mammal. As a general matter, single, daily, biweekly or weekly 
30 dosa«s of 0.00001-1000 mg of a morphogen are sufficiem with 0.0001-100 mg being preferable, 
and 0.001 to 10 mg being even more preferable. Alternatively, a single, daily, biweekly or weekly 
dosage of 0.01-1000 Mg/kg body weight, more preferably 0.01-10 mg^cg body weight, may be 

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advantageously employed. The present effective dose can be administered in a single dose or in a 
plurality (two or more) of installment doses, as desired or considered appropriate under the 
specific circumstances. A bolus injection or diffusable infusion formulation can be used. If 
desired to facilitate repeated or frequent infusions, implantation of a semi-permanent stent {e g., 

5 intravenous, intraperitoneal, intracisternal or intracapsular) may be advisable. In Example 2 
below, intracisternal administration of 6-240 Mg/kg of the reference morphogen (hOP-1) 
conferred clearly detectable levels of restoration of lost or impaired central nervous system 
function. It should be noted that no obvious morphogen induced pathological lesions arise when 
mature morphogen (e.g., OP-1, 20 mg) is administered daily to normal growing rats for 

10 21 consecutive days. Moreover, 10 mg systemic injections of morphogen {e.g., OP-1) injected 
daily for 1 0 days into normal newborn mice does not produce any gross abnormalities. 

The morphogens, inducers or agonists of the invention may, of course, be administered 
alone or in combination with other molecules known to be beneficial in the treatment of the 
conditions described herein. For example, various well-known growth factors, hormones, 

15 enzymes, therapeutic compositions, antibiotics, or other bioactive agents can also be administered 
with the morphogen. Thus, various known growth factors such as NGF, EOF, PDGF, IGF, FGF, 
TGF-a , and TGF-P, as well as enzymes, enzyme inhibitors, antioxidants, anti-inflammatory 
agents, free radical scavenging agents, antibiotics and/or chemoattractant/chemotactic factors, can 
be included in the present administratable morphogen formulation. To facilitate uptake by central 

20 nervous system tissue, the morphogens, inducers or agonists provided herein can be derivatized or 
conjugated to a lipophilic moiety or to a substance that is actively transported across the blood- 
brain barrier. 

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

Example 1: Preparation of Soluble Morphogen Protein Solutions for In Vivo 
Administration 

A. Aqueous Solutions 

While the mature dimeric morphogenic proteins defined herein typically, are substantially 
30 only sparingly soluble in physiological buffers, they can be solubilized to form injectable solutions. 
One exemplary aqueous solution containing a morphogen can be made, for example, by dissolving 



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or dispersing the morphogen in 50% ethanol containing acetonitrile in 0. 1% trifluoroacetic acid 
(TFA) or 0. 1 % HCl, or in an equivalent solvent. One volume of the resultant solution then is 
added, for example, to ten volumes of phosphate buffered saline (PBS), which further may include 
0. 1-0.2% human serum albumin (HSA) or a similar carrier protein. The resultant solution 
5 preferably is vortexed extensively to produce a physiologically acceptable morphogen 
formulation. 

In another embodiment, the morphogen, including OP-1. can be solubilized by reducing 
the pH of the solution. In one currently preferred formulation, the protein is solubilized in 0.2 
raM acetate buffer. pH 4.5. containing 5% mannitol, to render the solution more isotonic. Other 
10 standard means for creating physiologically acceptable formulations arc contemplated to be within 
the skill of the art. 

B. Soluble Complex Formulations 

Another currently preferred form of the morphogen useful herein, having improved 
solubility in aqueous solutions, is a dimeric morphogenic protein comprising at least the 

15 C-terminal seven cysteine domain characteristic of the morphogen family, complexed with a 
peptide comprising a pro region of a member of the morphogen family, or a solubUity-enhancing 
fragment thereof, or an alleUc. species or other sequence variant thereof. Solubility-enhancing 
fragment can be any N-terminal or C-teiminal fragment of the pro region of a member of the 
morphogen family that complexes with the mature polypeptide dimer to enhance the stability of 

20 the soluble complex. Preferably, the dimeric morphogenic protein is complexed with two pro 
region peptides. 

As described above and in published appUcation WO 94/03600, the teachings of which are 
incorporated herein by reference, the soluble complex form can be isolated from the cell culture 
media (or a body fluid) under appropriate conditions. Alternatively, the complex can be 
25 formulated in vitro. 

Soluble morphogen complexes can be isolated from conditioned media using a simple, 
three step chromatographic protocol performed in the absence of denaturants. The protocol 
involves rum^ng the media (or body fluid) over an affmity column, followed by ion exchange and 
gel filtration chromatographies generally as described in WO 94/03600. The affinity column 
described below is a Zn-IMAC column. The example used OP-l and is not intended to be 
limiting. The present protocol has general applicability to the purification of a variety of 

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morphogens, all of which are anticipated to be isolatable using only minor modifications of the 
protocol described below. An alternative protocol also envisioned to have utility includes an 
immunoaffinity column, created using standard procedures and, for example, using antibody 
specific for a given morphogen pro domain (complexed, for example, to a protein A-conjugated 
5 Sepharose colunm). Protocols for developing immunoaffinity columns are well described in the 
art (see, for example, Guide to Protein Purification, M. Deutscher, ed.. Academic Press, San 
Diego, 1990, particularly sections VII and XI thereoO- 

In this example, OP-1 was expressed in mammalian (CHO, Chinese hamster ovary) cells 
as described in the art (see, for example, international application US90/05903 (WO 91/05802). 

10 The CHO cell conditioned media containing 0.5% FBS is initially purified using Immobilized 

Metal-Ion Affinity Chromatography (IMAC). The soluble OP-1 complex fi-om 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 
purified soluble OP-1 is next applied to an S-Sepharose action-exchange column equilibrated in 

15 20 mM NaP04 (pH 7.0) with 50 mM NaCl. The protein then is applied to a Sephacryl S-200HR 
column equilibrated in TBS. Using substantially the same protocol, soluble morphogens also can 
be isolated fi-om one or more body fluids, including serum, cerebrospinal fluid or peritoneal fluid. 

The soluble OP- 1 complex elutes with an apparent molecular weight of 1 1 0 kDa. This 
agrees well with the predicted composition of the soluble OP-1 complex with one mature OP-1 
20 dimer (35-36 kDa) associated with two pro-domains (39 kDa each). Purity of the final complex 
can be verified by running the appropriate fi^action in a reduced 15% polyacrylamide gel. 

As an alternative to purifying soluble complexes fi^om culture media or a body fluid, 
soluble complexes can be formulated from purified pro domains and mature dimeric species. 
Successful complex formation apparently requires association of the components under 

25 denaturing conditions sufficient to relax the folded structure of these molecules, without affecting 
disulfide bonds. Preferably, the denaturing conditions mimic the environment of an intracellular 
vesicle sufficiently such that the cleaved pro domain has an opportunity to associate with the 
mature dimeric species under relaxed folding conditions. The concentration of denaturant in the 
solution then is decreased in a controlled, preferably step-wise manner, so as to allow proper 

30 refolding of the dimer and pro regions while maintaining the association of the pro domain with 
the dimer. Useful denaiurants include 4-6M urea or guanidine hydrochloride (GuHCl), in 



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buffered solutions of pH 4-10. preferably pH 6-8. The soluble complex then is formed by 
controlled dialysis or dilution into a solution having a final denaturant concentration of less than 
0.1-2M.urea or GuHCl. preferably 1-2 M urea of GuHCl, which then preferably can be diluted 
into a physiological buffer. Protein purification/renaturing procedures and considerations are well 
described in the art, and details for developing a suitable renaturing protocol readily can be 
determined by one having ordinary skill in the art. One useful text on the subject is Guide to 
Protem Purification, M. Deutscher. ed., Academic Press, San Diego, 1990, particularly section 
V. Complex formation also may be aided by addition of one or more chaperone proteins. 

The stability of the highly purified soluble morphogen complex in a physiological buffer. 
e g Tris-buffered saline (TBS) and phosphate-buffered saline (PBS), can be enhanced by any of a 
number of means, including any one or more of three classes of additives. These additives include 
basic amino acids (e.g., L-arginine, lysine and betaine); nonionic detergents (e.g.. Tween 80 or 
Nonldet P-120); and carrier proteins {e.g.. serum albumin and casein). Usefiil concentrations of 
these additives include 1-100 mM, preferably 10-70 mM. including 50 mM, basic amino acid;. 
5 0.01-1.0%, preferably 0.05-0.2%. including 0.1% (v/v) nonionic detergent;, and 0.01-1.0%, 
preferably 0.05-0.2%, including 0.1% (w/v) carrier protein. 

Example 2: Stroke Model Involving Surgical Occlusion of the Cerebral Artery 

The middle cerebral artery (MCA) occhision model is a well accepted model of a focal 
ischemic episode or stroke (Gotti, et al, (1990) Brain Res. 522: 290-307). Focal ischemia is 
>0 produced by obstructing blood flow through the MCA, resulting in infarction of the brain locus 
supplied by this artery The MCA model is reasonably predicUve of the ability and efficacy of 
drugs such as morphogen, to alter functional recovery in humans in whom central nervous system 
tissue has been damaged or lost due to stroke. For example, the MCA model is deemed 
reasonably predictive of drug efficacy to restore or detectably improve motor coordination, 
25 sensory perception, speech or any other central nervous system function naturally contnbuted to 
by tissue within the territory of the MCA. 

Animals that were treated with OP-l. beginning 24 hours after occlusion. of the MCA, 
performed significantly better than vehicle-treated animals in a variety of fimctional/behavioral 
tests as described below. 



wo 97/34626 PCT/US97/04177 
/. Surgical Occlusion Procedure 

The animals used in this study were male Sprague-Dawley rats weighing 250-300 grams 
(Charles River). For surgical procedures, the animals were anesthetized with 2% haJothane in 
70% NO2/30% O7 The tail artery was cannulated in order to monitor blood gases and blood 
5 glucose. Body temperature was monitored using a rectal probe and was maintained at 37 + O.S^C 
with a heating pad. The proximal right middle cerebral artery (MCA) was occluded permanently 
using a modification of the method of Tamura, ei al, (1981, J. Cereb, Blood Flow Metab, J : 
53-60). Briefly, the proximal MCA was exposed transcranially without removing the zygomatic 
arch or transacting the facial nerve. The artery was then electrocoagulated using a bipolar 

10 microcoagulator from just proximal to the olfactory tract to the inferior cerebral vein, and was 
then transected (Bederson, et ai, (1986) Stroke 17: 472-476). Rats were observed until they 
regained consciousness and were then returned to their home cages. Cefazolin sodium (40 
nig^gj i p ), an antibiotic, was administered to all animals on the day before and just after stroke 
surgery in order to prevent infection. During stroke surgery, there were no differences in the 

15 levels of blood gases or glucose among animals that subsequently received OP-1 or vehicle 
treatment. 

//. Administration of Morphogen 

Animals in the treatment group received OP-1 intracistemally at a dose of 1 or 
10 ng/injection. Control animals received vehicle solutions lacking OP-1 but with all other 

20 components at equivalent final concentrations. 

To administer the injection, the animals were anesthetized with halothane in 70% 
N02/30% O2 and placed in a stereotaxic frame. The procedure for intracistemal injection of OP-1 
containing solutions or vehicle-only solutions was identical. Using aseptic technique, OP-1 (1 or 
10 ^ig/injection ) or an equivalent volume of vehicle were introduced by percutaneous injection 

25 (10 ^il/injection) into the cisterna magna using a Hamilton syringe fitted with a 2^ gauge needle 
(Yamada, etal, (1991) J. Cereb. Blood Flow Metab. 11\ 472-478). Before each injection, 1-2 |il 
of cerebrospinal fluid (CSF) was drawn back through the Hamilton s>Tinge to verify needle 
placement in the subarachnoid space. Preliminary studies demonstrated that a dye, 1% Evans 
blue, delivered in this fashion diffused freely through the basal cisterns and over the cerebral 

30 cortex within one hour of injection. Animals were randomly assigned to either of the OP-1 
treatment groups or to the vehicle treatment group. 



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In a first study, intracistemal injections (10 ng/injection OP-1 or vehicle) were made 
biweekly for four weeks, starting 24 hours after stroke {i.e.. on post-stroke days 1 , 4. 8. 1 1, 1 5, 
18, 22, and 25). In a second study, animals received two intracistemal injections 
(2 X 1 ^:g/injection OP-1, 2X10 ng/injection OP-1 , or 2 X vehicle); the first injection was 
5 administered 24 hours after stroke and the second injection was administered 4 days after stroke. 
In a third study, a single injection (10 ^g/mjection OP-1 or vehicle) was administered 24 hours 
after stroke. 

///. Behavioral TesAng 

To accustom the animals to handling, which would be necessary for behavioral/fiinctional 
10 testing, they were handled for three days before surgery; for 10 minutes each day. Following 
surgery, they were housed in individual cages. Four standard fimctionalA)ehavioral tests were 
used to assess sensorimotor and reflex function after infarction. The tests have been fiiUy 
described in the literature, including Bederson, et ai, (1986) Stroke 17. 472-476; DeRyck, et al, 
(1992) Brain Res. 573, 44-60; Markgraf, ai, (1992) Brairt Res. 575: 238-246; and Alexis, ei 
15 a/., (1995) 5fro/te2<5: 2338-2346. 

A. The Forelimb Placing Test 
Briefly, the forelimb placing test is comprised of three subtests. Separate scores are 
obtained for each forelimb. For the visual placing subtest, the animal is held upright by the 
researcher and brought close to a table top. Normal placing of the limb on the table is scored as 
20 "0." delayed placing (< 2 sec) is scored as "1." and no or very delayed placing (> 2 sec) is scored 
as "2." Separate scores are obtained first as the animal is brought forward and then again as the 
animal is brought sideways to the uble (maximum score per limb=4; in each<:ase higher numbers 
denote greater deficits). For the tactile placing subtest, the animal is held so that it camiot see or 
touch the table top with its whiskers. The dorsal forepaw is touched lightly to the table top as the 
25 animal is first brought forward and then brought sideways to the table. Placing each time is 

scored as above (maximum score per hmb=4). For the proprioceptive placing subtest, the ammal 
is brought forward only and greater pressure is applied to the dorsal forepaw; placing is scored as 
above (maximum score per limb=2). These subscores are added to give the total forelimb placmg 
score per limb (range=01 0) In some ammals. the whisker placing subtest was done, in which the 
30 ability of the animal to place the forelimb in response to whisker stimulation by the tabletop was 



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tested (tnaximum score per limb = 2). Then subscores were added to give the total forehmb 
placing score per limb (range = 0-10, 0-12 with whisker subtest. 

A The Hindlimb Placing Test 

The hindlimb placing test is conducted in the same manner as the forehmb placing test but 
5 involves only tactile and proprioceptive subtests of the hindlimbs (maximal scores 4 and 2, 
respectively; total score range=0-6). 

C The Modified Balance Beam Test 

The modified balance beam test examines vestibulo motor reflex activity as the animal 
balances on a narrow beam (30 x 1 .3 cm) for 60 seconds. Ability to balance on the beam is 
10 scored as follows: 1 -animal balances with all four paws on top of beam; 2-animal puts paws on 
side of beam or wavers on beam; 3 -one or two limbs slip off beam; 4-three limbs slip off beam; 
5-animal attempts to balance with paws on beam but falls off; 6-animai drapes over beam, then 
falls oflF, 7-animal falls off beam without an attempt to balance. Animals received three training 
trials before surgery: the score of the last of these was taken as the baseline score. 

15 D. The Postural Reflex Test 

The postural reflex test measures both reflex and sensorimotor function. Animals are first 
held by the tail suspended above the floor. Animals that reach symmetrically toward the floor 
with both forelimbs are scored "0." Animals showing abnormal postures (flexing of a limb, 
rotation of the body) are then placed on a plastic-backed sheet of paper. Those animals able to 
20 resist side-to-side movement with gentle lateral pressure are scored " 1 while those unable to 
resist such movement are scored "2." All functionaL^ehavioral tests were administered just 
before stroke surgery and then every other day from post-stroke day 1 to day 3 L At each 
session, animals were allowed to adapt to the testing room for 30 minutes before testing was 
begun. 

25 IV, Histological Analysis 

On day 3 1 after MCA occlusion, animals were anesthetized deeply with pentobarbital and 
perfused transcardially with heparinized saline followed by 10% buffered formalin. Brains were 
removed, cut into three pieces, and stored in 10% buffered formalin before dehydration and 
embedding in paraffin. Coronal sections (5 ^im) were cut on a sliding microtome, mounted onto 
30 glass slides, and stained with hematoxylin and eosin. The area of cerebral infarcts on each of 



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seven slices (+4.7. +2.7, +0.7, -1.3, -3.3, -5.3, and -7.3 compared to bregma) was determined 
using a computer interfaced imaging system (Rioquant, R&M Biometnix, Inc., Nashville, TN). 
Total infarct area per slice was determined by the "indirect method" as (the area of the intact 
contralateral hemisphere] - [the area of the intact ipsilateral hemisphere] to correct for brain 
5 shrinkage during processing (Swanson, et al, (1990) J. Cereh. Blood Flow Metab. JO: 290-293) 
Infarct volume was then expressed as a percentage of the intact contralateral hemispheric volume 
The volumes of infarction in cortex and striatum were also determined separately using these 
methods. 

The practitioner performing intracistemal injections, behavioral tesUng, and histological 
10 analysis was blinded to the treatments assigned until all data had been collected. Data were 
expressed as means ± SD or means + SEM and were analyzed by repeated measures analysis of 
variance (ANOVA) followed by appropriate unpaired two tailed tests, with the Bonferroni 
correction for multiple comparisons. 
V. Results 

15 Difference in total infarct volume and body weight between OP-l-treated or 

vehicle-treated animak 

The right lateral cerebral cortex and underlying striatum of both OP-l-treated animals and 
vehicle-treated animals showed large infarcts in the territory of the MCA. Brain regions severely 
damaged by infarcts included parietal cortex, areas 1 and 2 (Pari, Par2) and granular insular 
20 cortex (GI). Regions partially damaged by infarcts included frontal cortex, areas 1 , 2, and 3 
(FRl, FR2, FR3); a granular insular cortex (Al); temporal cortex, areas 1 and 3 (Tell, Tel3); 
lateral occipital cortex, area 2 (Oc2L); the cortical forelimb area (FL), and the caudoputamen 
(cPu; Paxinos and Watson, 1986). The cortical hindlimb area (HL) was generally spared by 
infer cts. 

25 There was no difference in total infarct volume between animals treated with a series of 

OP-1 intracistemal administrations (8 X 10 ng /injection) and vehicle-treated animals (26.3 + 
2.5% vs. 28.0 + 2.0% of intact contralateral hemispheric volume, respectively, t=0.538, p-n.s.). 
Moreover, there was no difference in cortical or striatal infarct volume among the OP-l-treated 
animals and the vehicle-treated animals, when these volumes were calculated separately (cortex: 

30 30.9 + 3.1% vs. 3 1 .9 + 2.9% of intact contralateral cortex volume, respectively, t=0.254, p-n.s. 
striatum: 66,0 + 3.0% vs. 66.5 + 2.9% of intact contralateral striatum volume, respectively. 



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t=0. 121, p-n.s.) Funher, inspection of hematoxylin and eosin-stained sections showed no 
evidence of abnormal cell proliferation in the brains of OP- 1 -treated animals. Similarly, the total 
infarct volume of animals receiving a single OP-1 injection or two OP-1 injections did not differ 
significantly from the corresponding vehicle-treated animals (data not shown). 

5 The time course of body weight during the month after infarction of vehicle-treated 

animals did not differ significantly from: (a) animals treated with a series (8X10 ng/animal) of 
OP-1 administrations (FIG. 4; F=0.56, p-n.s ); (b) animals treated with two injections (High 
dose=2 X 10 i^g/animal; Low dose=2 X 1 ug/animal) of OP-1 (FIG. 7; F=0.417, p-n.s.); and 
(c) animals treated with a single injection (10 |ig/animai) of OP-1 (FIG. 10; F=0.693, p-n.s.). 

10 Functional Performance of OP-1 -Treated Animals and 

Vehicle-Treaied Animals 

Following infarction, all animals showed severe disturbances of sensorimotor and reflex 
function on all four behavioral tests. For the limb placing tests, deficits were confined to the 
contralateral (lefi) limbs. Animals receiving the vehicle showed partial recovery on all four 
15 behavioral tests during the first month after stroke (see FIGS. 2A-2B, 3A-3B, 5A-5B, 6, 8A-8B, 
and 9). 

(i) Animals Receiving Biweekfy OP'l Administrations 

Animals receiving biweekly OP- 1 administrations (8 X 1 0 ^ig /injection) recovered more 
rapidly and to a greater degree than vehicle-treated rats. Improved recovery of OP-1 vs. vehicle- 

20 treated animals was most pronounced for the forelimb (FIG. 2A; F= 1 09.0, p=0.000 1 ) and 
hindlimb placing tasks (FIG. 2B; F=34.8, p=0.0001), and less pronounced, although still 
significant, for the beam balance (FIG. 3 A; F=I 1 .7, p=0.005 1 ). However, there was no 
significance among the two groups in the postural reflex tests (FIG. SB; F=3 .7, p-n.s ). 
Enhanced recovery was seen on all subtests of the limb placing tests (visual, tactile, and 

25 proprioceptive) following OP- 1 treatment (data not shown). 

Enhancement of recovery by OP-1 was most pronounced on tests of sensorimotor 
ftmction of the affected limbs and less pronounced on tests of reflex and postural fijnctioh. The 
MCA infarcts did not completely damage forelimb and hindlimb conical areas, which is 
compatible with recovery on limb placing tests following focal infarction in the MCA t<erritory. 



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(ii) Animals Receiving Two OP-1 Administrations 

Animals receiving two OP-1 administrations (on post-stroke days 1 and 4) recovered 
more rapidly and to a greater degree than vehicle-treated rats during the month of behavioral 
testine. OP-1 (2 X 1 or 10 ^g /injection) induced significant enhancement of recovery of: 

5 (a) foreiimb placing without whisker (FIG. 5A; F=31.835. p=0.0001; High dose vs. vehicle, 
p<0.0001 , Low dose vs. vehicle, p<0.0001); (b) foreiimb placing with whisker (FIG. 5B; 
F=27.462, p=0.0001; High dose vs. vehicle, p<0.0001; Low dose vs. vehicle, p<0.0001); and 
(c) hindlimb placing (FIG. 6; F=14.867, p=0.0001; High dose vs. vehicle, p<0 0001; Low dose vs. 
vehicle, p=O.0036). Although the High dose produced a trend toward better recovery than the 

10 Low dose in all three behavioral tests, the differences between the two OP- 1 -treated groups were 
non-significant. 

ftii) Animals Receiving a Single OP-1 Administrations 

Long-term enhancements of fimctional recovery also were seen with a single 
administration of OP-1 . Animals receiving 10 ^g of OP-1 intracisteraally 24 hours after the 
15 occlusion of the MCA recovered more rapidly and to a greater degree during the month of 

behavioral testing than vehicle-treated rats. OP-1 induced significant enhancement of recovery of: 
(a) foreiimb placing without whisker (FIG. 8A; F=10.853, p=0.0064); (b) foreiimb placing with 
whisker (FIG. 8B; F=10.629, p=0.0068); and (c) hindlimb, placing (FIG. 9; F=15.343, p=0.002). 

In the present invention, treatment of an ischemic injury of the central nervous system with 
20 OP-1 enhanced both the rate and degree of fimctionalrecoveiy during the first month after 

infaraion. A single administration of an effective dose of OP-1 was sufficiem to induce long-term 
enhancement of functional recovery. 

Improved behavioral recovery was seen without a change {e.g., vnXhoux a decrease) in 
infarct volume in OP-l-treated compared to vehicle-weated animals In all these groups, the OP-1 
25 administration commenced one day after ischemia, beyond the apparent "therapeutic window" 
during which OP-1, according to the teachings of WO 93/04692 and/or WO 94/03200, can 
reduce infarct size. The current findings are among the first demonstrations that an exogenously 
administered, biologically active factor can enhance behavioral recovery without a reduction in 
infarct size in an animal model of stroke. 



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Similar routine modifications can be made in other accepted models of stroke or traumatic 
central nervous system injury, to confirm efficacy of morphogen treatment to restore impaired or 
lost CNS function. 

Equivalents 

5 The invention may be embodied in other specific forms without departing from the spirit 

or essential characteristics thereof The foregoing embodiments are therefore to be considered in 
all respects illustrative rather than limiting on the invention described herein. Scope of the 
invention is thus indicated by the appended claims rather than by the foregoing description, and all 
changes which come within the meaning and range of equivalency of the claims are intended to be 
10 embraced therein. 



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SEQUENCE LISTING 

(1) GENERAL INFORMATION: 

(i) APPLICANT: CHARETTE, Marc F. 

FINKLESTEIN, Seth P. 

(ii) TITLE OF INVENTION: METHODS FOR ENHANCING FUNCTIONAL 

RECOVERY FOLLOWING CENTRAL NERVOUS SYSTEM ISCHEMIA OR 
TRAUMA 

(ili) NUMBER OF SEQUENCES: 9 

^iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: CREATIVE BIOMOLECULES , INC 

(B) STREET: 45 SOUTH STREET 

(C) CITY: HOPKINTON 

(D) STATE: MA 

(E) COUNTRY: USA 

(F) ZIP: 01748 

(V) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

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

(D) S0FTW3\RE: Patentin Release #1.0, Version #1.30 

(vi) CURRENT APPLICATION DATA: 

(A) APPLICATION NUMBER: 

(B) FILING DATE: 

(C) CLASSIFICATION: 

(viii) ATTORNEY /AGENT INFORMATION: 

(A) NAME: FENTON, GILLIAN M 

(B) REGISTRATION NUMBER: 36,508 

(C) REFERENCE/DOCKET NUMBER: CRP-069CP 

(ix) TELECOMMUNICATION INFORMATION: 
<A) TELEPHONE: (617) 24B-7000 
{B) TELEFAX: < 617) 248-7100 

(2) INFORMATION FOR SEQ ID N0:1: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: 

(D) TOPOLOGY: linear 

Hi) MOLECULE TYPE: protein 

Ux) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1 . . 97 ^ . 
(D) OTHER INFORMATION: /label= Generic-Seg-7 

/note- "wherein each Xaa is independently selected from a group 
of one or moire specified amino acids as defined in the 
specification. " 

ixi) SEQUENCE DESCRIPTION: SEQ ID N0:1: 

Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa Xaa Xaa Xaa Xaa 

Add " — 'J- ^* ' • 

20 . 25 



Pro xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pi 

20 ■ 25 

Xaa Xac ^aa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa 
35 40 

xaa xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro 

50 55 60 

xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
65 "70 

val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys 
85 90 

Xaa 



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(2) IIIFORMATION FOR SEQ ID NO: 2: 

ii) SEQUENCE CHARACTERISTICS: 

{A) LENGTH: 102 anano acids 
(B) TVPE: anvino. acid 
fC) STRANDEDNESS : 
(D) TOPOLOGY: linear 

Ui) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

(D) OTHER INFORMATION: /label= Generic-Sea-8 
/note= "wherein each Xaa is independently selected from a group 
of one or more specified amino acids as defined in the 
specification." 

(xi) SEQUENCE DESCRIPTION: SEO ID NO: 2: 

Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Cly Trp Xaa 
. 1 5 10 15 

Xaa Xaa 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 
35 40 45 

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

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

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 
100 

(2) INFORMATION FOR SEQ ID N0:3: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(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. " 

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

Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xaa Asp Leu Gly Trp Xaa 
15 10 15 

Asp Trp Xaa He Ala Pro Xaa Glv 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 
35 40 45 

He Xaa Gin Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys 
50 55 60 

Xaa Cys Cvs Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 
65 * 70 75 BO 

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 



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201 



100 

{2) INFORMATION FOR SEQ ID N0:4: 

U) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1822 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : single 
it) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

(VI) ORIGINAL SOURCE: 

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

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 49. .1341 ^ ^ 

(C) IDENTIFICATION METHOD: experimental 

(D) OTHER INFORMATION: /functlon== "OSTEOGENIC PROTEIN 
/product" "OPl" 
/evidence- EXPERIMENTAL 
/5tandard_name« "OPl" 

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

GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG 57 

Met nis vai 
1 

CGC TCA CTG CGA GCT GCG GCG CCG CAC AGC TTC GTG GCG CTC TGG GCA 105 
X±i Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala 
5 10 1^ 

CCC CTG TTC CTG CTG CGC TCC GCC CTG GCC GAC TTC AGC CTG GAC AAC 153 
Pro Leu Phe Leu Leu Ara Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn 
20 25 30 3b 

GAG GTG CAC TCG AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG 
Glu Val His Ser Ser Phe He His Arg Aro Leu Arg Ser Gin Glu Arg 
40 45 =>y 

CGG GAG ATG CAG CGC GAG ATC CTC TCC ATT TTG GGC TTG ^CC CAC CGC 249 
Axq Glu Met Gin Arg Glu lie Leu Ser He Leu Gly Leu Pro His Arg 

55 ^60 

CCG CGC CCG CAC CTC CAG GGC AAG CAC AAC TCG GCA CCC ATG TTC ATG 297 
Pro irg Pro His Leu Gin Gly Lvs His Asn Ser Ala Pro Met Phe Met 
70 *5 BO 

CTG GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG GGC GGC GGG CCC GGC 345 
lIS Asp Leu ?yr ila Met Ala Val Glu Glu Gly Gly Gly Pro Gly 
85 90 

GGC GAG GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AGT ACC CAG GGC 393 
Gl^ Gil III sir T^r Pro Tyr Lys Ala Val Phe Ser Thr Gin Gl^ 

CCC eCT CTG GCC AGC CTG CAA <3AT 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 
X20 125 1-3" 

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

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

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 Giu Phe Arg He Tyr Lys Asp 
165 1"'0 

TAC ATC CGG GAA CGC TTC GAC AAT GAG ACG TTC CGG ATC AGC GTT TAT 633 
?yr lie A^g ^ Phe Asp Asn Glu Thr Phe Arg He Ser Val Tyr 
leo 185 I'O 

CAr rrr CTC CAG GAG CAC TTG GGC AGG GAA TCG GAT CTC TTC CTG CTC 681 
Gin vll llu lln ilu His llu Gly Arg Glu Ser Asp Leu Phe Leu Leu 
200 205 

GAC AGC CGT ACC CTC TGG GCC TCG GAG GAG GGC TGG CTG GTG TTT GAC 729 

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Asp Ser Arg Thr X.eu Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp 
215 22C 225 

ATC ACA GCC ACC AGC AAC CAC TGG GTG GTC AAT CCG CGG CAC AAC CTG "77 
lie Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arq His Asn Leu 
230 235 246 

GGC CTG CAG CTC TCG GTG GAG ACQ 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 =73 
Lys Leu Ala Gly Leu lie Gly Arg His Gly Pro Gin Asn Lys Gin Pro 
260 265 270 275 

TTC ATG GTG GCT TTC TTC AAG GCC ACG GAG GTC CAC TTC CGC AGC ATC -21 
Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser He 
280 285 290 

CGG TCC ACG GGG AGC AAA CAG CGC AGC CAG AAC CGC TCC AAG ACG CCC 969 
Arg Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Ser Lvs Thr Pro 
295 300 3^)5 

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

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 T^ ATC ATC GCG CCT GAA GGC TAC GCC 1113 
Ara Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly Tyr Ala 
340 345 350 355 

f?^ l^^ l^^ ^ GCC TTC CCT CTG AAC TCC TAC ATG 1161 

Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met 
360 365 370 

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

Sr^ ^ SE*^ "^^"^ ACG CAG CTC AAT GCC 1257 

Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala 
390 395 400 

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

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

GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT CCATTGCTCG CCTTGGCCAG 1411 

GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA ACTTTAAAGG 1471 

TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CAGTGGCAGC 1531 

ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AAAAAACAAC 1591 

GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TGCACGGACT 1651 

CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GGAGGAAGGG 1711 

GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTGAC CCGGAAGTTC 1771 

CTGTAATAAA TGTCACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A 1S22 

(2) INFORMATION FOR SEQ ID NO:S: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 431 amino acids 

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

(ii) MOLECULE TYPE; protein 

(Xi) SEQUENCE DESCRIPTION: SEO ID N0:5: 

Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 
* h 10 IS 

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Leu Trp Ala Fro Leu Phe Leu Leu Arg 5er Aia Leu Ala Asp Phe Ser 
20 2S 

Leu ASP Asn Glu Val His Ser Ser Phe lie His Arg Ara Leu Arg Ser 
35 40 

Gin Glu Ara Arg Glu Met Gin Aj:g Glu lie Leu Ser He Leu Gly Leu 



50 ■ ' 55 60 

Pro His Arg Pro Arg Pro His Leu Gin Giy Lvs His Asr. Ser Ala Pro 
65 70 

Met Phe Met Leu As| Leu Tyr Asn Ala Met Ala Val Glu Glu Gl^ Gly 

Gly Pro Gly Gl^ Gin Gly Phe Ser Tgr Pro Tyr Lys Ala Val Phe Ser 

Thr Gin Gly Pro Pro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr 
115 120 '■^^ 

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

Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu 

145 150 '-^^ 

ser Lys He Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Ara He 

165 I'O 

Tyr Lys Asp T^r lie Arg Glu Axg Phe Asp Asn Glu Thr Phe Arg He 
ser Val Tyr Gin Val Leu Gin Glu His Leu Gly Arg Glu Ser Asp Leu 



195 



Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu 

210 215 
val Phe Asp lie Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg 
225 230 ''JS 

His Asn Leu Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser 

lie Asn Pro Lys Leu Ala Gly Leu lie Gly Arg His Gly Pro Gin Asn 

260 265 
Lys Gin Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe 

^ 275 280 

Arg ser He Arg Ser Thr Glv Ser Lys Gin Arg Ser Gin Asn Arg Ser 

290 295 
Lys Thr Pro Lys Asn Gin Glu Ala Leu Arg Met Ala Asn Val Ala Glu 
305 310 ■''■^ 

Asn ser. ser Ser As| Gin Arg Gin Ala C^s Lys Lys His Glu Leu Tyr 

val ser Phe Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu 

Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn 

355 360 ■'o^ 

ser Tvr Met Asn Ala Thr Asn His Ala He Val Gin Thr Leu Val His 

370 3^- 
Phe lie Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin 
385 -59b 

Leu Asn Ala lie Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val He 

Leu Lvs Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
420 

(2) INFORMATION FOR SEQ ID N0:6: 

(i) SEQUENCE CHARACTERISTICS: 

(Al LENGTH: 97 amino acids 

(Bl TYPE: amino acid 

(C) STRANDEDNESS: single 

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(D) TOPOLOGY: linear 
Ui; MOLECULE TYPE: protein 

iix) FEATURE: 

(A} NAME/KEY: Protein 

(E) LOCATION: 1 . . 97 

(D) OTHER INFORMATION: /label«= Generic-Seq-9 
/note= "wherein each Xaa is independent iy selected from a group 
of one or more specified amino acids as defined in the 
specification . *' 

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

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
lb 10 15 

Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly 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 Xaa Cys Xaa Pro 

- 50 55 60 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

£5 70 . 75 80 

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: 7: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE; amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

(D) OTHER INFORMATION: /label« Generic-Seq-10 
/note« "wherein each Xaa is independently selected from a group 
of one or more specified amino acids as defined in the 
specification." 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7: 

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

Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Glv 
20 25 30 

Xaa Cys 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 Xaa Xaa Xaa Xaa 

50 55 60 

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

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
65 90 95 

Xaa Xaa Cys Xaa Cys Xaa 
100 

(2) INFORMATION FOR SEQ ID NO: 8: 

ii) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 5 amino acids 

(B) TYPE: amino acid 



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(C) STRANDEDNESS: 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 



(iX) FEATURE:. 

(A) NAME/KEY: Protein 



D OTHER INFORMATION: ^note- "wherein each Xaai- 
independently selected from a group of one or more specified 
amino acids is defined in the specification 

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

cys Xaa Xaa Xaa Xaa 

1 S 

(2) INFORMATION FOR SEQ ID N0:9: 

ii) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B} TYPE: amino acid 

(C) STRANDEDNESS : 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(ixi FEATURE: 

(A) NAME/KEY: Protein 

' \l] S'^Sfo^TION: /note^ "wherein each xaa is 
independently selected from a 9^^^?^^^ specified 
amino acids Is defined in the specification 

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

Cys Xaa Xaa Xaa Xaa 
1 5 



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What is claimed is: 

A method for enhancing recovery of central nervous system function in a mammal, 
comprising the step of: 



3 administering an effective amount of a morphogen to a mammal afflicted with a central 

4 nervous system injury selected from ischemia or trauma, 

5 wherein said morphogen comprises a dimeric protein having the property of inducing 

6 tissue-specific morphogenesis in said mammal and comprising a pair of folded 

7 polypeptides, each having an amino acid sequence having at least 70% homology with the 

8 C-terminal seven-cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: 5. 

1 2. The method of claim 1 , wherein said functional recovery comprises an improvement in a 

2 central nervous system function selected from motor coordination function, sensory 

3 perception, and speech. 

1 3 The method of claim 2, wherein said motor coordination function is selected from posture, 

2 balance, grasp and gait. 

1 4. The method of claim 2, wherein said sensory perception is selected from vision, hearing, 

2 touch, taste, proprioception, and olfaction. 

1 5. The method of claim 1 , wherein said mammal is a human. 

1 6. The method of claim 1 , wherein the effective amount of a morphogen is provided in a 

2 single administration. 

1 7. The method of claim 1 , wherein the effective amount of a morphogen is provided in a 

2 phirality of administrations. 

1 8 The method of claim 6, wherein the effective amount of a morphogen is provided in two 

2 administrations. 

1 9. The method of claim 1 , 6, 7 or 8 wherein the effective amount of a morphogen is 

2 administered at least 6 hours afler the onset of said injury. 

1 10. The method of claim K 6, 7 or 8 wherein the effective amount of a morphogen is 

2 administered at least 24 hours after the onset of said injury 

. 49 . 



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1 1 L The method of ciaim 1 , 6, 7 or 8 wherein the effective amount of a morphogen is 

2 administered at least 48 hours after the onset of said injury. 

1 12. The method of claim 7, wherein the morphogen is administered daily, 

i 13. The method of claim 7, wherein the morphogen is provided biweekly. 

1 14. The method of claim 7, wherein the morphogen is provided weekly. 

1 15. A method for enhancing recovery of central nervous system iunction in a mammal, 

2 comprising the step of: 

3 administering an effective amount of a morphogen to a mammal afflicted with a central 

4 nervous system injury selected from ischemia or trauma, 

5 wherein said morphogen comprises a dimeric protein having the property of inducing 

6 tissue-specific morphogenesis in said manunal and comprising a pair of folded 

7 polypeptides, each having an amino acid sequence selected from the group consisting of: 

8 (a) Generic Sequence 7 defined by SEQ ID NO: 1 , 

9 (b) Generic Sequence 8 defined by SEQ ID NO: 2; 

10 (c) Generic Sequence 9 defined by SEQ ID NO: 6; and 

1 1 (d) Generic Sequence 1 0 defined by SEQ ID NO: 7; 

1 16. The method of claim 1 or 15, wherein said amino acid sequence is selected from the group 

2 consisting of: 

3 (a) a sequence having greater than 60% amino acid sequence identity with the 

4 C-terminal seven-cysteine domain of human OP-1 , residues 38-139 of SEQ © NO: 5; and 

5 (b) OPX sequence defined by SEQ. ID NO: 3 . 

1 17. The method of claim 1 or 1 5, wherein said wherein said amino acid sequence is that of the 

2 C-terminai seven-cysteine domain of human OP-K residues 38-139 of SEQ ID NO: 5 or a 

3 conservative substitution variant thereof 

1 18. The method of claim 1 or 1 5, wherein said wherein said amino acid sequence is that of the 

2 G-terminal seven-cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: 5 or a 

3 naturally-occurring variant thereof 

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1 19. A method for enhancing recovery of central ner\'ous system funaion in a mammal, 

2 comprising the step of 

3 administering an effective amount of a morphogen to a mammal afflicted with a central 

4 nervous system injury selected from ischemia or trauma, 

5 wherein said morphogen is selected from the group consisting of human OP- 1 , mouse 

6 human OP-2, mouse OP-2, 60A, GDF-L BMP-2A, BMP-2B, DPP, Vgl, Vgr-l, 

7 BMP-3. BMP.5, and BMP-6. 

1 20 A method for enhancing recovery of central nervous system fiinction in a mammal, 

2 comprising the step of: 

3 . administering an effective amount of a morphogen to a mammal afflicted with a central 

4 nervous system injury selected from ischemia or trauma, 

5 wherein said morphogen is a conservative substitution variant of a morphogen selected 

6 from the group consisting of human OP-1, mouse OP- 1, human OP-2, mouse OP-2, 60 A, 

7 GDF- 1 , BMP-2A, BMP-2B, DPP, Vgl, Vgr-l , BMP-S, BMP-5, and BMP-6 

1 21, The method of claim 1, 6, 7, 8, 15, 19 or 20 wherein said morphogen is complexed with at 

2 least one pro-domain peptide comprising an N-terminal 18 amino acid peptide selected 

3 from the group consisting of N-termini of the pro domains of OP- 1 , OP-2, 60 A, GDF- 1 , 

4 BMP.2A, BMP-2B, DPP, Vgl, Vgr- 1 , BMP-3, BMP-5, and BMP-6. 

1 22, The method of claim 1 , 6, 7, 8, 1 5, 1 9 or 20 wherein said morphogen is complexed with at 

2 least one pro-domain polypeptide that is a conservative substitution variant of a pro- 

3 domain polypeptide selected from the group consisting of the pro-domains of OP- 1 , OP-2, 

4 60A, GDF- 1 , BMP-2A, BMP-2B, DPP, Vgl, Vgr- 1 , BMP.3, BMP-5, and BMP.6 

1 23. The method of claim 1, 6, 7, 8, 15, 19 or 20 wherein said morphogen is noncovalently 

2 complexed with at least one solubility-enhancing fragment of a pro-domain polypeptide 

3 selected from the pro-domains of naturally-occurring morphogens. 

1 24. The niethod of claim 23, wherein said morphogen is complexed with a pair of said 

2 fragments 

1 25. The method of claim I, 6, 7, 8, 15, 19 or 20 wherein said morphogen is obtained from 

2 culture supernatant of a morphogen secreting host cell. 

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1 26 The method of claim 1 , 6, 7, 8. 1 5, 1 9 or 20 wherein said morphogen is administered 

2 intracistemally, intraventricularly, intrathecally or intravenously. 



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% Amino Acid Sequence Similarity, Identity to Human OP-1 
7-Cysteine Domain 



>=70% 
Sequence 



hOP-1 (hBMP-7) 
raOP-1 (mBMP-7) 
hOP-2 (hBMP-8) 
mOP.2 (mBMP-8) 
hBMP.5 
hBMP-6 (Vgr-l) 
Vgr-l (PT) 
OP-3 
d60A 

BMP-4 (BMP-2b) 
BMP-2 (BMP.2a) 
dpp 

sUNTVIN 
xVg-l 

hCDMP-1 (mGDF-5) 

hCDMP-3 (mGDF-y, hBMP.12) 

mGDF.3 (hVgr-2) 

hCDMP.2 (mGDF-6. hBMP.13) 

cDORSALIN 

hGDF-1 

mGDF-10 

rBMP.3b 

hBMP-10 

hBMP-3 

dSCREW 

ADMP 

mODF-l 

hBMP-9 

mNODAL 

hBMP-I5 



* Similarity 


% IdentitA- 


1 nn 


100 


1 AA 
iUU 


99 


at 

yl 


72 


97 


75 


97 


88 


96 


87 


94 


85 


91 


66 


90 


69 


90 


58 


89 


60 


87 


57 


87 


63 


86 


58 


85 


50 


83 


54 


83 


50 


82 


53 


79 


50 


78 


49 


78 


40 


78 


41 


78 


47 


78 


43 


77 


49 


77 




73 


50 


73 


52 


71 


41 


7! 


41 



Species Labels: 

n=Human. m^Mouse. x=Xenopus. c=Chicken. s*Sea Urchin. d»Drosoptiilia. r^rat 



FIG. 1 



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FIG. 2A 



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FIG. 2B 



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FIG. 3A 



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FIG. 3B 



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SUBSTITUTE SHEET (RULE 26) 



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FIG. 5 A 



T 



J. 



-o- 



or- 



7 



11 



15 



Days After Stroke 



"7" 

23 



27 



31 



High Dose (1 0 jig/ injection) 



Low Oose injection) 



Vehicle 



FIG. SB 



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FIG. 6 



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500-1 




FIG. 7 



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PCr/US97/04177 




FIG. 8A 



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FIG. 8B 



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FIG. 9 



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FIG. 10 



INTERNATIONAL SEARCH REPORT 



Intenu J Appiiution No 

PCT/US 97mV7 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 A61K38/18 



Acconiifig to tntemationri Patent Qatnficttion (IPC) or to both ntoottfl dagification and IPC 



B. FIELDS SEARCHED 



Minimum documentaDon searched (classification system followed by dassncafion symbols) 

IPC 6 A61K 



Documeniaoon uarchcd other than minimum documentation to the extent thai such documents are mchided in the fields searched 



Electronic data base consulted during the international search (name of data base and, where practical, search terini used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category ' Qtaiion of document, with indication, wbae appropriate, of the relevant passages 



Relevant to claim No. 



NEUROSCIENCE LETTERS, 

vol. 187. 1995. 

pages 21-24. XP002G37656 

PERIDES ET AL: •NEUROPROTECTIVE EFFECT OF 

HUMAN OSTEOGENIC PROTEIN- 1 IN A RAT MODEL 

OF CEREBRAL HYPOXIA/! SCHEMIA" 

see the whole document 

WO 94 03200 A (CREATIVE BIONOLECULES INC] 

17 February 1994 

cited in the application 

see page 8, line 4 - page 9, line 5 

see page 17. line 28 - page 19, line 3 

see page 97 - page 99; example 10 

-/-- 



1-26 



1-26 



CD 



Further doctancntt are listed in the oontinuabon of box C. 



0 



Patent family members arc listed in annex. 



* Special categories of cited documents : 

'A* document defining the general suu of the art which is not 

oofifidervd to be of partcular relevance 
'E* earlier document but published on or after the tntemationaJ 

filing date 

'L' document which may throw doubts on priority claini(t) or 
which is ated to csubltsh the publication datt of another 
dtation or other special reason (as spcofied) 

'O' doctnnent refemng to an oral disclosure, use, exhibition or 
other means 

'P' document published pnor to the international filing date but 
later than tiie pnoril^ date claimed 



'T* later document publtshed alter the international filing date 
or pnonly date and not in oonflict with the af^icabon but 
cited to understand the phnciple or theory underlying (he 
mvention 

'X' document of particular relevance; the daimed invention 
cannot be considered novel or cannot be considered to 
mvolve an inventive step when the document is taken alone 

* Y' documecrt of partcular relcvanor. the claimed invention 
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 an 

'A' document member of the same patent family 



Date of the actual completion of the mtcriMtional search 



13 August 1997 



Dau of mailing of the international search report 

25.08.97 



Name and mailing address of the ISA 

Etiropcan Patent Office, P.B. 5818 Pauntlaan 3 
KL • 2380 HV Rijswijk 
Td. < 4 31 70) 340- 2040, Tx, 31 651 epo nl. 
Fax 31-70)540 3016 



Authorized officer 



Sitch, W 



Form PCT.lSAatO (Mcontf ihaet) <July 1993) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



Inteii. .lal ApplicaSon No 

PCT/US 97/04177 



C^ContimuOon) DOCUMENTS CONSIDERED TO BE RELEVANT 



Catetory * I Quson of documtni. wiih indicitton, wtoe ippropnstt, of the rtlevmt pasisu 



WO 93 G4692 A (CREATIVE BIOMOLECULES INC) 

18 March 1993 

cited in the application 

see page 9, line 3 - page IG, line lO 

see page 55, line 31 - page 56, line 22 

see page 75 - page 78; example 6 

WO 92 15323 A (CREATIVE BIOMOLECULES INC) 

17 September 1992 

cited in the application 

see page 6, line 3 - page 7, line 27 

see page 61, line 12 - line 17 

see page 65, line 7 - line 33 



1-26 



1-26 



Form PCT-1SA-21II (cojiiiftttJtion of mcdaO ib«*«) (J»»y 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 



cmaitonal application No. 

PCT/US 97/04177 



Box i Observations where certain claims were found unsearchable (Continuation of itcro I of first sheet) 

This Iniernational Search Report has noi betn esublishtd in respect of certain claims under Article 17(2)(i) for the following reasons: 

1. \T\ Claims Nos.: 1-26 

because they relate to subject matter not required to be searched by this Authority, namely: 

Remark: Although claims 1 to 26 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. 

Claims Nos.: 

because they relate to paru 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: 



3. Q Claims Nos.: 

because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(aX 

Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) 
This International Searching Authority found multiple inventions in this international application* as follows: 



1. As alt required additiona] search Tees were timely paid by the applicant, this International Search Report covers all 
— ' searchable dainu. 

2. I I As all searchable ciaims could be searched without effort justifying an additional fee. this Authority did not invite payment 

of any additional fee. 



3. I At only some of the required additional search fees were timely paid by the applicant, this International Search Report 
■ ' covers only those claims for which fees were paid, specifically claims Nos.: 



4- I I No required additional search fees were timely paid by the applicant. Consequently, this International Search Report is 
restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 



Remark on Protest j | The additional search fees were accompanied by the applicant's protest 

j I No protest accompanied the payment of additional search fees. 



Form PCT;ISA,710 (continuation of first sheet (1)) (July 1992) 



INTERNATIONAL SEARCH REPORT 



IMonnibon on patent family mcmben 



Inh ional Applicalion No 

PCT/US BJ/om 



Pucnt document 
dud in March repoit 



Publication 
dau 



Patent faroiiy 
mcinbei(i) 



Publication 
date 



WO 940320G A 



17-02-94 



AU 

AU 

AU 

AU 

AU 

AU 

AU 

AU 

AU 

CA 

CA 

CA 

CA 

EP 

EP 

EP 

EP 

EP 

EP 

EP 

OP 

JP 

JP 

JP 

JP 

JP 

JP 

WO 

WO 

WO 

WO 

WO 

WO 

WO 

US 

US 



678380 B 
4795193 A 
4797193 
4995593 
5129293 
5129393 
5162393 
529G893 
559G094 
2141554 
2141555 
2141556 
2147598 
0652953 
0653942 
0661933 
0665739 
0661987 
0680334 
0672064 
7509611 
7509720 
7509721 
8501779 
8501558 
8501315 
8503198 
94036G0 
9403G75 
9406447 
9406399 
9406449 
9406420 
9410203 
5652337 
5652118 



A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
T 
T 
T 
T 
T 
T 
T 
A 
A 
A 
A 
A 
A 
A 
A 
A 



WO 9304692 A 



18-03-93 



AU 669127 B 
AU 2564592 A 
AU 670558 B 



29-05-97 
03-03-94 
03-03-94 
03-03-94 
12-04-94 
12-04-94 
12-04-94 
12-04-94 
24-05-94 
17-02-94 
17-02-94 
17-02-94 

11- 05-94 
17-05-95 
24-05-95 

12- 07-95 
09-08-95 

12- 07-95 

08- 11-95 
20-09-95 
26-10-95 
26-10-95 

26- 10-95 

27- 02-96 
20-02-96 

13- 02-96 

09- 04-96 
17-02-94 
17-02-94 
31-03-94 
31-03-94 
31-03-94 
31-03-94 
11-05-94 
29-07-97 
29-07-97 



30-05-96 
05.-04-93 
25-07-96 



Form PCT./t&A/210 <p«unt family uitMk] (J«ly 1992) 



page I of 2 



INTERNATIONAL SEARCH REPORT 



Inromubon on patent family members 



Inter, jnal Application No 

PCT/US 97/04177 



Patent document 
rit^ in search reoort 


T ' 

1 Publkaiion 


Patent famiJy 


Publication 


date 




membcr(s) 




WO 9304692 A 




AU 


3176293 A 


27-04-93 




CA 


2104678 A 


12-09-92 






CA 


2116559 A 


01-04-93 






CA 


2116562 A 


18-03-93 






t r 


0601106 A 


15-06-94 






EP 


0601135 A 


15-06-94 






JP 


6510989 T 


08-12-94 






JP 

w r 


7502021 T 


02-03-95 






WO 


9305751 A 


01-04-93 








5650276 A 


22-07-97 






All 
nU 




29-05-97 






AU 


2862492 A 


05-04-93 






CA 


2116560 A 


18-03-93 






FP 


0601129 A 


15-06-94 






IP 

Or 




24-11-94 






nU 


Q305172 A 


18-03-93 






IK 


565P337 A 


29-07-97 








5fi<)?1 Ifi A 


29-97-97 


WO 9215323 A 


17-09-92 


AU 


66G019 B 


08-06-95 






All 

AU 


l/b4j9^ A 


06-10-92 






CA 


2104678 A 


12-09-92 






EP 


0575555 A 


29-12-93 






JP 


6506360 T 


21-07-94 






US 


5652337 A 


29-07-97 






US 


5652118 A 


29-07-97 



Fonn PCT/lSA.aiO (paufit famUy anncv) (July 1993] 



page 2 of 2 



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